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Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

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Original Article

Acta Pharmacologica Sinica (2010) 31: 821–830; doi: 10.1038/aps.2010.67; published online 28 June 2010
Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

Chen Zhang1, Kun-zheng Wang1, Hui Qiang1, Yi-lun Tang1, Qian Li2, Miao Li2 and Xiao-qian Dang1

1. 1Department of Orthopedic Surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China
2. 2Department of Ultrasound, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China

Correspondence: Xiao-qian Dang, E-mail dang_xiaoqian@sohu.com

Received 22 February 2010; Accepted 6 May 2010; Published online 28 June 2010.
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Abstract
Aim:

To investigate the therapeutic potential of adeno-associated virus (AAV)-mediated expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP).
Methods:

Four experimental groups were administered the following AAV vector constructs: rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF165-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP). VEGF165 and BMP-7 gene expression was detected using RT-PCR. The VEGF165 and BMP-7 protein expression was determined by Western blotting and ELISA. The rabbit ischemic hind limb model was adopted and rAAV was administered intramuscularly into the ischemic limb.
Results:

Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were cultured and infected with the four viral vectors. The expression of GFP increased from the 7th day of infection and could be detected on the 28th day post-infection. In the AAV-VEGF/BMP group, the levels of VEGF165 and BMP-7 increased with prolonged infection time. The VEGF165 and BMP-7 secreted from BMSCs in the AAV-VEGF/BMP group enhanced HUVEC tube formation and resulted in a stronger osteogenic ability, respectively. In rabbit ischemic hind limb model, GFP expression increased from the 4th week and could be detected at 8 weeks post-injection. The rAAV vector had superior gene expressing activity. Eight weeks after gene transfer, the mean blood flow was significantly higher in the AAV-VEGF/BMP group. Orthotopic ossification was radiographically evident, and capillary growth and calcium deposits were obvious in this group.
Conclusion:

AAV-mediated VEGF and BMP gene transfer stimulates angiogenesis and bone regeneration and may be a new therapeutic technique for the treatment of avascular necrosis of the femoral head (ANFH).
Keywords:

adeno-associated virus; vascular endothelial growth factor; bone morphogenetic protein (BMP); avascular necrosis of the femoral head (ANFH); gene therapy
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Introduction

Recent insight into the pathogenesis of avascular necrosis of the femoral head (ANFH) has not identified satisfactory methods to increase blood circulation in necrotic areas of the femoral head, to promote bone regeneration, or to prevent osteonecrosis. The rapid development of gene therapy technology is increasingly recognized as a new therapeutic option for the treatment of ANFH, especially through therapeutic neovascularization and bone formation. Among growth factors, vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP) play important roles and have been extensively studied.

The VEGF family of growth factors is one of the most important cytokine families involved in angiogenesis. These factors promote the division of vascular endothelial cells and induce angiopoiesis. VEGF growth factors are essential for bone formation and repair during the bone regeneration process, which directly attracts endothelial cells and osteoclasts and enhances the differentiation of osteoblasts1, 2. BMP growth factors are the only signaling molecules that are individually sufficient for the induction of bone formation at orthotopic and heterotopic sites. They have defined roles in stimulating the proliferation and differentiation of mesenchymal and osteoprogenitor cells and have efficient bone induction activity3, 4. Because bone formation is a coordinated process involving the BMP and VEGF growth factors5, 6, orchestrating the timing with which these two factors are expressed may greatly enhance this process.

Choosing a safe and effective vector system to transfer and correctly express a target gene during gene therapy is important. Several different strategies have been examined for the delivery of genes of interest, including the use of naked DNA or an adenoviral vector. Treatment with naked DNA is simple and well tolerated by the recipient organism due to its low toxicity and weak induction of immune responses. However, the transduction efficiency is significantly lower when compared with other methods. The adenovirus has frequently been the vector of choice for gene transfer because it is able to transduce a variety of cells with high efﬁciency. However, adenoviral vectors have major limitations, including a lack of sustained expression, the antigenicity of viral proteins that are targeted by both humoral immunity and cytotoxic T lymphocytes, and possible toxicity at high doses. However, there are many inherent features of the adeno-associated virus system that make it an attractive option as a human viral vector. AAV is a non-pathogenic, defective human parvovirus that requires the presence of a helper virus, such as adenovirus or herpes virus, for productive infection7, 8. Other advantages of this vector system include its low immunogenicity, its ability to transduce both dividing and non-dividing cells, the potential to integrate into specific sites, its ability to achieve long-term gene expression (even in vivo), and its broad tropism, allowing for the efficient transduction of diverse organs9. These features make AAV attractive and efficient for gene transfer in vitro and local injection in vivo.

To enhance neovascularization and bone regeneration during osteonecrosis therapy, we constructed adeno-associated viruses co-expressing hVEGF165 and hBMP-7 (rAAV-VEGF165-IRES-BMP-7) and detected their effect on gene expression and biological activity in vitro and in vivo. These data demonstrate the synergistic action of these two genes and may provide a new therapeutic option for ANFH.
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Materials and methods
Materials and reagents

The rAAV-hVEGF165-IRES-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF165-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP) plasmids were constructed by Dr Xiang-hui HUANG. Human embryonic kidney cells-293 (HEK-293) and human umbilical vein endothelial cells (HUVECs) were obtained from the Department of Orthopedic Surgery in the Second Affiliated Hospital of Xi'an Jiaotong University. Male New Zealand rabbits (two months old, weighing 2.0–3.0 kg) were obtained from the experimental animal center of Xi'an Jiao Tong University. All animal protocols followed the recommendations and guidelines of the National Institutes of Health and were approved by the Xi'an Jiao Tong University Animal Care and Use Committee. The AAV helper-free system was obtained from Stratagene (La Jolla, CA, USA). A schematic representation of the structure of the plasmids in the AAV Helper Free System is provided in Figure 1A.
Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

(A) Schematic representation of the structure of plasmids in AAV Helper Free System. (B) Conceptual diagram of construction of pAAV-hVEGF165-IRES-hBMP-7. hVEGF165 gene (600 bp) and hBMP-7 gene (1300 bp) were respectively inserted into upstream MCS and downstream MCS located on either side of IRES sequence (631 bp). The length of the bicistronic frame is 2.5 kb.
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rAAV vector production

The construction of the rAAV-hVEGF165-IRES-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF165-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP) vectors was carried out as previously described10. The structure of the pAAV-hVEGF165-IRES-hBMP-7 vector is shown in Figure 1B. IRES sequences were incorporated into the pAAV MCS to construct a bicistronic vector with two multiple cloning sites. Then, the hVEGF165 (Pubmed NM-003376) and hBMP-7 (Pubmed NM-001719) genes were inserted into the upstream and downstream MCS, respectively. The length of the bicistronic frame is 2.5 kb, which is within the capacity of the vector. The AAV helper-free system was used to generate recombinant AAV. HEK-293 cells were cultured in H-DMEM supplemented with 10% fetal bovine serum containing 20 mg/mL penicillin-streptomycin and incubated with 5% CO2 at 37 oC. The AAV vector was co-transfected with the pAAV-helper and pAAV-RC vectors into HEK 293 cells by a calcium phosphate method according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). A primary virus stock was collected 72 h after transfection and further concentrated and purified by chloroform/PEG8000 protocols11. The recombinant adeno-associated virus had a titer of 5.5×1011 vp/mL.
Rabbit bone marrow-derived mesenchymal stem cells (BMSC) culture and rAAV infection in vitro

Male New Zealand rabbits were used to obtain rabbit BMSCs. The cells were harvested by gently flushing the tibiae and femora with L-DMEM. Density gradient centrifugation and adherent screening methods were used to isolate BMSCs as previously described12. The cells were cultured in L-DMEM supplemented with 10% fetal bovine serum containing 20 mg/mL penicillin-streptomycin and incubated with 5% CO2 at 37 oC. Following the 3rd passage, BMSCs (5×104 cells/well) were seeded onto 24-well plates 24 h before rAAV infection. By taking into account the cytopathogenic effect, infection efficiency, and cost of recombinant virus, we determined that the best multiplication of infection (MOI) for infecting rabbit BMSCs with rAAV was 5×104 vp/cell. The four rAAV virus variants were introduced into BMSCs using this MOI. Cells were incubated as above and were swirled gently at 30-min intervals. One hour later, the medium was replaced with L-DMEM supplemented with 10% fetal bovine serum. Medium was then completely replaced every three days.
Rabbit hind limb ischemia model and rAAV infection in vivo

Male New Zealand rabbits were kept under specific pathogen free conditions and supplied with sterile food and acidified water. The hind limb ischemia model was developed as described previously13. Rabbits were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Under a surgical microscope, a vertical longitudinal incision was made in the right hind limb. The right femoral arteries were separated from the origin of the external iliac artery, ligated, and completely excised. Immediately after ligation of the femoral artery, the four rAAV virus variants were each injected into five different sites14 on the three major thigh muscles of each rabbit (5.5×1011 vp/20 μL per site), including the adductor (two sites), the quadriceps (two sites), and the semimembranous (one site) muscles. Subsequently, the skin was sutured. After surgery, all animals were housed under standard conditions (temperature: 21±1 °C; humidity: 55%–60%) with food and water continuously available. The hind limbs were mobilized without any fixation. To prevent infection, animals received prophylactic injections of gentamicin (0.03 mg·kg−1·d−1, im) within 3 days after surgery.

Rabbits were sacrificed at various time points post-injection to characterize gene expression efficiency and the effects on angiopoiesis and bone regeneration in vivo. Each group contained 30 rabbits and was divided into four experimental subgroups: group A (n=6) was examined at week 2 for GFP expression (n=3) and immunoblotting (n=3), group B (n=6) at week 4 for GFP expression (n=3) and immunoblotting (n=3), group C (n=9) at week 6 for GFP expression (n=3), immunoblotting (n=3), and ELISA (n=3), and group D (n=9) at week 8 for GFP expression (n=3) and for blood flow measurement, X-ray radiography, and immunohistochemistry (n=6).
Reporter gene (GFP) expression in vitro and in vivo

Following 3, 7, 14, and 28 days of infection with AAV-GFP virus in vitro, the expression of GFP protein was observed by inverted fluorescence microscopy. At 2, 4, 6, and 8 weeks post-injection in vivo, the muscles injected with the AAV-GFP virus were sliced by the frozen section method and the expression of the GFP protein was observed as above. Each assay was performed in triplicate.
Preparation of culture medium and assessment of VEGF165 and BMP-7 gene expression

Total cellular RNA was isolated at 1, 2, 3, 7, 14, 21, and 28 days following infection with the AAV-GFP, AAV-VEGF, AAV-BMP or AAV-VEGF/BMP viruses using TRIzol Reagent (Invitrogen). Extracted RNA was treated with DNase I (Takara, Tokyo, Japan) to eliminate DNA contamination, and first-strand cDNA was synthesized with random hexamer primers using the reverse first-strand cDNA synthesis kit from MBI Fermentas (Glen Burnie, MD, USA). PCR was performed to amplify humanVEGF165 (forward primer 5′-CCATCGATATGAACTTTCTGCTGTCTTG-3′; reverse primer 5′-CGGAATTCTCACCGCCTCGGCTTGTC-3′) and BMP-7 (forward primer 5′-GGCCGGATCCATGCACGTGCGCTCACTGCG-3′; reverse primer 5′-GGCCGTCGACCTAGTGGCAGCCACAG-3′). β-actin (forward primer 5′-GAGGGAAATCGTGCGTGAC-3′; reverse primer 5′-TAGGAGCCAGGGCAGTAATCT-3′) was detected by RT-PCR as an internal control. PCR was performed using the following program: 94 °C for 3 min for one cycle and 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s. The PCR products were electrophoresed on ethidium bromide-stained 2.0% agarose gels. Each assay was performed in triplicate.
Muscle extract preparation and assessment of VEGF165 and BMP-7 gene expression

At 2, 4, and 6 weeks following injection with the AAV-GFP, AAV-VEGF, AAV-BMP, or AAV-VEGF/BMP viruses, the frozen muscles were pulverized in liquid nitrogen and homogenized in 3 mL of ice-cold lysis buffer (1% Nonidet P-40; 50 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 200 U/mL aprotinin; 1 mmol/L phenylmethylsulfonyl fluoride, PMSF). The tissue lysates (50 mg of protein) were separated by 12% polyacrylamide gel electrophoresis and blotted onto polyvinylidene diﬂuoride membranes. Immunoblotting was performed with anti-human VEGF165 and BMP-7 antibodies and the speciﬁc binding of the antibody was visualized with an ECL detection system. At 6 weeks post-injection, muscle extracts were measured with an enzyme-linked immunosorbent assay (ELISA) kit using the Biotrak ELISA system (R&D, Minneapolis, MN, USA) according to the manufacturer's instructions. Each assay was performed in triplicate.
Angiogenic and osteogenic in vitro assays
Tube formation assay

HUVECs were cultured as previously described15. Basement membrane matrigel matrix (BD, Bedford, MA, USA) was diluted by serum-free medium, added to a 24-well plate, and incubated at 37 °C for 30 min to allow solidification to occur. HUVECs (5×104 cells/well) were seeded on the matrigel and fresh L-DMEM medium supplemented with 10% FBS was added. Next, 1 mL of culture supernatant was harvested from the AAV-GFP, AAV-VEGF, AAV-BMP, or AAV-VEGF/BMP groups 14 days post-infection and added to the 24-well plate. The plate was then incubated at 37 oC with 5% CO2 for 12 h. The images of tube formation were captured under a light microscope from three random fields, and quantification of the tubes was analyzed by image processing software (Media Cybernetics, USA) to assess the biological activity of VEGF in vitro.
Mineralization assay

BMSCs were infected with the four virus groups above. The cells were then cultured in L-DMEM supplemented with 10% fetal bovine serum containing 20 mg/mL penicillin-streptomycin with 5% CO2 at 37 °C (the culture medium did not contain osteogenic induction factors, such as ascorbic acid, β-glycerophosphate, or dexamethasone). Mineralization effects were detected by von Kossa and alizarin red (AZR) staining16 for calcium deposits 4 weeks post-infection and observed using an inverted phase contrast microscope. The images of mineral nodules were captured under a light microscope from three random fields, and quantification of the mineral nodules was analyzed by image processing software to assess the biological activity of BMP in vitro.
Blood flow measurement and orthotopic bone formation in vivo

Eight weeks after injection, rabbits in the four groups were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Blood flow in the anterior tibial artery of ischemic and normal hind limbs was measured at rest with an Aspen Advanced Doppler ultrasound device from Acuson (Siemens Medical Solutions, Mountain View, CA, USA) using a perivascular flow probe and calculated by the inlay automatic processing software. The data were expressed as a percentage of the contralateral limbs. Three separate measurements were performed for each rabbit at every time point and the results were averaged. In addition, rabbits in the four groups were subjected to X-ray radiography to assess orthotopic bone formation.
Histological assessment

Eight weeks after injection, thigh muscle tissue sections of ischemic limbs from the four groups were harvested and ﬁxed in 10% neutral-buffered formalin. To identify the proliferation of capillary endothelial cells, tissue sections were immunostained for CD34. The monoclonal antibody against CD34 was applied at a 1:500 dilution after blocking with 1% normal bovine serum. Subsequent incubation with biotinylated horse anti-mouse IgG and an ABC Elite kit (Santa Cruz) was performed. The number of CD34-positive vessels was counted at a magnification of 200×, and twenty fields from each typical slide were counted (mean number of capillaries per square millimeter). To assess orthotopic bone formation, the slides were stained by von Kossa staining to detect mineralization.
Statistical analysis

The results are reported as means±standard deviation. The normality of the data distribution was assessed with the Shapiro-Wilk (W) test. ANOVA followed by the Fisher's test was conducted to assess differences among treatment groups. Statistical significance was set at a P-value less than or equal to 0.05. The SPSS mathematical statistics software used for this analysis was purchased from SPSS Inc (version 8; SPSS Inc, Chicago, IL, USA).
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Results
Animal condition after rAAV infection

There were no symptoms of local or systemic toxicity after rAAV infection. In the region of the injection sites, no inflammatory reaction, such as rubeosis, engorgement, or abscessus, was observed. The activities of all animals were normal. There was no systemic toxicity, such as nutation, instability of gait, anhelation, retardation, cyanosis, or convulsion. No animals died before the end of the experiments.
GFP gene expression

In vitro: GFP protein expression could be detected on the third day post-infection. However, the efficiency and density of infection were unstable. The expression of GFP protein increased from the 7th day and could be detected at 28 days post-infection (Figures 2A, 2B). In vivo: With prolonged infection time, GFP protein expression increased from the 4th week and could be detected at 8 weeks post-infection (Figures 2C, 2D).
Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Representative images of GFP protein expression. (A–B) on the 3rd, 7th, 14th, and 28th days after rAAV-IRES-GFP virus transfection in vitro. (A) Magnification×100; (B) Magnification×200; (C–D) on the 2nd, 4th, 6th, and 8th weeks after rAAV-IRES-GFP virus injection in vivo. (C) Magnification×100; (D) magnification×200.
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Efficient genes expression of hVEGF165 and hBMP-7

To confirm hVEGF165 and hBMP-7 gene expression in vitro, RT-PCR assays were performed. As shown in Figure 3A–3D, the sizes of the PCR products for VEGF165, BMP-7, and β-actin were 600 bp, 1300 bp, and 340 bp, respectively. With prolonged infection time, the intensity of the VEGF165 and BMP-7 bands increased in the AAV-VEGF/BMP group. Together, these data demonstrate that VEGF165 was expressed in the AAV-VEGF and AAV-VEGF/BMP groups but not in the AAV-BMP and AAV-GFP groups and that BMP-7 was expressed in the AAV-BMP and AAV-VEGF/BMP groups but not in the AAV-VEGF and AAV-GFP groups. Protein expression of 2, 4, and 6 weeks following injection with AAV-VEGF/BMPin vivo is shown in Figure 3E–3H. Expression of the VEGF165 and BMP-7 proteins was visualized by Western blot analysis. Strong staining at the expected molecular weights of 23 kDa (hVEGF165), 55 kDa (hBMP-7), and 43 kDa (β-actin) was observed. With prolonged infection time, the intensity of the VEGF165 and BMP-7 bands increased. These data demonstrate that VEGF165 was expressed in the AAV-VEGF and AAV-VEGF/BMP groups but not in the AAV-BMP and AAV-GFP groups and that BMP-7 was expressed in the AAV-BMP and AAV-VEGF/BMP groups but not in the AAV-VEGF and AAV-GFP groups. As shown in Figure 3I, 3J, the production of hVEGF165 and hBMP-7 was quantified in relevant muscle extracts 6 weeks post-injection. The average amounts of hVEGF165 protein in the AAV-VEGF/BMP and AAV-VEGF groups were significantly higher than those in the AAV-GFP and AAV-BMP groups (P<0.05, n=30). The average amounts of hBMP-7 protein in the AAV-VEGF/BMP and AAV-BMP groups were significantly higher than those in the AAV-GFP and AAV-VEGF groups (P<0.05, n=30). Figure 3. Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author Expression of hVEGF165 and hBMP-7. (A–D) Representative images of RT-PCR assay of AAV-GFP group (A), AAV-VEGF group (B), AAV-BMP group (C) and AAV-VEGF/BMP group (D). The size of the PCR products for hVEGF165, hBMP-7, and β-actin were 600 bp, 1300 bp, and 340 bp, respectively. With prolonged infection time, the brightness of the VEGF165 or BMP-7 bands increased in AAV-VEGF/BMP group. No hVEGF165 or hBMP-7 band could be detected in AAV-GFP group. Track 1–7 stands for the 1st, 2nd, 3rd, 7th, 14th, 21st, and 28th days post-transfection. (E–H) Representative images of Western blotting assay of AAV-GFP group (E), AAV-VEGF group (F), AAV-BMP group (G) and AAV-VEGF/BMP group (H). The molecular weights of hVEGF165, hBMP-7, and β-actin were 23 kDa, 55 kDa and 43 kDa respectively. Strong staining with the expected molecular weight was observed in AAV-VEGF/BMP group, and no hVEGF165 or hBMP-7 band was observed in AAV-GFP group. (I) ELISA assay for VEGF protein expression. The data is expressed as the mean±SD from three independent experiments. bP<0.05 vs AAV-GFP group, eP<0.05 vs AAV-BMP group. (J) ELISA assay for BMP protein expression. The data is expressed as the mean±SD from three independent experiments. bP<0.05 vs AAV-GFP group. eP<0.05 vs AAV-VEGF group. Full figure and legend (53K) Biological activity of hVEGF165 and hBMP-7 in vitro As shown in Figure 4A, hVEGF165 secreted from BMSCs in the AAV-VEGF/BMP group enhanced HUVEC migration, proliferation, and tube formation in comparison with the other three groups. The number of tubes in the AAV-VEGF/BMP group was significantly higher than that in the AAV-GFP and AAV-BMP groups. However, there was no statistical difference between the AAV-VEGF/BMP group and the AAV-VEGF group (Figure 4B). In addition, the mineralization effect of hBMP-7 was detected by von Kossa (Figure 5A) and alizarin red staining (Figure 5B). The AAV-VEGF/BMP group displayed stronger osteogenic activity than all the other groups. The number of mineralized nodules in the AAV-VEGF/BMP group was significantly higher than that in the AAV-GFP and AAV-VEGF groups. However, there was no statistical difference between the AAV-VEGF/BMP group and the AAV-BMP group (Figure 5C). Figure 4. Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author Tube formation experiment with HUVECs. (A) Representative images of tube formation. The HUVECs of AAV-VEGF/BMP group displayed stronger migration, proliferation, and tube formation ability than other three groups (magnification×200). (B) The data is expressed as the mean±SD from three independent experiments. bP<0.05 vs AAV-GFP group. eP<0.05 vs AAV-BMP group. Full figure and legend (50K) Figure 5. Figure 5 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author Osteogenic assay with BMSCs. (A) Representative images of von Kossa staining. BMSCs were stained by the von Kossa method and the mineralization is seen as black dots. (B) Representative images of alizarin red staining. BMSCs were stained by the alizarin red method and the mineralization is seen as mineralized nodules. (C) The data is expressed as the mean±SD from three independent experiments. (magnification×400). bP<0.05 vs AAV-GFP group. eP<0.05 vs AAV-VEGF group. Full figure and legend (104K) Biological activity of hVEGF165 and hBMP-7 in vivo The ability of hVEGF165 and hBMP-7 to induce tube formation and mineralization in vitro correlated well with their in vivo role in neovascularization and bone regeneration. Blood flow in the anterior tibial artery of ischemic and normal hind limbs was measured 8 weeks post-injection to assess the neovascularization capability of hVEGF165 in vivo. As shown in Figure 6, the ratio of mean ischemic/normal blood flow in the AAV-VEGF/BMP group was highest when compared with the AAV-GFP, AAV-VEGF, and AAV-BMP groups. However, there was no statistical difference between the AAV-VEGF/BMP group and the AAV-VEGF group. In addition, rabbits in the four groups were subjected to X-ray radiography to assess the bone regeneration activity of hBMP-7 in vivo. As shown in Figure 7, orthotopic ossification was radiographically evident in the AAV-VEGF/BMP group eight weeks post-injection. In contrast, no radiographic evidence of bone formation was observed in the AAV-GFP group or the AAV-VEGF group. Figure 6. Figure 6 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author Blood flow in the rabbit hind limb. Blood flow in the anterior tibial artery of ischemic and normal hind limbs was measured at rest with an Aspen Advanced Doppler ultrasound device using a perivascular flow probe 8 weeks post-injection. (A) Representative record images of blood flow in AAV-GFP, AAV-VEGF, and AAV-BMP and AAV-VEGF/BMP group. (B) Mean blood flow in the ischemic limbs 8 weeks post-injection. Values are expressed as a percentage of contralateral limbs and are shown as mean values±SEM from three independent experiments. bP<0.05 vs AAV-GFP group. eP<0.05 vs AAV-BMP group. Full figure and legend (106K) Figure 7. Figure 7 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author Representative images of orthotopic ossification in the rabbit hind limb. Orthotopic ossification was radiographically evident in AAV-VEGF/BMP group eight weeks post injection (indicated as arrows). However, no radiographic evidence of bone formation was observed in AAV-GFP group and AAV-VEGF group. Full figure and legend (31K) Histological assessment To further assess vascularity in rAAV-infected muscle, immunostaining for CD34, a marker of vessel endothelial cells, was performed to detect the number of capillaries. As shown in Figure 8A, muscle from the AAV-VEGF/BMP group contained significantly more capillaries when compared with the other three groups at 8 weeks post-injection. The mean density of capillaries in the AAV-VEGF/BMP group was significantly higher than that in the AAV-GFP and AAV-BMP groups. However, there was no statistical difference between the AAV-VEGF/BMP group and the AAV-VEGF group (Figure 8C). To analyze bone formation, von Kossa staining was adopted to assess calcium deposits 8 weeks post-injection. As shown in Figure 8B, calcium deposits stained black. The osteogenic ability of the AAV-VEGF/BMP group was significantly enhanced compared with that found in the other three groups. Figure 8. Figure 8 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author Histological assessment. (A) Representative images of rabbit muscle tissues using immunostaining for CD34 (capillaries were indicated as arrows). (B) Representative images of rabbit muscle tissues using von Kossa staining for calcium deposits. The calcium deposits were stained as black (indicated as arrows). (C) Capillary density in rabbit muscle tissues. The number of capillaries was counted in twenty different fields of one muscle section, and capillary density was calculated. Data are shown as mean values±SEM from three independent experiments. (magnification×200). bP<0.05 vs AAV-GFP group. eP<0.05 vs AAV-BMP group. Full figure and legend (112K) Top of page Discussion The packaging capacity of the rAAV vector (5 kb, including the inverted terminal repeats) remains one of its primary limitations in terms of gene delivery. However, substantial progress has recently been made to overcome this restriction. Among the several different strategies to co-express multiple genes, the incorporation of an IRES into this gene therapy vector represents one of the more promising strategies17, 18, 19. The IRES functions as a ribosome landing pad for the efficient internal initiation of translation, ensuring coordinated expression of several genes. The IRES initiates ribosome binding and translation in the absence of a 5'CAP, thus overcoming the main disadvantage of traditional strategies that express two different genes. This characteristic is especially useful for AAV production due to the packaging size limitation imposed by the AAV vectors. In our current study, an IRES sequence was incorporated into the pAAV MCS to construct a bicistronic vector. Then, the hVEGF165 and hBMP-7 genes were inserted upstream and downstream of the MCS, located on either side of the IRES to create a bicistronic frame of 2.5 kb in length, which is within the capacity of the vector. In our study, we reveal that AAV-mediated hVEGF165 and hBMP-7 gene transfer in vitro and in vivo induces the expression and secretion of the hVEGF165 and hBMP-7 proteins. These results demonstrate that the IRES sequence may be a superior strategy for co-expressing multiple genes in rAAVs. An important characteristic of rAAV is that when the host cell is infected with rAAV, the efficiency of infection cannot immediately be determined. The expression of the gene of interest will not be activated until the double-stranded nucleic acid version of the virus has been synthesized by DNA synthetase. The time required for this to occur may be several days, weeks, or months and is dependent on the infection surrounding the host cells20. For this reason, it is essential to detect the timing of gene expression in vitro and in vivo. The results of GFP expression and RT-PCR analysis in vitro and of Western blotting and ELISA assays in vivo revealed expression of the genes of interest, indicating that the rAAV vector has superior gene expressing ability. The key aim of gene therapy for osteonecrosis disease is bone and vessel regeneration. Bone restoration is a complicated process involving many kinds of cytokines, and VEGFs and BMPs play important roles during renovation that have been studied extensively. VEGF is one of the most important cytokines in angiogenesis. It specifically promotes the division and growth of vascular endothelial cells and ultimately induces angiopoiesis2, 4, 18, 20, 21. BMPs are the only signaling molecules that can singly induce de novo bone formation at orthotopic and heterotopic sites. BMPs have discrete effects on the proliferation and differentiation of mesenchymal cells and osteoprogenitor cells and also have efficient bone induction activity3, 22. Thus, orchestrating the timing of expression of these two factors may greatly enhance this process. We performed angiogenic and osteogenic assays to identify the biological effect of VEGF165 and BMP-7 in vitro and in vivo. The results indicated that at the dosage used, the rAAV-hVEGF165-IRES-hBMP-7 virus had excellent biological activity and could properly mediate biological activity both in vitro and in vivo. However, one interesting finding of our study was that VEGF alone is not sufficient to improve bone formation and that BMP alone is not sufficient to improve vessel regeneration. We conclude that these findings were not due to improper dosage, but reflect the fact that expression of VEGF or BMP alone is not sufficient to initiate the cascade of bone formation or vessel regeneration, respectively. These findings also demonstrate that orchestrating the expression of these two factors is essential for effective therapy of osteonecrosis disease. An additional interesting finding in our studies was that there was no statistical difference between the AAV-VEGF/BMP group and the AAV-VEGF group in terms of angiogenesis. Similarly, there was no statistical difference between the AAV-VEGF/BMP group and the AAV-BMP group in terms of bone formation. We conclude that there may be a requirement for the proper ratio of VEGF to BMP. As shown by a previous study23, the proper ratio of VEGF to BMP is critical to ensure synergistic effects. In addition, the unequal expression of the VEGF and BMP genes located upstream and downstream of the IRES may be responsible24. Thus, comparison of the expression of genes located upstream and downstream of the IRES and the identification of the best ratio of VEGF and BMP for treatment of osteonecrosis will be imperative in future experiments. In summary, we used rAAV as a gene transduction system by successfully inserting the VEGF165 and BMP-7 genes in this vector, allowing them to be efficiently and stably co-expressed. The VEGF165 and BMP-7 proteins that were expressed from the rAAV-hVEGF165-IRES-hBMP-7 vector enhanced angiogenesis and bone regeneration in vitro and in vivo. Our experiments establish a foundation for investigating the synergistic biological effects of VEGF165 and BMP-7 in vitro and in vivo and provide theoretical support for gene therapy of ANFH with our recombinant virus. Top of page Author contribution Chen ZHANG designed and performed the experiments; Kun-zheng WANG, Hui QIANG, Yi-lun TANG contributed to the in vitro studies; Qian LI and Miao LI contributed to the blood flow measurement studies; Xiao-qian DANG assisted in the design of the study, reviewed all data, and assisted in writing the manuscript. All authors have read and approved the final manuscript. Top of page References 1. Dai J, Rabie AB. VEGF: an essential mediator of both angiogenesis and endochondral ossification. J Dent Res 2007; 86: 937–50. | Article | PubMed | ChemPort | 2. Clarkin CE, Emery RJ, Pitsillides AA, Wheeler-Jones CP. Evaluation of VEGF-mediated signaling in primary human cells reveals a paracrine action for VEGF in osteoblast-mediated crosstalk to endothelial cells. J Cell Physiol 2008; 214: 537–44. | Article | PubMed | ChemPort | 3. Hu J, Qi MC, Zou SJ, Li JH, Luo E. Callus formation enhanced by BMP-7 exvivo gene therapy during distraction osteogenesis in rats. J Orthop Res 2007; 25: 241–51. | Article | PubMed | ChemPort | 4. White AP, Vaccaro AR, Hall JA, Whang PG, Friel BC, McKee MD. Clinical applications of BMP-7/OP-1 in fractures, nonunions and spinal fusion. Int Orthop 2007; 31: 735–41. | Article | PubMed 5. Hou H, Zhang X, Tang T, Dai K, Ge R. Enhancement of bone formation by genetically-engineered bone marrow stromal cells expressing BMP-2, VEGF and angiopoietin-1. Biotechnol Lett 2009; 31: 1183–9. | Article | PubMed | ChemPort | 6. Young S, Patel ZS, Kretlow JD, Murphy MB, Mountziaris PM, Baggett LS, et al. Dose effect of dual delivery of vascular endothelial growth factor and bone morphogenetic protein-2 on bone regeneration in a rat critical-size defect model. Tissue Eng Part A 2009; 15: 2347–62. | Article | PubMed | ChemPort | 7. Merten OW, Gény-Fiamma C, Douar AM. Current issues in adeno-associated viral vector production. Gene Ther 2005; 12: S51–61. | Article | PubMed | ChemPort | 8. Grieger JC, Choi VW, Samulski RJ. Production and characterization of adeno-associated viral vectors. Nat Protoc 2006; 1: 1412–28. | Article | PubMed | ChemPort | 9. Tu L, Xu X, Wan H, Zhou C, Deng J, Xu Q, et al. Delivery of recombinant adeno-associated virus-mediated human tissue Kallikrein for therapy of chronic renal failure in rats. Hum Gene Ther 2008; 19: 318–30. | Article | PubMed | ChemPort | 10. Huang X, Shi Z, Wang K, Dang X, Yang P, Yu P. Construction of recombinat adeno-associated virus vector co-expressing hVEGF165 and hBMP-7 genes. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2008; 22: 807–13. Chinese. | PubMed | ChemPort | 11. Yan H, Guo Y, Zhang P, Zu L, Dong X, Chen L, et al. Superior neovascularization and muscle regeneration in ischemic skeletal muscles following VEGF gene transfer by rAAV1 pseudotyped vectors. Biochem Biophys Res Commun 2005; 336: 287–98. | Article | PubMed | ChemPort | 12. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284 (5411): 143–7. | Article 13. Silvestre JS, Tamarat R, Ebrahimian TG, Le-Roux A, Clergue M, Emmanuel F, et al. Vascular endothelial growth factor-B promotes in vivo angiogenesis. Circ Res 2003; 93: 114–23. | Article | PubMed | ISI | ChemPort | 14. Byun J, Heard JM, Huh JE, Park SJ, Jung EA, Jeong JO, et al. Efﬁcient expression of the vascular endothelial growth factor gene in vitro and in vivo, using an adeno-associated virus vector. J Mol Cell Cardiol 2001; 33: 295–305. | Article | PubMed | ChemPort | 15. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J Clin Invest 1973; 52 (11): 2745–56. | Article 16. Bellows CG, Aubin JE, Heersche JN, Antosz ME. Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif Tissue Int 1986; 38: 143–54. | Article | PubMed | ISI | ChemPort | 17. Pelletier J, Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 1998; 334: 320–5. | Article 18. Li W, Thakor N, Xu EY, Huang Y, Chen C, Yu R, et al. An internal ribosomal entry site mediates redox-sensitive translation of Nrf2. Nucleic Acids Res 2010; 38: 778–88. | Article | PubMed | ChemPort | 19. Jeffrey S. Kieft. Viral IRES RNA structures and ribosome interactions.Trends Biochem Sci 2008; 33: 274–83. 20. Tang X, Fu DH, Yang SH, Chen YC, Li Q, Yu CN, et al. Assessment of the expression profile during the entochondrostosis of vascular endothelial growth factor in bone morphogenetic protein 2 induced osteogenesis. Zhonghua Wai Ke Za Zhi 2008; 46: 614–7. | PubMed | 21. Samee M, Kasugai S, Kondo H, Ohya K, Shimokawa H, Kuroda S. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) transfection to human periosteal cells enhances osteoblast differentiation and bone formation. J Pharmacol Sci 2008; 108: 18–31. | Article | PubMed | ChemPort | 22. Stöve J, Schneider-Wald B, Scharf HP, Schwarz ML. Bone morphogenetic protein 7 (bmp-7) stimulates proteoglycan synthesis in human osteoarthritic chondrocytes in vitro. Biomed Pharmacother 2006; 60: 639–43. | Article | PubMed | ChemPort | 23. Peng H, Wright V, Usas A, Gearhart B, Shen HC, Cummins J, et al. Synergistic enhancement of bone formation and healing by stem cell–expressed VEGF and bone morphogenetic protein-4. J Clin Invest, 2002; 110: 751–9. | Article | PubMed | ISI | ChemPort | 24. Kapturczak M, Zolotukhin S, Cross J, Pileggi A, Molano RD, Jorgensen M, et al. Transduction of human and mouse pancreatic islet cells using a bicistronic recombinant adeno-associated viral vector. Mol Ther 2002; 5: 154–60. | Article | PubMed | ISI | ChemPort | Top of page Acknowledgements This work was supported by the National Natural Science Foundation of China (No 30600624) and (No 30772189). We acknowledge the support of Xi'an Jiao Tong University. We are grateful to Dr Xiang-hui HUANG (Department of Orthopedics, Shaanxi Provincial People's Hospital, Xi'an 710068, Shaanxi Province, China) for his help in the construction of the rAAV-hVEGF165-IRES-hBMP-7 viral vector

The Biotechnology Industry By Richard Graydon, M.D.

21september1978@gmail.com (Unknown) — Thu, 28 Apr 2011 08:18:00 +0000

The primary goal of the biotechnology industry is to invent new biologically-active substances, for the treatment of disease, the pharmaceutical industry, and the management of agriculture.

The U.S. is the world leader in biotechnology, currently providing employment for more than 300,000 people in over 6,000 U.S. biotech companies, with an estimated market capitalization of approximately USD 281 billion in 2008. The biotechnology industry has more than tripled in size since 2000, with revenues increasing from USD 25 billion in 2000 to more than USD 80 billion in 2008. In 2007, Amgen, Inc., the world's largest biotechnology company, achieved total sales of USD 14.7 billion. Expectations are that the biotechnology market will increase to more than USD 130 billion by 2011.

Geographically, the U.S. accounts for 65% of the biotechnology market, Europe 23%, Canada 7%, Australia 3%, and the rest of the world, 2%.

Today, genomics and bioinformatics development both are being pursued within major pharmaceutical companies. There have been key collaborations with traditional IT technology companies such as IBM, Hitachi, Ltd., Samsung, SK Telecomm and Motorola. The U.S. biotechnology research and pharmaceutical industry spent a record USD 8.5 billion in 2007 on the R&D of new medicines and vaccines. As of 2006, the biotechnology industry compared favorably with the pharmaceutical industry in terms of R&D expenditures per employee. Venture capital investments in bioscience companies reached USD 11.6 billion in 2007.

Ernst & Young reported that in 2006, 82 publicly-traded Canadian biotech firms claimed S3.2 billion in revenue, accounting for 4.4% of global biotech revenues. Human health accounts for the largest segment of the Canadian biotech industry, representing more than half the interest of all biotech companies, approximately 70% of all biotech revenues and nearly 90% of all biotech R&D. Bioinforrnatics provides an important capability for the Canadian biotech industry, encompassing genomics and other "omics", tissue engineering and drug discovery technology.

Canadian bioinformatics companies include Bioinformatics Solutions, Inc., DNA LandMarks, Inc., and Kinexus Bioinformatics Corporation. Montreal, Quebec, has Canada's leading biotech cluster, followed by Toronto, Ontario; Vancouver, British Columbia; and Winnipeg, Manitoba.

Five European Union (E.U.) member states-Denmark, Finland, Germany, Sweden and the U.K.-continue to be world leaders in biotech innovation along with the U.S. and Japan. In the second half' of 2007, European biotech companies reached USD 765.3 million in venture capital investment. In January and February of 2008, venture capital funds invested a total of USD 145.6 million into new European biotech companies; however, with the economic stresses of the latter part of the year, many European biotech firms may find it harder to raise money in early 2009.

The Biotechnology Industry Organization of Germany (BlO Deutschland), located in Berlin, has more than 180 members, including companies, BioRegions, and sector service providers. The goal of the organization is to support and promote a stable economy through innovation in bioscience. Among the German biotech companies are the Biotechnology Research and Information Network (BRAIN AG), 4SC. AG, Bionas, UmbEl and Direvo, which recently (September 2008) was purchased by the German pharmaceutical and chemicals giant Bayer HealthCare. In view of' the burgeoning German biotech sector, traditional German scientific companies like Eppendorf, AG, Sartorius, AG and Qiagen also have gotten into the act.

Dr. Richard Graydon, http://www.medauthor.com, trained as an Oncologist, holding both M.D. and PhD degrees, specializing in molecular genetics and cancer research. His education and experience have provided him analytical and clinical skills for keen insight into diagnosis, treatment, and care of cancer patients. See http://www.medauthor.com for further information

Article Source: http://EzineArticles.com/?expert=Richard_Graydon,_M.D.

Article Source: http://EzineArticles.com/6161504

Motivated research

21september1978@gmail.com (Unknown) — Wed, 27 Apr 2011 23:59:00 +0000

Motivated research

Antoine Danchin

Three years ago, a senior politician attended his country's Annual Congress for the Advancement of Science to give the introductory lecture. He asked the attending scientists to make science and research more attractive to young students and the general public, and asked his countrymen to support scientists to address the urgent challenges of global climate change, energy needs and dwindling water resources.

if you student or college in university you must have

Motivated research for case ctuy, discustion and sharing

Press release Polaris Software Lab Limited (POLARIS.NS)(532254.BO)

21september1978@gmail.com (Unknown) — Wed, 27 Apr 2011 23:47:00 +0000

ts release from http://www.businesswireindia.com if you share release gaian or new release you can email in here
Source: Polaris Software Lab Limited (POLARIS.NS)(532254.BO)
Wednesday, April 27, 2011 11:49 AM IST (06:19 AM GMT)
Editors: General: Economy; Business: Business services, Defence & security, Financial Analyst, Information technology, Stock exchanges; Technology

Polaris Enters Cloud Computing Space through Strategic Investment in IdenTrustTM
Investment in US-based Global Leader in Trusted Identity Solutions uniquely positions Polaris to drive the most robust digital security infrastructure in the US & European markets

Chennai, Tamil Nadu, India and New York, United States, Wednesday, April 27, 2011 -- (Business Wire India)

Polaris Software Lab Limited (POLS.BO), a leading global Financial Technology Company today announced a strategic investment in IdenTrustTM, a global leader in trusted identity solutions recognized by global financial institutions and one of the premier providers of digital identity authentication services to several key banks, United States federal identity programs as well as supply chain markets. This strategic investment will mark Polaris’ entry into the cloud computing space for Financial Technology solutions.

IdenTrust™ Inc. was founded in 1999 by a group of financial institutions that included Citigroup, Bank of America, Chase, Barclays, HSBC, and Deutsche. As the only bank-developed identity authentication system, IdenTrustTM provides a unique legally and technologically interoperable environment for authenticating and using identities worldwide. IdenTrustTM provides applications that use electronic identities that establish contracts, grant authority, support secure encrypted data and information storage and secure online interactions/transactions.

IdenTrustTM shareholders include 20 banks, and the company brings with it an existing customer base of ten of the world’s largest banks; a portfolio of patents, and the associated intellectual property and expertise around identity, authentication, encryption, and electronic signatures on a global basis; and strong operational and hosting capabilities, led by a solid management team. Digital security is key for next generation banking, and IdenTrust’s existing products such as Trust SignTM in the certificate space and Trust PrimeTM in the eBAM space are supported by a globally interoperable legal and technology framework to deliver the highest levels of security on a cloud.

For IdenTrustTM, Polaris’ global customer base is a logical channel and network accelerator, and for its existing customers, the investment by Polaris reinforces their belief in the company as the leading global identity platform, while giving them a direct access to a comprehensive suite of next generation financial technology solutions.

On choosing Polaris as its lead investor, Mr. John Sculley, Chairman of the Board of Directors, IdenTrustTM said, “IdenTrust and Polaris are both innovative and powerful players in the banking space, and I am confident that, together, they will be able to drive even more compelling new products and services on a global basis.”
IdenTrustTM is also an active player in several US Government programs, including the External Certificate Authority (ECA) policy under the auspices of the Defense Information Systems Agency (DISA), and the Access Certificates for Electronic Services (ACES) under the auspices of the General Services Administration (GSA). IdenTrustTM is authorized to operate in both spaces, and issues digital certificates to non-government employees or entities that are required to comply with either the ACES or ECA policies.

Zions Bancorporation, one of US' premier financial services companies, will be a significant equity holder in IdenTrustTM. Polaris has a significant majority in the shareholding pattern, while Zions will maintain a substantial minority position. Speaking on the occasion, Mr. Doyle Arnold, Vice Chairman and CFO, Zions Bancorporation said, “We have been associated with IdenTrustTM and its predecessor companies since 1996 and have seen them grow tremendously over the years. They have revolutionized the identity authentication solutions business across the US and we are confident that the addition of Polaris’ experience and reach in the BFSI sector would only add to its success globally.”

Ms. Karen J. Wendel, Chief Executive Officer, IdenTrustTM said, “As a global leader in identity solutions with scalable and globally interoperable identity authentication solutions, we occupy a premier position in our field of operations. We needed a partner with scale and reach who can fuel our growth and provide us access to various global markets and we are very excited to join the Polaris family that has a legacy of providing cutting edge solutions to large financial institutions globally.”

Mr. Arun Jain, Founder, Chairman & CEO, Polaris Software said, “With increasing dependency on the Internet, security solutions that incorporate identity management are a key element in any cloud offering. We believe that IdenTrustTM, being a proven provider of bank-grade identity authentication in 175 countries, will bring in the required expertise in this area allowing us to create more value for our clients by providing the most secure Financial Infrastructure solutions. We are excited about getting into a new area with this significant investment that shows our long term commitment to being a one-stop shop for all FinTech needs.”

About Polaris Software Lab

Polaris Software Lab (POLS.BO) is a leading Financial Technology company, with its comprehensive portfolio of products, services and consulting. Polaris has a talent strength of over 10,000 solution architects, domain and technology experts. The company owns the largest set of Intellectual Properties in the form of a comprehensive product suite, IntellectTM Global Universal Banking (GUB) 10.0. IntellectTM is the first pure play SOA based application suite for Retail, Corporate, Investment banking and Insurance. Polaris is headquartered in Chennai and has offices in all global financial hubs including Tokyo, Sydney, Hong Kong, Singapore, India, Dubai, Bahrain, Riyadh, London, Belfast, Zurich, Frankfurt, Toronto, New York, Chicago, Fremont, Pittsburgh and Chile. For more information, please visit http://www.polarisFT.com/

About IdenTrustTM

IdenTrustTM is a leader in trusted identity solutions recognized by financial institutions, government agencies and businesses around the world. The only bank-developed identity authentication system, IdenTrustTM provides a legally and technologically interoperable environment for authenticating and using identities in more than 175 countries. IdenTrustTM enables end-users to have a single identity that can be used with any bank, any application, and across any network. IdenTrustTM identities are globally interoperable under uniform private contracts. For more information, please visit www.identrust.com

About Zions Bancorporation

Zions Bancorporation is one of the nation's premier financial services companies, consisting of a collection of great banks in select high growth markets. Zions operates its banking businesses under local management teams and community identities through approximately 500 offices in 10 Western and Southwestern states: Arizona, California, Colorado, Idaho, Nevada, New Mexico, Oregon, Texas, Utah and Washington. The company is a national leader in Small Business Administration lending and public finance advisory services. In addition, Zions is included in the S&P 500 and NASDAQ Financial 100 indices. Investor information and links to subsidiary banks can be accessed at www.zionsbancorporation.com

Conference Call

Polaris Software Lab Ltd. will host a Conference Call, where the Senior Management of Polaris will comment on the strategic investment and respond to questions from participants. The conference call will take place at 11:30 Hrs IST on Wednesday, April 27, 2011.

To participate in the conference call, please dial the numbers given below five minutes ahead of schedule.

The dial-in numbers to join the conference call are:

Conference code: 9144282524#
Security Code: 5638#

Access Numbers:
Delhi: 01130360201
Mumbai: 02230360201
Kolkata: 03330360201
Chennai: 04430360201
Hyderabad: 04030360201
Bangalore: 08030360201
Australia: 1800210897
US & Canada: 18663944524
Singapore: 8001011904

For press backgrounder on Polaris Software Lab Limited click here

Media contact details

Dwaipayan Deb,
Polaris Software Lab Limited,
+91 9962536442,
dwaipayan.d@polaris.co.in

KEYWORDS: ECONOMY, BUSINESS SERVICES, DEFENCE, Financial Analyst, IT, STOCK EXCHANGES, TECHNOLOGY, POLARIS.NS, 532254.BO

Beberapa tips memilih emas

21september1978@gmail.com (Unknown) — Mon, 25 Apr 2011 08:34:00 +0000

Beberapa tips pemilihan emas:

Update Kurs Emas Update kurs emas bisa anda dapatkan setiap hari, pada pukul 09.30 atau anda bisa menghubungi langsung ke PT Antam, Tbk.
Perhatikan Dua Faktor Penentu yaitu faktor harga emas dunia dan faktor kurs rupiah terhadap dolar. Oleh karena itu disarankan untuk selalu meng-update setiap saat, informasi dua faktor tersebut.
Perhatikan Keaslian Emas Keaslian emas dapat mengacu pada sertifikat yang diperoleh pada saat transaksi emas batangan (lempengan). Dimana sertifikat itu harus dikeluarkan oleh PT Antam, Tbk. (khusus Indonesia) yang berstandar internasional dan telah diakui oleh London Bullion Market Association (LBMA). Sertifikat asli memiliki nomor seri yang juga terdapat pada lempengan emas, dan ukuran 5 x 6 cm. Sedangkan untuk memastikan keaslian emas lempengan, anda dapat melihat logo LM berbentuk segi lima yang tertera, serta terdapat tulisan Fine Gold .9999, dan apabila nilai emas lebih dari 5 gram disertai nomor seri pada lempengan (yang biasanya diawali dengan dua karakter huruf dan tiga digit angka).
Pastikan Kadar Kemurnian Emas, sesuai dengan Standar Internasional Emas 24 Karat (emas murni) berkomposisi 99.99% emas, Emas 22 Karat berkomposisi 91.7% emas dan 8.3% campuran bahan lain (perak), Emas 20 Karat berkomposisi 83.3% emas, Emas 18 Karat berkomposisi 75.0% emas, Emas 16 Karat berkomposisi 66.6% emas, Emas 14 Karat berkomposisi 58.5% emas, dan Emas 9 Karat berkomposisi 37.5% emas.
Biaya Produksi Biaya produksi yang dikenakan berkisar antara Rp 33.500,- sampai dengan Rp 102.000,-/keping emas.
Simpan Bukti Pembelian dan Bukti Keaslian Emas Hal ini adalah sebagai bukti keaslian bilamana anda menjual emas kembali ke tempat Anda membelinya, karena tentu akan lebih mudah dan tidak ada banya pertanyaan seputar emasnya.

Posted in: Emas Batangan, Investasi Emas, logam mulia.
Tagged: Cek Keaslian Emas · emas 14 karat · emas 18 karat · Emas 22karat · emas 9 karat · Investasi Emas · investasi logam mulia · Kadar Kemurnian Emas · keping emas · Kurs Emas · logam mulia · London Bullion Market Association · PT Antam · tips investasi

Switching Careers - 7 Key Steps By Alotta Candor Article Source: EzineArticles.e

21september1978@gmail.com (Unknown) — Wed, 20 Apr 2011 22:38:00 +0000

Are you thinking about switching careers? If you are, you're not alone. Most Americans switch careers three times in their lifetime. Nevertheless, switching careers is scary. And it's especially paralyzing the older you get. But making a career switch is very possible and much more common than you might think. Before you're ready to leap, realize that it's a heavyweight decision that deserves some time and solid thought. Here are seven steps to help you on your way.

1. Gain insight from your current situation.

When considering a career switch, the first thing you should do is learn from your current situation. To do this, take a step back and study what you do for a living today and why you do it. Examine the reasons that you are in your current job or career. Was it what you went to school for? Was it what your parents wanted you to do? Was it the "hot career" at one time? Did you just "fall into it"? Did you love it at one time? Did you do it for the money you could make? Was it just to pay the bills? The answers to these questions can provide valuable insight into the core reasons that you want or need to change.

Now examine why you want to leave your current career field. Remove any company or management related politics that are specific to your current employer from the picture. See your situation for what it is and ask yourself why you are looking to switch. Are you being forced out because of market shifts of business trends? Are you burnt out? Do you want to make more money? Are you miserable doing what you do? Have you tried your best but found that your career is "just not a good fit"? Have you decided its time to pursue a long lost career love?

Inspecting your current situation and reasons for your desire to change careers will provide a foundation for your next step.

2. Look inside

Whether you already have a career in mind or you are searching for a new career, you must look inward. In order to gain the most from your reflection, it is essential to start with a clean slate. Set aside any notions (real or imagined) about what type of money certain careers offer. Discard any stereotypes or judgments of occupations. Distance yourself from any pre-conceived ideas about what you are right or destined for.

Now seriously examine what you truly love. First start with the obvious. Look at your hobbies and interests. List out the things you are passionate about or in which you have talent. Give yourself credit for things you are good at and don't be afraid to write things down that you love, but are not yet good at. Write them all down, even if you think they may not be a possible career path. You're just brainstorming at this point and you should not eliminate anything right out of the gate. And it's important to bear in mind that what you may think are your interests are not necessarily all of your interests. To help you get a good look at your interests, observe the simple things. What kinds of news stories perk your interest? What kinds of TV shows do you enjoy? What kinds of books magazines do you find yourself drawn to? What kind of people do you like to associate with or find interesting? What parts of your current career have brought you the most satisfaction?

Next, remind yourself of what you wanted to be when you grew up. Is it something you still want to be? Do you still get stars in your eyes when you think about it? This may give you some real clues. And of course, depending on what you wanted to be, that young dream may be out of reach. Or...is it? Think about it. If your ideal career aspiration at the age of 10 was to be an astronaut and you are now over the age limit or are not physically able to, you can rule it out. But what about other careers associated with astronauts or astronomy? There is a wide array of careers that touch upon astronomy from teaching, to marketing telescopes, to writing for a science magazine, to building models or sets for movies to working at a museum on a space exhibit! When you look at your passion and then use a little imagination, the sky (or should I say space) becomes the limit.

Lastly, look at what type of person you are. Be honest with yourself. Do you enjoy working with your hands? Do you enjoy working alone? Do you enjoy a social work setting? Do you enjoy being part of a team? Do you enjoy working at night? These are all examples of questions that will lead you down the path to discovering and evaluating whether a given career path is right for you.

As you are going through the exercise of looking inside, it is important to avoid cluttering your mind or list with any "buts". If your answer to the question "do you enjoy working with your hands" was "yes", leave it at "yes". Don't append any knee jerk reactions to your answers such as "yes, but I am clumsy" or "yes, but those jobs don't pay as much". Leave your mind open and you will be pleasantly surprised at how easily any natural human discouragement subsides.

3. Explore what's out there

Now that you're armed with a list of personal interests and talents, sit on them for a few days and let them cook. Let yourself get used to your newfound list. You may find yourself adding a few more during this time or even crossing a few out.

Begin your next step by opening your eyes to what's out there (not what you perceive to be out there, but rather what is out there). Pick up your local community college catalog and flip through both credit and continuing education courses. Look online for education or career programs. Make a list of the careers of your friends and family. On your next ride to work or to the store, turn off the radio, look around and take notice of the buildings and businesses around you. Look at the people you see outside and start piecing together what their days are like.

The object of this exercise is to compare what is out there, with what interests you. Let's stick with the astronomy example. You're interested in astronomy. So what? Well...now you've begun looking through the local community college catalog and there, you see a continuing education course on astronomy. You've now found something concrete, a class that you can take that will allow you to pursue your interest. But what is a continuing education course going to get you? A couple of things. One, you will meet other people who share your interest. These people bring information to the table. They may know of groups or clubs that you can join. Or perhaps, they may have friends or relatives who are looking for someone to do research work or work part-time in their science store. Two, you will be able to further your interest...or be able to rule it out as a career path. You may learn that you really love astronomy and would like to pursue it further. On the other hand, you may learn that it really isn't what you thought it was and you really don't care enough about it to pursue it as a career. Any way you slice it, you will learn something about yourself and at the very least will have met others who share your interest.

Let's try another example. Perhaps, on your way to work, you start to notice a road construction worker. The first day you see him, you're in a suit, he's in jeans and he's joking with a coworker as he shovels asphalt under a sunny sky. You think to yourself, "Boy it'd be nice to get out of this suit, work outside...break a sweat for once! Maybe I'd like to do that..." The next day you see him and you watch as a driver leans out his window and curses at him. "Hmmm", you think. The third day you see him, it's raining and cold and he's out braving the elements while you're dry and warm inside your car. "Cross that one off the list", you think. If you had only noticed the man on the first day, you'd only have seen him on a good day. If you had only noticed him on the last day, you'd have seen him at the worst. Either way, without really opening your eyes full time, you may have a fragmented impression on what it means to be this or that. The point here is not to look for distinctly negative or positive things about a given occupation, but to begin to see it as a whole. With this type of information, you will be able to form an opinion on whether a given occupation could be a possibility for you.

In addition to concrete and mindful exploration, talking to your friends and family is an invaluable type of investigation. When you start bringing up your interests or ideas for possible career paths in conversations or e-mails, you will no doubt hear a lot of "Oh! Susan's son teaches astronomy at the university, he's writing a book on the Hubble telescope this year." or "Oh John does construction on the side, he loves it!" By talking to other people, you may make connections or gain insight into the experiences and opinions of people connected with your interest areas. It will also trigger some more ideas for you. Perhaps it never occurred to you to pair a love of writing with a love of astronomy until you talked with your cousin.

4. Do your homework

So, you've looked inside. You've come up with several interests and you've taken steps to explore what's out there. By this time, you've come up with a few things you might like to do or have found one you've decided you want to pursue. Now it's time to get to work. It's time to delve into what it really means to have a job in a particular career field. To accomplish this part of your journey, you need to do serious research.

Your research homework consists of concrete exploration of available paths for your career options. For the majority of careers, you will need to embark on some type of structured educational path. Examples of this are things like sponsored career programs, college degrees, certification programs, professional designations, internships or apprenticeships. Even if your chosen career path does not require ordered training or education, you will no doubt have to "put in your time" and you will need to find out what and how much time you will realistically be expected to "put in".

So how do you find out? Let's say you've decided you want to seriously explore being a pharmacist. Wonderful! How do you get to be a pharmacist? For starters, inquire with your friends and family to see if anyone knows a pharmacist that you can speak with. Talk to your neighborhood pharmacist, find out where she went to school and ask her about any professional designations she holds or ongoing education she may be taking. If you're brave, ask her what kind of salary pharmacists can expect to earn. In addition, pick up that college course catalog again and inspect the pharmacology program. Look at the prerequisites and notice how long the program will take to complete and how much the courses cost. Read the course descriptions. Do they peak your interest or do they make you want to throw the book down? A great supplement to all of your research is the internet. There are plenty of newsgroup, blog, forum and professional association sites out there. Any of these can give you a solid peak into what it means and takes to be a given occupation.

For each career path you are interested in, you will want to know the following:

* What are the education requirements?
* Does it require any job certification?
* Is there an apprenticeship required?
* Does it require travel?
* How much money can you make?
* Where are the regions of the country that people in this career field are more successful? more in supply? more in demand? make more money?
* Will it require you to relocate?
* Will it require regular, ongoing education?
* Will it require you to have your own business or work for another company?
* Are there yearly fees? (e.g. license renewal, union dues, association membership, equipment, etc.)
* How many hours will you normally work in a week?
* Are there any age requirements or cut-offs?
* How much will it cost you to become gainfully employed in the field?
* How long will it take you to become gainfully employed in the field?

The answers to these questions will help you narrow your career choices further and may even get a fire burning under your feet.

5. Take financial stock

Okay. You've done it! You've come up with one or more career paths that you'd like to embark on. You have looked into what it means to be employed in the career field(s) and you are now powerfully armed with the knowledge of what it takes to get you where you want to go! But like everything in life, it's going to cost you. You now need to list out all the costs, add them up and compare them with what you will be able to swing. There's no doubt you will be able to accomplish your goals, no matter what your financial situation. It may take you longer than you like or may come at the expense of some other items or conveniences in your life, but you can do it if you set your mind to it and approach it methodically. This may mean you have to call upon your research skills again and explore financial options such as grants, scholarships, payment plans, or 401K or private institution education loans. You even may be able to pay for education or experience by doing an internship or by performing work using a skill that you currently have in exchange for training or experience.

When you are taking financial stock, allow yourself breathing room. Perhaps your goals are not financially feasible at this moment in time. Perhaps you just had a baby, your youngest son needs braces, or you've just paid an absorbent amount of money to repair your car. It's okay. Rejoice in the fact that you've come this far. You can put a plan together and start saving or start applying for aid or other means of financing. There may even be things you can start to do that will be free or cheap, such as volunteering in the field or reading books to prepare you for your studies. Most people don't have the luxury of quitting their jobs while they switch careers, so most likely you will be living a "double life" while you prepare for the switch. Whatever you do, don't break the bank, because you'll inevitably find yourself right back where you started.

6. Check your calendar

Great! You've got it all under control. You know where you're going, how you're going to get there and how you're going to pay for it. But can you afford the time? Do you have enough free time or flexibility to meet the educational requirements? Are you young enough or old enough to fall within any professional age requirements? If you have the time and the age, skip this step. If not, the last step of your journey is to shuffle your calendar!

Shuffling your calendar may be easy. You may know right away what to eliminate or move in order to make room for your new career path. Conversely, finding the time might be downright complicated. Maybe you have small children, maybe you have an ailing parent, maybe you have church or community commitments...any of these things would make working towards a new career an uphill battle. Here again, it may mean that you have to post-pone your journey for a little while. Or, it could mean that you have to settle for beginning your education informally by reading books or taking online courses when you can. Whatever your challenge, don't lose hope! Faith in yourself and perseverance will get you where you want to go.

7. Take the plunge

You've arrived at the last step. It's time to jump in! Time to register for that first class, accept that internship, or apply for that entry level or part-time job in your chosen field. Whatever you've found to be the first step towards your new career, delight in the fact that you've come a long way and you're doing it! You've done a lot of hard, thorough work and you have a lot to be proud of. Start down that path with your chin up and your shoulders back!

Food for your trip

You can't take this trip without packing! If you leave home with one thing, leave home with the comforting knowledge that your approach to a new career is circular. This means that you can always go back to the beginning of the circle or to any point within the circle. If you've started a class or program and found that you hate it. So what? You've learned to cross it off you list and go back and take another look. Even if you get all the way to end of the path and decide it is not for you, take comfort that you did your best and its time to go back to another number in the circle. There is no shame in that.

So go on now...get out of here! Your train is leaving and you better be on it! Just don't forget to take some pictures along the way, stop and smell the flowers, buy a few souvenirs and by all means, don't forget where you came from!

Alotta Candor is a staff writer and commentator for JobSchmob.com, the "lighter" side of the working world. She is proud to be a liberated ex-corporate office worker.

http://www.JobSchmob.com

Article Source: http://EzineArticles.com

Article Source: http://EzineArticles.com/24663

Biology Terms: Countershading By Gwen Nicodemus Diamond Quality Author Article Source:

21september1978@gmail.com (Unknown) — Wed, 20 Apr 2011 22:08:00 +0000

Many marine creatures are countershaded, which is a type of camouflage for them. What exactly is countershading?

Imagine that you are in a boat on the ocean and looking down. The ocean water looks dark blue. If you were an animal in the ocean, what color should you be to hide from animals in the sky, or higher up in the ocean, than you are?

Now, imagine you are SCUBA diving on the bottom of the ocean floor and look up, toward the sky. During the daytime, the sunlight filters into the water. What color should an animal be so it can blend into the water and hide from the animals below it in the water?

Animals selected for countershading

Animals that are darkly colored on their top part and lightly colored on their underside are said to be countershaded.

Animals that are darkly colored on their top part, where their dorsal fins are located, are less likely to be found and eaten by sky creatures or ocean animals higher up in the ocean than they are. Their dark colored skin blends into the color of the ocean around them and they have higher survival rates than animals that are lightly colored on the top.

Animals that are lightly colored on their underside blend into the ocean better when looking from the ground upward. They are found and eaten less often by the fish and animals underneath them in the water, so they have better survival rates than animals with darker colored under sides.

So, animals with both of these features have an even better chance of living to an age that they can reproduce.

What kind of animals are countershaded?

Countershading of the type, where the animal blends into its background, is most prevalent with marine animals. Sharks, dolphins, porpoises, fish, and penguins all present this kind of countershading.

Another type of countershading has the same type of principles, but the animals don't blend completely into their background. Instead, their coloring makes it difficult for predators to see where their bodies start and end. Some lizards and caterpillars present this type of countershading.

What have we learned from the animals?

An artist and naturalist named Abbott Thayer studied countershading. He described and published his nature studies regarding countershading in 1892. In fact, sometimes countershading is called Thayer's law. Thayer made his contribution in World War I by suggesting that the military paint their ships using countershading techniques.

Countershading and camouflage techniques are used frequently by the military. Clothing fashions are also influenced by these types of color schemes.

I'm an engineer who quit full-time work and now freelance so I can homeschool my two children. I also teach science classes at a local homeschooling cooperative. As a temporarily retired engineer, I try to keep my brain active by reading, writing, and teaching. Check out the free resources, including unit studies, videos, tutorials, and little books at Unit Studies By Gwen or her blog at GwenOnline.
©2011, Gwen Nicodemus

Article Source: http://EzineArticles.com/?expert=Gwen_Nicodemus

Gwen Nicodemus - EzineArticles Expert Author

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Fun Science Experiment For Kids - How To Make Giant Bubbles! By Kalpana Nair

21september1978@gmail.com (Unknown) — Wed, 20 Apr 2011 22:03:00 +0000

Fun Science Experiment For Kids - How To Make Giant Bubbles!
By Kalpana Nair

Fun Science experiments are not only liked by children but adults as well. Personally, I have tried many Science experiments when I was a kid and later on with my own family members. Making giant bubbles is an absolute favorite of mine and you can do this experiment too in a matter of minutes.

Things You Will Need

In order to make giant bubbles, you need to get your hands on the following items -

1. Washing liquid.
2. Glycerine or corn syrup.
3. Jug
4. Water.
5. A large bucket.
6. A spoon.
7. A wire coat hanger.
8. A ball of string.
9. An electrical tape.

How To Make Your Own Giant Bubbles

1. Fill the large bucket with water.
2. Mix the washing liquid and glycerine/corn syrup in the jug. In order to make the best bubbles, try using one part of washing liquid and a quarter part of glycerine/corn syrup to every 15 parts of water. This solution can be really sticky!
3. Pour this mixture into the large bucket.
4. Shape the wire coat hanger in the form of a tennis racquet i.e, an arm handle with a large circle on top.
5. The arm of the wand and the circle should be tightly wound with the string.
6. You can fasten the string onto its place by using the electrical tape.
7. Insert your wand into the large bucket filled with pre-made mixture of solutions.
8. Swirl your wand in the air and see what happens!

Due to surface tension the water molecules are held together. The presence of washing liquid tends to weaken the surface tension and this is why giant bubbles are formed.

Science projects can be a lot of fun to do especially when you are idle. Not only are some of the experiments absolutely entertaining, but each of them have something to teach you. It is always recommended that an adult be always present for guidance whenever a tough Science experiment is being performed. Like for example, making an electric motor requires the usage of wires and bulbs. Additionally the experiment also involves cutting off pieces of wire in order to create the equipment. This is to be done under the strict surveillance of an adult!

By encouraging your child to do Science experiments, you are only helping them to understand Science concepts in a more clear and precise manner. You can find numerous experiments on the Web which you can perform in the comfort of your own home without the need of purchasing any expensive materials. You will be surprised to see how many high quality education materials are available online -- all for free! Take your time out in noting all these experiments down and then encourage your child to try them out at home.

Kalpana Nair is into Science big time! She has written many Science articles and has published various pages on Science experiments online which have benefitted many students worldwide. One of her best Science pages include - Making Fun Science Models.

The Biotechnology Industry By Richard Graydon, M.D.

21september1978@gmail.com (Unknown) — Wed, 20 Apr 2011 22:01:00 +0000

The 10th Annual Multi-Unit Franchising Conference.

21september1978@gmail.com (Unknown) — Wed, 20 Apr 2011 22:00:00 +0000

The 10th Annual Multi-Unit Franchising Conference.

For ten years, the Multi-Unit Franchising Conference has been the premier conference for multi-unit franchisees. This annual event continues to be the nexus point of learning, networking, and deal-making for multi-unit franchising.
THIS YEAR'S CONFERENCE - WHAT'S NEXT

We all know how to improve our performance by learning from the past. But today's complex business decisions require the ability to see what's on the horizon and anticipate what's next. At this year's conference we'll explore the changes and trends affecting the future of multi-unit franchising and the strategies you need to set in motion today to maintain your competitive advantage.
KEYNOTE SPEAKERS


Sean Tuohy Entrepreneur, NBA Broadcaster & Subject of The Blind Side	Jim Carroll Futurist, Trends & Innovation Expert	J. Patrick Doyle President and CEO, Domino's Pizza	John DiJulius Customer Experience Speaker, Author & Consultant

WHO SHOULD ATTEND

Expected attendance to this event will be over 700 multi-unit franchisees, franchisors, and franchise business solution providers.

Who should attend:

* Multi-Unit Franchisees
* Multi-Concept Franchisees
* Area Developers

* Area Representatives
* Chain Store Operators
* Franchisors

The 10th Annual Multi-Unit Franchising Conference.

* Franchise Investors
* Real Estate Professionals
* Finance Professionals

Special Seminar: Agricultural Biotechnology in Indonesia

21september1978@gmail.com (Unknown) — Sun, 17 Apr 2011 02:32:00 +0000

Thu, January 13, 2011. 12:00-13.30
Prof. Hadi Susilo Arifin attended a Special Seminar of Agricultural Biotechnology in Indonesia
By Dr. M. Herman, Senior Scientist, ICABIOGRAD Biotechnology Center, Bogor, Indonesia.
Agenda
Day/Date: Thursday, January 13, 2011
Time: 12:00 noon – 1:30 p.m.
Venue: 271 Plant and Soil Sciences Building, Michigan State University
Seminar is organized by: WorldTAP/College of Agriculture and Natural Resources and ISP/Asian Studies Center
Pizza Lunch will be served.
Prof. Hadi Susilo Arifin and Dr. Muhammad Herman are colleagues in the team member of POKJA Ahli – Dewan Ketahanan Pangan Nasional RI (Taskforce Expertist - National Board of Food Security, the Republic of Indonesia) – 2010/2013. In the end of the seminar, they have talked together regarding collaboration program in education and capacity building among MSU – IPB – UNPAD. Through an informal discussion, Prof. Karim (Biotechnology – MSU), Prof. Siddharth Chandra (Director of Asian Studies Center), and Prof. Hadi Susilo Arifin (IPB) have expected that for the future collaboration in education and capacity building between MSU and IPB should be expanded not only in biotechnology, but included climate change, environmental management and other actual issues. Some modules and curricula could be established together and delivering them for short courses, workshops, or summer school. These programs should be involved more universities in Indonesia.

How to Choose Speaker

21september1978@gmail.com (Unknown) — Sat, 02 Apr 2011 02:48:00 +0000

This article can be used for reference in selecting speakers. Choosing speakers is tricky. Easy in the sense that the speaker now has a quality that is reliable. difficult, too many outstanding speakers, So making our confused to choice. Therefore here I will share some experience about choosing the speakers.
Here are some points to selecting the speakers:

How to Choose Speaker

Set option for what the speaker to used, whether used indoors or outdoors, because of different types. For speakers who used outside the room, select the type of full range of means all tone is created by the speakers. By using this type of full range, a tone that is created will not be lost, either low or high-pitched tone. But the shortcomings, the speakers full-range tone that created less tender. Meanwhile, if you want to buy speakers for use in the room chose the type woofer (not a sub woofer, because different species), with the woofer low tone will be heard by more soft and smooth. If speakers are used outside the room then the low tone will be lost.
The larger diameter of the speaker, the lower the tone that can be produced, but certainly require a large power amplifiers. 80-10 inches is suitable for home, but the size of 14 inches is not big enough to use a small stage. At least 18 inches, if you want to use in the room, then 10 inches is more than enough.
The speakers that have a large magnet usually have good quality, but the price usually also follow, that meaning is more expensive.
Look for speakers with great power. Do not be fooled by the big power contained labeled (sticker). Look more closely. There are speakers that include the ability to direct the magnetic power, but many were written on a paper sticker. Find a written directly in the magnet, because it is the original power of the speaker. You better choose the power of 150 watts is written directly in the magnet from the power of 300 watts but written on a paper sticker.
The speakers will not produce good sound if it stands alone, so even if you buy any expensive speakers, if you will not be accompanied by Midle range and tweeter will be useless.
large spool having a better quality and will produce a louder sound.
Note the flexibility of the leaves of the speakers, the more flexible will usually produce a low tone more soft and smooth.
Notice the brand, Look for brands that are trusted for speaker manufacturers.

So what can I share with you all, may be useful for visitors, thank you.

How to Choose Speaker

UNITED THERAPEUTICS IS AMONG THE COMPANIES IN THE BIOTECHNOLOGY INDUSTRY WITH THE HIGHEST EPS GROWTH (UTHR, GENZ, ALXN, CELG, CEPH)

21september1978@gmail.com (Unknown) — Wed, 30 Mar 2011 13:19:00 +0000

Mar 30, 2011 (SmarTrend(R) News Watch via COMTEX) -- Below are the top 5 companies in the Biotechnology industry ranked by the year-over-year expected EPS growth rate. The long-term growth rate is the expected annual increase in operating EPS over the next three to five years.
United Therapeutics (NASDAQ:UTHR) EPS is expected to grow 283.1% year-over-year, better than the company's long-term growth rate of 60%. Based on the forward P/E of 12.7x its PEG ratio is 0.21, which signifies a discount in value relative to growth.
Genzyme (NASDAQ:GENZ) EPS is expected to grow 234.8% year-over-year, better than the company's long-term growth rate of 19.3%. Based on the forward P/E of 18.6x its PEG ratio is 0.96, which signifies a discount in value relative to growth.
Alexion Pharmaceuticals (NASDAQ:ALXN) EPS is expected to grow 118.3% year-over-year, better than the company's long-term growth rate of 38.8%. Based on the forward P/E of 43.9x its PEG ratio is 1.13, which signifies a premium valuation given for growth.
Celgene (NASDAQ:CELG) EPS is expected to grow 60% year-over-year, better than the company's long-term growth rate of 25.7%. Based on the forward P/E of 16.4x its PEG ratio is 0.64, which signifies a discount in value relative to growth.
Cephalon (NASDAQ:CEPH) EPS is expected to grow 49.3% year-over-year, better than the company's long-term growth rate of 10.2%. Based on the forward P/E of 7x its PEG ratio is 0.68, which signifies a discount in value relative to growth.
SmarTrend currently has shares of Genzyme in an Uptrend and issued the Uptrend alert on July 23, 2010 at $60.20. The stock has risen 26.3% since the Uptrend alert was issued.

Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

21september1978@gmail.com (Unknown) — Wed, 30 Mar 2011 13:18:00 +0000

Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

Chen Zhang¹, Kun-zheng Wang¹, Hui Qiang¹, Yi-lun Tang¹, Qian Li², Miao Li² and Xiao-qian Dang¹

¹Department of Orthopedic Surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China
²Department of Ultrasound, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China

Correspondence: Xiao-qian Dang, E-mail dang_xiaoqian@sohu.com

Received 22 February 2010; Accepted 6 May 2010; Published online 28 June 2010.

Top

Abstract

Aim:

To investigate the therapeutic potential of adeno-associated virus (AAV)-mediated expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP).

Methods:

Four experimental groups were administered the following AAV vector constructs: rAAV-hVEGF₁₆₅-internal ribosome entry site (IRES)-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF₁₆₅-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP). VEGF₁₆₅ and BMP-7 gene expression was detected using RT-PCR. The VEGF₁₆₅ and BMP-7 protein expression was determined by Western blotting and ELISA. The rabbit ischemic hind limb model was adopted and rAAV was administered intramuscularly into the ischemic limb.

Results:

Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were cultured and infected with the four viral vectors. The expression of GFP increased from the 7th day of infection and could be detected on the 28th day post-infection. In the AAV-VEGF/BMP group, the levels of VEGF165 and BMP-7 increased with prolonged infection time. The VEGF₁₆₅ and BMP-7 secreted from BMSCs in the AAV-VEGF/BMP group enhanced HUVEC tube formation and resulted in a stronger osteogenic ability, respectively. In rabbit ischemic hind limb model, GFP expression increased from the 4th week and could be detected at 8 weeks post-injection. The rAAV vector had superior gene expressing activity. Eight weeks after gene transfer, the mean blood flow was significantly higher in the AAV-VEGF/BMP group. Orthotopic ossification was radiographically evident, and capillary growth and calcium deposits were obvious in this group.

Conclusion:

AAV-mediated VEGF and BMP gene transfer stimulates angiogenesis and bone regeneration and may be a new therapeutic technique for the treatment of avascular necrosis of the femoral head (ANFH).

Keywords:

adeno-associated virus; vascular endothelial growth factor; bone morphogenetic protein (BMP); avascular necrosis of the femoral head (ANFH); gene therapy

Top

Introduction

Recent insight into the pathogenesis of avascular necrosis of the femoral head (ANFH) has not identified satisfactory methods to increase blood circulation in necrotic areas of the femoral head, to promote bone regeneration, or to prevent osteonecrosis. The rapid development of gene therapy technology is increasingly recognized as a new therapeutic option for the treatment of ANFH, especially through therapeutic neovascularization and bone formation. Among growth factors, vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP) play important roles and have been extensively studied.

The VEGF family of growth factors is one of the most important cytokine families involved in angiogenesis. These factors promote the division of vascular endothelial cells and induce angiopoiesis. VEGF growth factors are essential for bone formation and repair during the bone regeneration process, which directly attracts endothelial cells and osteoclasts and enhances the differentiation of osteoblasts^{1, 2}. BMP growth factors are the only signaling molecules that are individually sufficient for the induction of bone formation at orthotopic and heterotopic sites. They have defined roles in stimulating the proliferation and differentiation of mesenchymal and osteoprogenitor cells and have efficient bone induction activity^{3, 4}. Because bone formation is a coordinated process involving the BMP and VEGF growth factors^{5, 6}, orchestrating the timing with which these two factors are expressed may greatly enhance this process.

Choosing a safe and effective vector system to transfer and correctly express a target gene during gene therapy is important. Several different strategies have been examined for the delivery of genes of interest, including the use of naked DNA or an adenoviral vector. Treatment with naked DNA is simple and well tolerated by the recipient organism due to its low toxicity and weak induction of immune responses. However, the transduction efficiency is significantly lower when compared with other methods. The adenovirus has frequently been the vector of choice for gene transfer because it is able to transduce a variety of cells with high efﬁciency. However, adenoviral vectors have major limitations, including a lack of sustained expression, the antigenicity of viral proteins that are targeted by both humoral immunity and cytotoxic T lymphocytes, and possible toxicity at high doses. However, there are many inherent features of the adeno-associated virus system that make it an attractive option as a human viral vector. AAV is a non-pathogenic, defective human parvovirus that requires the presence of a helper virus, such as adenovirus or herpes virus, for productive infection^{7, 8}. Other advantages of this vector system include its low immunogenicity, its ability to transduce both dividing and non-dividing cells, the potential to integrate into specific sites, its ability to achieve long-term gene expression (even in vivo), and its broad tropism, allowing for the efficient transduction of diverse organs⁹. These features make AAV attractive and efficient for gene transfer in vitro and local injection in vivo.

To enhance neovascularization and bone regeneration during osteonecrosis therapy, we constructed adeno-associated viruses co-expressing hVEGF₁₆₅ and hBMP-7 (rAAV-VEGF₁₆₅-IRES-BMP-7) and detected their effect on gene expression and biological activity in vitro and in vivo. These data demonstrate the synergistic action of these two genes and may provide a new therapeutic option for ANFH.

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Materials and methods

Materials and reagents

The rAAV-hVEGF₁₆₅-IRES-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF₁₆₅-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP) plasmids were constructed by Dr Xiang-hui HUANG. Human embryonic kidney cells-293 (HEK-293) and human umbilical vein endothelial cells (HUVECs) were obtained from the Department of Orthopedic Surgery in the Second Affiliated Hospital of Xi'an Jiaotong University. Male New Zealand rabbits (two months old, weighing 2.0–3.0 kg) were obtained from the experimental animal center of Xi'an Jiao Tong University. All animal protocols followed the recommendations and guidelines of the National Institutes of Health and were approved by the Xi'an Jiao Tong University Animal Care and Use Committee. The AAV helper-free system was obtained from Stratagene (La Jolla, CA, USA). A schematic representation of the structure of the plasmids in the AAV Helper Free System is provided in Figure 1A

Insanity Workout - Pushing Workout To The Extreme

21september1978@gmail.com (Unknown) — Sun, 27 Mar 2011 15:07:00 +0000

Are you sick and tired of the standard workout and diet that does not cause you to lose weight whatsoever? Then how about using a different sort of exercise method that will definitely make you shed the unwanted fats you long to take away for years. Shaun T’s insanity workout could be the answer to your desired body figure. It’s a 60-day workout program that will absolutely help you get the body weight and figure you’ve been wishing for in years. Compared to the other intense routines out in the market, this program only takes shorter time however still enable you to do the extensive movements that will ensure to make you sweat off the extra fat away. This program will be suitable for those who rarely have the time and chance to visit the gym.
insanity workout In case you are unhappy with shorter time frame of workout, you might try out something with similar intense and complex exercise moves which you could find in px90. It’s a 90-day intense workout program created by Tony Horton. The said program includes intense workout, complex actions that needs the use of resistance bands and yoga mat to further attain your weight goal. Both this programs will surely help you obtain the body figure and weight you always wished to have. insanity workout,

Insanity Workout - Pushing Workout To The Extreme

Cell therapy strategies and improvements for muscular dystrophy

21september1978@gmail.com (Unknown) — Sat, 19 Mar 2011 09:58:00 +0000

Edited by R De Maria

M Quattrocelli1, M Cassano1, S Crippa1, I Perini1 and M Sampaolesi1,2

1. 1Translational Cardiomyology, SCIL Katholieke Universiteit Leuven, Herestraat 49 bus 814, Leuven 3000, Belgium
2. 2Human Anatomy, University of Pavia, Via Forlanini 8, Pavia 27100, Italy

Correspondence: M Sampaolesi, Translational Cardiomyology, Stem Cell Research Institute, University Hospital Gasthuisberg, Herestraat 49, Leuven B-3000, Belgium. Tel: +32 0163 30295; Fax: +32 0163 30294; E-mail: maurilio.sampaolesi@med.kuleuven.be

Received 9 July 2009; Revised 8 September 2009; Accepted 21 September 2009; Published online 30 October 2009.
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Abstract

Understanding stem cell commitment and differentiation is a critical step towards clinical translation of cell therapies. In past few years, several cell types have been characterized and transplanted in animal models for different diseased tissues, eligible for a cell-mediated regeneration. Skeletal muscle damage is a challenge for cell- and gene-based therapeutical approaches, given the unique architecture of the tissue and the clinical relevance of acute damages or dystrophies. In this review, we will consider the regenerative potential of embryonic and somatic stem cells and the outcomes achieved on their transplantation into animal models for muscular dystrophy or acute muscle impairment.
Keywords:

muscular dystrophy; animal models; cell therapy; stem cells
Abbreviations:

ABCG2, ATP-binding cassette, sub-family G (WHITE), member 2; ALP, alkaline phosphatase; αsg, α-sarcoglycan; bg, natural killer (NK) cell-deficient mice (beige); BM, bone marrow; BMD, Becker muscular dystrophy; BMSCs, bone marrow-derived stem cells; BMT, bone marrow transplantation; βsg, β-sarcoglycan; c-MYC, cellular myelocytomatosis oncogene; cDNA, complementary DNA; Cxcr4, chemokine (C-X-C motif) receptor 4; DMD, Duchenne muscular dystrophy; ESCs, embryonic stem cells; FACS, fluorescence-activated cell sorting; Flk1, fetal liver kinase 1; GFP, green fluorescent protein; GLP–GMP, good laboratory practice–good manufacturing practice; GRMD, Golden Retriever muscular dystrophy; Gy, Gray (unit); HCT, haemopoietic cell transplantation; hMADS, human multi-potent adipose-derived stem cells; HLA, human leukocyte antigen; Hmgb1, high mobility group box 1; IGF1, Insulin-like growth factor 1; iPS, induced pluripotent stem cells; KLF4, Kruppel-like factor 4; KO, knockout; KSN, mice strain with high natural killer activity; LGMD, limb-girdle muscular dystrophy; MABs, mesoangioblasts; Mac1, integrin alpha M; MagicF1, cMet-activating genetically improved factor1; MAPCs, multipotent adult progenitor cells; mdx, muscular dystrophy X-linked (?); μDYS, micro-dystrophin; MGF, mechano growth factor; MMP9, matrix metallopeptidase 9; mRNA, messenger ribonucleic acid; MyoD, myoblast determination protein; Myf5, myogenic factor 5; NCAM, neural cell adhesion molecule; NG2, chondroitin sulfate proteoglycan 4; NICD, Notch1 intracellular domain; NOD, non-obese diabetic; OCT4, octamer-binding transcription factor 4; Pax3, paired box gene 3; PDGFRα, platelet derived growth factor receptor alpha; PIGF, phosphatidylinositol glycan anchor biosynthesis, class F; PTC124, 3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid (C15H9FN2O3); RT-PCR, retrotranscription-based polymerase chain reaction; Sca1, stem cell antigen 1; scid, severe combined immuno-deficiency; SDF1, stromal-derived factor 1; SMPs, skeletal muscle precursors; SOX2, SRY (sex determining region Y)-box 2; SP, side population; TNF-α, tumor necrosis factor-α; VEGFR2, vascular endothelial growth factor receptor 2; Wnt3a, wingless-type MMTV integration site family, member 3A

Muscular dystrophies are a heterogeneous group of inherited diseases, primarily characterized by severe and chronic skeletal muscle degeneration. Duchenne muscular dystrophy (DMD) is the most severe disease among similar dystrophic diseases and is caused by frame-shift deletions, duplications, or point mutations in dystrophin gene. Patient mobility is highly affected, usually resulting in wheelchair dependency, and death occurs due to respiratory or cardiac failure.1, 2

New strategies for the treatment of this disease are currently being investigated and are categorized by two approaches: endogenous activation and exogenous delivery (Figure 1). The first strategy consists of re-activating endogenous cells to achieve muscle hypertrophy, counteracting the mass/force loss in inflamed fibres. To reach this goal, a growing range of small molecules or recombinant proteins has been tested, including insulin-like growth factor 1 (IGF1),3, 4 MagicF15 or valproic acid.6 The second strategy, on the contrary, relies on exogenous tools (gene and/or cell therapies) to improve muscle regeneration, thus providing new, functional fibres to the dystrophic muscle. Gene therapy targets the genetic defects, attempting to overcome pathological mutations by providing the muscle with the correct form of the gene7 or by correcting the splicing through the exon-skipping vectors8, 9 or drugs, such as PTC124.10 Cell therapy, however, is based on stem cell-driven muscle regeneration, by systemic or local injections.
Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Combining endogenous activation and exogenous delivery to enhance muscle regeneration. Currently, two therapeutical approaches are investigated for the regeneration of the skeletal muscle: the endogeneous activation of physiological repair potential, such as targeting of satellite, circulating or perycite cells through small molecules, or the exogenous delivery of stem cells or genetic tools, such as antisense oligonucleotides (ODNs), drugs (PTC124) or viral vectors. M-SP, muscle side population; BM-SP, bone marrow side population; BMSCs, bone marrow-derived stem cells; MABs: mesoangioblasts; MADS, multipotent adipose-derived stem cells; iPS, induced pluripotent stem cells, not yet injected in animal models for muscular dystrophy; ESCs, embryonic stem cells
Full figure and legend (192K)

This review will focus on in vivo cell therapy strategies and improvements in the treatment of sarcoglycan/dystrophin complex-related dystrophies, such as Duchenne or limb-girdle muscular dystrophies.
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Animal Models for Sarcoglycan/Dystrophin Complex-Related Muscular Dystrophies

Sarcoglycan/dystrophin complex-related muscular dystrophies are caused by disruption of the sarcoglycan–dystrophin complex that normally anchors the actin fibres to the sarcolemma, generally resulting in chronic muscle wastage, progressive fibrotic infiltrations and force decrease. Moreover, cardiac involvement is often described, in terms of chronic dilatative cardiomyopathy, scar infiltrations, aneurisms and repeated microinfarctions.

DMD is the most severe form and it is caused by frame-shift mutations or huge deletions in the dystrophin gene. It is one of the largest gene in the human genome and encodes a 14-kb mRNA. In this type of dystrophy, the protein is completely or partially lost. A less severe phenotype is observed in Becker dystrophy, in which mutations still affect the dystrophin gene, but myofibres retain a truncated and low-active isoform of the protein. Some forms of limb-girdle muscular dystrophy (LGMD2) are also caused by mutations in sarcoglycan complex proteins, for example, α- or β-sarcoglycan (αsg or βsg) depletion, and can result in severe pathological phenotypes.

Several animal models have been developed to study muscular dystrophies, particularly for DMD and LGMD2. The most widely used model for dystrophy is the mdx mouse that carries an X-linked mutation in the dystrophin gene, thus mimicking, at least in principle, the DMD genotype in humans. In mdx mice, the effects of degeneration are less severe, mainly due to the presence of relatively high numbers of revertant fibres (1–3%)11 and an upregulation of utrophin. Utrophin is a smaller analogue of the dystrophin and may account for the partial compensatory effect on muscle wastage.12, 13

Recently, it has been demonstrated that mdx satellite cells undergo telomere erosion, which may also contribute to the inability of these cells to continuously repair DMD muscle. It is possible that muscle stem cells or myogenic progenitors that maintain telomerase activity until late passages, may contribute in part to the muscular regeneration, that provides mdx mice with a normal life span.14

Feasible models of LGMD2 are α-sarcoglycan- and β-sarcoglycan-knockout mice. These mice are very close to the human phenotype of LGMD2D and LGMD2E, respectively, as they show chronic skeletal muscle degeneration and, in the case of βsg-KO mice, dilatative cardiomyopathy.15, 16 In α-sarcoglycan-KO mice, αsg gene is disrupted through a neomycin cassette insertion between exons 1 and 9, through homologous recombination of the flanking regions. Similarly, in the β-sarcoglycan-KO mice, the region encompassing exons 3–6 of βsg gene is disrupted. LGMD mice are considered a better animal model than mdx mice because of their lack of revertant fibres, which often render mdx mice-related results controversial.17

Canine models of DMD are being also extensively studied.18, 19 The larger fibres in canine muscles mimic the human dystrophy effects better than that of the mouse. At present, there are two major colonies of dystrophic dogs all over the world, bearing the same mutation in different genetic backgrounds; a colony of Golden Retrievers and one of Beagle dogs. These animals are both derived through cross-breeding of a naturally born, affected founder because transgenic creation would be unethical in these animal models. The mutation lies in intron 6 of dystrophin gene and results in aberrant splicing that causes a premature transcription stop codon. Dystrophic dogs, from both varieties, show extremely affected motility, posing, salivation, severe chronic scar infiltrations and skeletal muscle degeneration. In these animals, revertant fibres are also almost undetectable, thus providing a good model to analyze regeneration effects of cell and genetic therapies.
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Cell Models for In Vivo Skeletal Muscle Regeneration

To date, the only clinical treatments for muscular dystrophy are steroid administration and non-invasive intermittent positive pressure ventilation. These treatments result in amelioration of symptoms and improved quality of life.20 Despite the benefits of slightly longer lifespan, alleviation of pain and surgical management of scoliosis, this therapy shows many side effects, such as weight increase, and has no real beneficial effects on skeletal muscle architecture and force.21 Thus, cell therapy represents a theoretical valuable alternative. The main goal of cell therapy is to directly regenerate wasted, adult muscle fibres through systemic or targeted injection of stem cells, which function to block muscle loss and restore, at least partially, the normal muscular activity. It is currently a difficult task to conjugate high efficiencies in cell motility, homing, engraftment and differentiation into the complex environment of a severely inflammed and degenerated muscle. Several cell models have been tested in vivo, with diverse results (Table 1). Three main classes of stem cells with a myogenic differentiation potential have been considered for cell therapy protocols in preclinical studies for the treatment of muscular dystrophy: (i) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSs); (ii) bone marrow-derived stem cells (BMSCs) and circulating progenitors; and (iii) local myogenic-committed progenitors.
Table 1 - Stem cell types for the treatment of chronic or acute skeletal muscle damage.
Table 1 - Stem cell types for the treatment of chronic or acute skeletal muscle damage - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorFull table

ESCs and iPS cells

Embryonic stem cells (ESCs) are generally considered the most promising natural source of pluripotent cells for cell therapy, but few attempts have been reported in muscle regeneration and efficiencies are still quite low. Importantly, ethical issues around their procurement and use, such as blastocyst disgregation and oocyte requirement, have raised a lot of concerns and limited the use of human ESCs (hESCs) for research purposes in several countries.

The critical step in using ESCs resides in cell conditioning before engraftment, a necessary step that increases the differentiation rate towards myogenesis and avoids teratoma formation. A heterogeneous suspension, derived from a co-culture of male embryoid bodies and female freshly isolated dystrophic satellite cells, has been demonstrated to produce few chimeric, dystrophin-positive myofibres in injured muscles of female mdx mice.22 Promising results have been obtained with hESCs, cultivated to enrich in mesenchymal precursors. The CD73+ NCAM+ sub-fraction was able to differentiate into myotubes in vitro and, remarkably, to regenerate up to 7% of injured skeletal muscle in immunodeficient mice.23 Recently, another strategy has been developed using paraxial mesoderm progenitors that are isolated from differentiating ESCs. The PDGFRα+/Flk1− fraction of Pax3-induced, embryoid body-derived cells shows activation of myogenic transcription factors in vitro and good differentiation in dystrophin+ fibres on transplantation into both cardiotoxin-injured and dystrophic muscle. Injected mice also show an amelioration of the contractility force. In this case, intra-arterial administration of cells resulted in higher engraftment than intravenous injection.24 In another study, paraxial mesoderm progenitors were isolated as PDGFRα+/VEGFR2+ cells, which also have been successfully tested in cardiotoxin-injured quadriceps of KSN nude mice.25

A new source of pluripotent cells comes from the reprogramming of adult murine or human somatic cells, by means of pluripotency transcription factor expression. Human iPS (induced pluripotent stem, hiPS) cells are reprogrammed from differentiated, adult cells, such as fibroblasts, to an ES-like status, by retroviral-mediated transduction of OCT4, SOX2, KLF4 and, even if dispensable, c-MYC.26, 27, 28 As hiPS cells are created by reprogramming adult cells into a flexible, embryonic-like state, they have been claimed as alternative pluripotent cells to overcome the ethical issues regarding the use of ESCs. However, some reservations exist regarding the in vivo safety of hiPS cells, which must be addressed in the future. Recently, a wide range of disease-specific hiPS cells has been generated from patients with various Mendelian or complex diseases, including Duchenne and Becker muscular dystrophy.29 These cells could represent a greater advancement in terms of plasticity and life span, in comparison with other cell lines tested to date. Moreover, these DMD- or BMD-specific hiPS cells show the same genetic background of the donor, thus representing a good model for in vitro drug testing and, if genetically corrected, a suitable cell line for extensive skeletal muscle repair.
Bone marrow-derived and circulating progenitors

Mesenchymal stem cells have been tested in acute and chronic muscle wastage, but results are still controversial. After bone marrow transplantation (BMT) in dystrophic mice, BMSCs are able to migrate and contribute to the formation of new Myf5+ fibres through repeated rounds of inflammation and regeneration, which is typical of the mdx mouse muscles.30 In humans, the clinical case of a young DMD patient (deletion of the exon 45 in the dystrophin gene) has been reported, which 12 years after BMT, showed donor nuclei fused to 0.5% of dystrophic myofibres.31 Real-time RT-PCR analysis detected a small amount of a truncated, in-frame isoform of dystrophin, lacking exons 44 and 45, and trace amounts of the wild-type gene (0.0005%), although a direct correlation between the BMT-derived nuclei and the dystrophin isoform expression was missing. In addition, human mesenchymal stem cells isolated from synovial membrane of adult donors, on intramuscular delivery into tibialis anterior of mdx mice, efficiently produce new, functional myofibres, without any sign of fusion. These cells also contribute to the long-term satellite cell population and restore mechano growth factor (MGF) expression in treated muscles.32 The myogenic potential of these cells seems to be strongly related to the microenvironment surrounding the delivered cells because when injected systemically in dystrophic mice, they are observed in almost all tissues.

Given the broad availability of the source, human multipotent adipose-derived stem cells (hMADS) have been investigated as a possible alternative for muscle regeneration. hMADS are CD44+, CD90+ and CD105+, confirming their mesenchymal lineage. Once injected intramuscularly into tibialis anterior of immunocompetent and immunosuppressed mdx mice, they fuse with host fibres, resulting, 6 months later, in a large number of chimaeric myofibres expressing human dystrophin. Interestingly, no differences are reported between immuno-competent and immuno-suppressed hosts and it also seems that hMADS significantly reduce necrosis in the dystrophic muscle.33 These promising results have since been enhanced by priming the adipose-derived mesenchymal cells by co-culturing with myoblasts34 or forced MyoD expression.35

A sub-population of circulating, haematopoietic stem cells expressing CD133, which constitutes another interesting and easily isolatable cell pool, has been reported to express early myogenic markers.36 Intramuscular or intra-arterial injection of genetically corrected CD133+ progenitors, isolated from both peripheral blood and muscles of DMD patients, results in a significant recovery of muscle morphology, function and re-expression of human dystrophin in scid/mdx mice.37

In contrast, several studies have reported of absent or incomplete muscle repair by mesenchymal or haematopoietic stem cells. On being intravenously injected into a mouse model for LGMD2F (mice lacking δ-sarcoglycan), bone marrow side population cells engraft and fuse into skeletal fibres, but do not restore δ-sarcoglycan expression.38 Green fluorescent protein-positive bone marrow (GFP+ BM) cells, delivered through retro-orbital injection, fuse with ~3% of fibres in the tibialis anterior of treated mdx mice, but almost no dystrophin expression is detected in GFP+ myofibres. However, where dystrophin is detected, its expression is spatially more limited than in revertant fibres.39 These data have been confirmed in another study, in which it has been demonstrated that greater than or equal to80% of BM-derived muscle-incorporated nuclei in the transplanted dystrophic mouse are ‘silent’. Incorporated nuclei fail to express myogenic factors, including dystrophin, and this ‘silencing’ is still retained even in the presence of strong chromatin-remodelling agents, such as 5′-azacytidine.40 It has also been demonstrated that haematopoietic cell transplantation (HCT) alone result neither in any skeletal fibre regeneration nor in expression of dystrophin or other muscle genes.41 Nevertheless, an interesting role for HCT in muscle regeneration may come from immunotolerance effects towards allogeneic myoblast engraftment. DMD dogs have been successfully treated using both peripheral HCT and freshly isolated myoblasts from the same healthy donor. Donor myoblast-related dystrophin expression increased up to ~7% of wild-type levels and was maintained for at least 24 weeks, without any pharmacological immunosuppression.42

Finally, bone marrow-derived multipotent adult progenitor cells (MAPCs) were observed to durably repair muscles in ischaemic limbs, by efficient revascularization of necrotic tissues.43
Local myogenic-committed progenitors

Satellite cells are quiescent unipotent myoprecursors, located between the fibre and the basal lamina; during embryogenesis, they form during the second wave of myogenesis and, after contributing massively to the first post-natal muscle growth, they stop proliferating and reach their niche.44 They can also be re-activated on muscle damage, re-entering cell cycle and contributing to the formation of new muscle fibres.45 Given their natural commitment, it's easy to imagine satellite cells as a major candidate for muscle regeneration in muscular dystrophies. On single fibre transplantation into radiation-ablated mdx tibialis anterior, donor satellite cells multiply and expand, re-populating the satellite cell pool and differentiate into functional myofibres.46 Pax7+ CD34+ GFP+ satellite cells, isolated from Pax3GFP/+ mice diaphragms, have also been demonstrated to be a good cell model for mdx irradiated muscle treatment, resulting in the restoration of dystrophin expression in many skeletal fibres and contributing to the resident satellite compartment.47 Injections were administered intramuscularly and, notably, two major problems arose; satellite cells have a very low migration capability and, furthermore, cells showed impaired engraftment capability when expanded in vitro, even if for few days.47

To test the regeneration capability of satellite cells at a clonal level, single-cell dilutions of CD34+ integrinα+ luciferase-expressing satellite cells were injected into the skeletal muscle of a NOD/SCID mouse depleted of resident satellite cells by 18 Gy irradiation. It was shown by in vivo imaging that a single satellite cell can reconstitute the satellite compartment and, on further damage, can rapidly re-enter a new proliferation wave, generating new myofibres.48

Recently, interesting findings resulted from a prospective isolation of skeletal muscle precursors (SMPs), consisting of a CD45− Sca1− Mac1− Cxcr4+ β1-integrin+ subset within the endogenous satellite compartment. When injected into cardiotoxin-injured muscles in immunodeficient mdx mice, SMPs robustly contributed to muscle regeneration (up to 94%) by fusing with pre-existing fibres or stimulating de novo myogenesis. Muscle histology and contractile force in treated mice were significantly better than those in untreated mice. Furthermore, SMPs contributed greatly to the endogenous satellite population, undergoing new cycles of re-activation on subsequent induced damage.49 However, as freshly isolated SMPs were injected, without in vitro expansion, the migration capability remains restricted in the areas surrounding the intramuscular injection site.

One of the major concerns about satellite cells is that, probably, they are not a homogeneous population. As argued by Collins et al.,46 the variable engraftment rate of satellite cells from a single myofibre transplantation could be due to the functional heterogeneity of the satellite cell pool and of their niche of origin. Satellite cell heterogeneity is still a contentious issue and has been extensively reviewed in several studies50, 51 and is still open for debate.52

Muscle side population (SP) cells are defined as Sca1+ CD45+ cells, able to rapidly efflux the Hoechst dye 33342, and are being investigated as potential myogenic progenitors. They are associated with the muscle vasculature and are spontaneously committed towards the haematopoietic lineage. On co-culture with myoblasts, they can form myotubes in vitro and, if injected intramuscularly into crushed tibialis of a scid/bg immunodeficient mouse, can give rise to up to 1% of regenerating fibres.53 The efficiency of SP-mediated muscle regeneration has been increased to 5–8% by injection into the femoral artery of mdx5cv DMD mice, resulting in Pax7 and desmin expression by donor cells, after extravasation and recruitment to inflammation sites.54 Impressive results have been obtained with the identification of a rare subset (0.25%) of SP cells, characterized by both satellite- and SP-related markers, such as Sca1+/ABCG2+/Syndecan4+/Pax7+, and found in the typical satellite compartment, under the basal lamina. Once sorted from the mononuclear fraction of the hind limb, they can grow in association with single muscle fibres and can robustly undergo myogenic differentiation in vitro. On intramuscular injection in the presence of 1.2% BaCl2, these satellite-SP cells have been shown to efficiently compete with endogenous satellite cells in regenerating the wild-type muscle. Injected cells resulted in 30% fibre regeneration and, strikingly, in up to 75% reconstitution of the endogenous satellite cell pool. The newly generated satellite cells were able to undergo new rounds of proliferation and muscle repair on subsequent injuries. Furthermore, the same long-term effects have been proven through injections into dystrophic mdx4cv tibialis anterior muscles, producing up to 70% regenerating fibres.55 However, BaCl2-induced muscle damage is not a widely used and accepted regeneration model, and this must be taken into consideration when interpreting these findings.

Recently, a new type of vessel-associated muscle-derived stem cells has been investigated as a suitable potential model for chronic muscle therapy, namely mesoangioblasts (MABs). They can be isolated from the dorsal aorta of E9.5 embryo56 or from adult skeletal muscle of mice, dogs and humans.57, 58, 59 MABs are CD34+, Sca1+, PDGFRα+, PDFGRβ+, NG2+ and ALP+, thus supporting the idea that they are a sub-group of the pericytic population.60 They show high proliferation rates in vitro, without transformation potential, and display, in in vitro and embryonic chimaera systems, multipotent differentiation capability towards myogenic, osteogenic, chondrogenic and adipogenic lineages. On intra-arterial injection into inflammated muscles of αsg-KO mice or DMD Golden Retriever dogs, they are capable of consistently regenerating (up to 50%) the muscle architecture, sarcoglycan/dystrophin expression and electrophysiological properties of wasted dystrophic muscle.57, 58 Similarly, good results have been achieved through human MAB transplantation into scid–mdx immunodeficient dystrophic mice.59

Treatment of dogs with MABs resulted in very good and long-term results in some animals, in terms of general motility restoration and whole muscle regeneration, whereas other animals did not show a good engraftment and any clinical improvement.58 This confirms the general idea that background variability in dystrophies in larger organisms, such as dogs or humans, has to be evaluated for effective cell therapies.

Furthermore, another class of myogenic precursors has been isolated from endothelial population of adult human muscle, through FACS-mediated prospective isolation of CD56+ CD34+ CD144+ cells. These myoendothelial progenitors, on injection into injured muscle of scid mice can achieve muscle engraftment and fibre neo-formation at an higher degree than CD56+ canonical myoprecursors.61
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Cell Conditioning and Priming

To increase engraftment specificity and differentiation potential, cell transplantation can be combined with pre-injection cell conditioning or with genetic manipulation.

Cell migration limitations are often a major cause of low engraftment efficiencies in skeletal muscle, which is a very complex tissue with severely limited cell motility, particularly in the presence of large fibrotic or necrotic areas. Thus, some attempts have been made to assist migration by conditioning cells with migration-enhancing soluble factors or by overexpressing commiting/mobilizing proteins before applying them to the degenerated muscle.

A lot of interest has been shown in Notch signalling, as a potential enhancer of myogenic commitment. Rat BMSCs, transfected with a plasmid for the intracellular domain of Notch1 (NICD) and injected locally or intravenously into injured muscles of rats or nude mice, account for a very high level of regenerating fibres (up to 89%).62 Given that NICD is the active signalling form of the receptor, it could be possible that finer genetic tools to activate Notch signalling could enhance myogenesis by donor cell injection, although recently it has been demonstrated that a temporal switch from Notch to Wnt3a signalling activation is necessary during normal adult myogenesis.63

Encouraging in vivo results have come from cell therapy experiments involving soluble factor-dependent cell conditioning. Hmgb1, a cytokine secreted by activated macrophages and monocytes, is able to increase the recruitment of MABs out of the vessels into the muscle. Heparin–Sepharose beads, loaded with Hmgb1 and injected into the femoral artery, were shown to promote the trans-endothelial migration of intra-arterial-injected embryonic MABs into non-injured tibialis anterior of wild-type mice.64 Pre-treatment of MABs with mobilization cytokines, such as SDF1 and TNFα, highly increase MAB homing into dystrophic muscle of αsg-null mice, thus reducing approximately 50% the aspecific homing into filter organs. Their regenerative effect was magnified by TNFα priming and α4-integrin overexpression.65 Furthermore, improving the angiogenic potential in necrotic areas could help cell therapy. For example, tendon fibroblasts expressing placenta growth factor (PIGF, an angiogenic factor) and matrix metalloproteinase 9 (MMP9), injected intramuscularly into aged αsg-KO mice, result in a dramatic increase in the extension of regenerated dystrophin+ muscle areas after intra-arterial delivery of wild-type MABs.66 Such results corroborate the idea that a deeper knowledge of cytokines and angiogenic factors regulating the inflammation-dependent recruitment of myoprecursors will serve to improve the benefits mediated by cell therapy.
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Genetic Manipulation for Autologous Cell Therapy

Correction of the dystrophic genotype in the transplanted cells could allow the usage of autologous cells, instead of heterologous wild-type cells, thus avoiding immunosuppressive drugs.

A widely used strategy relies on lentiviral transduction of muscle-regenerating cells to allow integration and expression of the disrupted gene. To correct DMD, several alternatives of the dystrophin gene (which is too long to be introduced in a lentiviral vector) exist, such as micro- or mini-dystrophin. These are shorter isoforms of the native protein, which retain a partial functionality, thus providing, in principle, a possible molecular rescue on differentiation towards newly formed myofibres. mdx5cv mice, injected intravenously with autologous muscle-SP cells that were previously transduced with human micro-dystrophin (hμDYS)-expressing lentiviruses, show some skeletal fibres positive for the human version of the lacking protein.67 Similarly, very good results came from transplantation of hμDYS-transduced human pericytes into mdx–scid mice.59 In GRMD dog treatment, autologous MABs, transduced with human micro-dystrophin, induced a quite widespread expression of dystrophin and other proteins of the sarcoglycan complex in analyzed muscles and a partial recovery of the histological architecture. However, treated dogs had poorly restored general motility and force.58 These results may support the idea that, especially in higher organisms, mini- or micro-genes are not so feasible, or, at least, show highly variable and only short-term effects. Genetic correction can be addressed also for treatment of other genetic defects resulting in dystrophy, as when αsg-KO MABs, carrying a lentiviral αsg cDNA construct under a constitutive promoter, are injected into LGMD2D mice, there is extensive fibre regeneration, mobility and muscle force recovery.57
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Conclusions and Perspectives

Stem cell-based therapy is the most attractive approach for the treatment of DMD and other muscular dystrophies, and research in this direction has moved rapidly in past few years. Experiments in small and large animal models are paving the way for clinical experimentation, but it would be imprudent to predict a ‘cure’ from these first attempts. Nevertheless, several clinical trials have been started or planned, involving myoblast or perycyte injection from HLA-matched donors and, given the growing variety of possible myogenic progenitors in literature, the number will increase during the near future. Other clinical trials are focusing on gene- or antibody-based strategies, such as adeno-associated viruses carrying γ-sarcoglycan or μ-dystrophin and antibody-triggered myostatin blockade.21

Furthermore, encouraging results have come from clinical trials related to exon-skipping technology. Specific exons carrying mutations can be skipped by antisense oligonucleotides administration to restore the reading frame and result in the production of internally deleted, but functional dystrophin. Recently, two clinical trials involving two different drugs, AVI-4658 (developed by the MDEX Consortium, United Kingdom and manufactured by AVI BioPharma, Bothell, WA, USA) and PRO051 (developed by University of Leiden, The Nederlands in collaboration with Prosensa B.V.), were performed on Duchenne patients. In both trials, biopsy data showed that injections of antisense oligonucleotides, to skip exon 51, into dystrophic muscles, successfully induced new dystrophin production, with no adverse events. These pioneering studies are now followed by randomized controlled trials of systemic therapies both in The Netherlands and the United Kingdom.

Therefore, it is reasonable to expect encouraging results that may also drive a combination of the stem cell- and gene-based therapies. It is critical to better understand the biological properties of stem cells and their paracrine role in the treatment of muscular diseases, to realize the potential positive effects of these new cures.

The scientific community largely accepts the presence of adult stem cells in all tissues but their origin is still controversial. We and other authors suggest pericytes as a source of stem cells present in skeletal and cardiac muscles.59, 60 They are influenced by their surrounding when maintaining a specific cell commitment, although this has to be clarified in pathological tissues.

Interestingly, other research groups have identified mesenchymal cells as stem cells for all tissues,68 raising questions regarding the possibility that the primary source of cell plasticity is confined to the bone marrow. It could be possible that cells move from bone marrow towards pericyte compartment, to adopt a specific cell fate, influenced by local niche. To further elucidate their origin, it is necessary to generate transgenic animals to track endogenous stem cells during muscle development and regeneration.

Despite the fact that current regulatory restrictions will defer the clinical translation of new approaches, that are successful in animal models, several trials have been started. Moreover, the actual huge costs of GLP–GMP (good laboratory and manufacture practice) stem cell technology limit the feasibility of cell therapy treatment for patients affected by muscular dystrophy. However, strategies to lower costs are being investigated to develop treatments that are available for large numbers of patients. In conclusion, it is critical to better understand the biological properties of stem cells and their paracrine role in the treatment of muscular diseases, to realize the potential positive effects of these new cures. Therefore, it is reasonable to expect encouraging results from the on-going trials that may also drive a combination of stem cell- and gene-based therapies for the treatment of muscular dystrophies.
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References

1. Emery AE. The muscular dystrophies. Lancet 2002; 359: 687–695. | Article | PubMed | ISI | ChemPort |
2. Cossu G, Sampaolesi M. New therapies for Duchenne muscular dystrophy: challenges, prospects and clinical trials. Trends Mol Med 2007; 13: 520–526. | Article | PubMed | ChemPort |
3. Abmayr S, Gregorevic P, Allen JM, Chamberlain JS. Phenotypic improvement of dystrophic muscles by rAAV/microdystrophin vectors is augmented by Igf1 codelivery. Mol Ther 2005; 12: 441–450. | Article | PubMed | ChemPort |
4. Pelosi L, Giacinti C, Nardis C, Borsellino G, Rizzuto E, Nicoletti C et al. Local expression of IGF-1 accelerates muscle regeneration by rapidly modulating inflammatory cytokines and chemokines. FASEB J 2007; 21: 1393–1402. | Article | PubMed | ChemPort |
5. Cassano M, Biressi S, Finan A, Benedetti L, Omes C, Boratto R et al. Magic-factor 1, a partial agonist of Met, induces muscle hypertrophy by protecting myogenic progenitors from apoptosis. PLoS ONE 2008; 3: e3223. | Article | PubMed | ChemPort |
6. Minetti GC, Colussi C, Adami R, Serra C, Mozzetta C, Parente V et al. Functional and morphological recovery of dystrophic muscles in mice treated with deacetylase inhibitors. Nat Med 2006; 12: 1147–1150. | Article | PubMed | ChemPort |
7. Hoshiya H, Kazuki Y, Abe S, Takiguchi M, Kajitani N, Watanabe Y et al. A highly stable and nonintegrated human artificial chromosome (HAC) containing the 2.4 Mb entire human dystrophin gene. Mol Ther 2008; 17: 309–317. | Article | PubMed | ChemPort |
8. Lu QL, Mann CJ, Lou F, Bou-Gharios G, Morris GE, Xue SA et al. Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse. Nat Med 2003; 9: 1009–1014. | Article | PubMed | ISI | ChemPort |
9. Benchaouir R, Meregalli M, Farini A, D'Antona G, Belicchi M, Goyenvalle A et al. Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice. Cell Stem Cell 2007; 1: 646–657. | Article | PubMed | ChemPort |
10. Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature 2007; 447: 87–91. | Article | PubMed | ISI | ChemPort |
11. Hoffman EP, Morgan JE, Watkins SC, Partridge TA. Somatic reversion/suppression of the mouse mdx phenotype in vivo. J Neurol Sci 1990; 99: 9–25. | Article | PubMed | ISI | ChemPort |
12. Weir AP, Burton EA, Harrod G, Davies KE. A- and B-utrophin have different expression patterns and are differentially upregulated in mdx muscle. J Biol Chem 2002; 277: 45285–45290. | Article | PubMed | ISI | ChemPort |
13. Hirst RC, McCullagh KJ, Davies KE. Utrophin upregulation in Duchenne muscular dystrophy. Acta Myol 2005; 24: 209–216. | PubMed | ChemPort |
14. Lund TC, Grange RW, Lowe DA. Telomere shortening in diaphragm and tibialis anterior muscles of aged mdx mice. Muscle Nerve 2007; 36: 387–390. | Article | PubMed | ChemPort |
15. Duclos F, Straub V, Moore SA, Venzke DP, Hrstka RF, Crosbie RH et al. Progressive muscular dystrophy in alpha-sarcoglycan-deficient mice. J Cell Biol 1998; 142: 1461–1471. | Article | PubMed | ISI | ChemPort |
16. Durbeej M, Cohn RD, Hrstka RF, Moore SA, Allamand V, Davidson BL et al. Disruption of the beta-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E. Mol Cell 2000; 5: 141–151. | Article | PubMed | ISI | ChemPort |
17. Cossu G. Fusion of bone marrow-derived stem cells with striated muscle may not be sufficient to activate muscle genes. J Clin Invest 2004; 114: 1540–1543. | PubMed | ChemPort |
18. Kornegay JN, Tuler SM, Miller DM, Levesque DC. Muscular dystrophy in a litter of golden retriever dogs. Muscle Nerve 1988; 11: 1056–1064. | Article | PubMed | ISI | ChemPort |
19. Sharp NJ, Kornegay JN, Van Camp SD, Herbstreith MH, Secore SL, Kettle S et al. An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy. Genomics 1992; 13: 115–121. | Article | PubMed | ISI | ChemPort |
20. Daftary AS, Crisanti M, Kalra M, Wong B, Amin R. Effect of long-term steroids on cough efficiency and respiratory muscle strength in patients with Duchenne muscular dystrophy. Pediatrics 2007; 119: e320–e324. | Article | PubMed
21. Cossu G, Sampaolesi M. New therapies for muscular dystrophy: cautious optimism. Trends Mol Med 2004; 10: 516–520. | Article | PubMed | ChemPort |
22. Bhagavati S, Xu W. Generation of skeletal muscle from transplanted embryonic stem cells in dystrophic mice. Biochem Biophys Res Commun 2005; 333: 644–649. | Article | PubMed | ChemPort |
23. Barberi T, Bradbury M, Dincer Z, Panagiotakos G, Socci ND, Studer L. Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nat Med 2007; 13: 642–648. | Article | PubMed | ChemPort |
24. Darabi R, Gehlbach K, Bachoo RM, Kamath S, Osawa M, Kamm KE et al. Functional skeletal muscle regeneration from differentiating embryonic stem cells. Nat Med 2008; 14: 134–143. | Article | PubMed | ChemPort |
25. Sakurai H, Okawa Y, Inami Y, Nishio N, Isobe K. Paraxial mesodermal progenitors derived from mouse embryonic stem cells contribute to muscle regeneration via differentiation into muscle satellite cells. Stem Cells 2008; 26: 1865–1873. | Article | PubMed | ChemPort |
26. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131: 861–872. | Article | PubMed | ChemPort |
27. Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 2008; 451: 141–146. | Article | PubMed | ChemPort |
28. Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 2008; 26: 101–106. | Article | PubMed | ChemPort |
29. Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A et al. Disease-specific induced pluripotent stem cells. Cell 2008; 134: 877–886. | Article | PubMed | ChemPort |
30. Bittner RE, Schöfer C, Weipoltshammer K, Ivanova S, Streubel B, Hauser E et al. Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice. Anat Embryol (Berl) 1999; 199: 391–396. | Article | PubMed | ChemPort |
31. Gussoni E, Bennett RR, Muskiewicz KR, Meyerrose T, Nolta JA, Gilgoff I et al. Long-term persistence of donor nuclei in a Duchenne muscular dystrophy patient receiving bone marrow transplantation. J Clin Invest 2002; 110: 807–814. | Article | PubMed | ISI | ChemPort |
32. De Bari C, Dell'Accio F, Vandenabeele F, Vermeesch JR, Raymackers JM, Luyten FP. Skeletal muscle repair by adult human mesenchymal stem cells from synovial membrane. J Cell Biol 2003; 160: 909–918. | Article | PubMed | ChemPort |
33. Rodriguez AM, Pisani D, Dechesne CA, Turc-Carel C, Kurzenne JY, Wdziekonski B et al. Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse. J Exp Med 2005; 201: 1397–1405. | Article | PubMed | ISI | ChemPort |
34. Di Rocco G, Iachininoto MG, Tritarelli A, Straino S, Zacheo A, Germani A et al. Myogenic potential of adipose-tissue-derived cells. J Cell Sci 2006; 119: 2945–2952. | Article | PubMed | ChemPort |
35. Goudenege S, Pisani DF, Wdziekonski B, Di Santo JP, Bagnis C, Dani C et al. Enhancement of myogenic and muscle repair capacities of human adipose-derived stem cells with forced expression of MyoD. Mol Ther 2009; 17: 1064–1072. | Article | PubMed | ChemPort |
36. Torrente Y, Belicchi M, Marchesi C, Dantona G, Cogiamanian F, Pisati F et al. Autologous transplantation of muscle-derived CD133+ stem cells in Duchenne muscle patients. Cell Transplant 2007; 16: 563–577. | PubMed | ChemPort |
37. Benchaouir R, Meregalli M, Farini A, D'Antona G, Belicchi M, Goyenvalle A et al. Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice. Cell Stem Cell 2007; 1: 646–657. | Article | PubMed | ChemPort |
38. Lapidos KA, Chen YE, Earley JU, Heydemann A, Huber JM, Chien M et al. Transplanted hematopoietic stem cells demonstrate impaired sarcoglycan expression after engraftment into cardiac and skeletal muscle. J Clin Invest 2004; 114: 1577–1585. | Article | PubMed | ChemPort |
39. Chretien F, Dreyfus PA, Christov C, Caramelle P, Lagrange JL, Chazaud B et al. In vivo fusion of circulating fluorescent cells with dystrophin-deficient myofibers results in extensive sarcoplasmic fluorescence expression but limited dystrophin sarcolemmal expression. Am J Pathol 2005; 166: 1741–1748. | PubMed | ChemPort |
40. Wernig G, Janzen V, Schäfer R, Zweyer M, Knauf U, Hoegemeier O et al. The vast majority of bone-marrow-derived cells integrated into mdx muscle fibers are silent despite long-term engraftment. Proc Natl Acad Sci USA 2005; 102: 11852–11857. | Article | PubMed | ChemPort |
41. Dell'Agnola C, Wang Z, Storb R, Tapscott SJ, Kuhr CS, Hauschka SD et al. Hematopoietic stem cell transplantation does not restore dystrophin expression in Duchenne muscular dystrophy dogs. Blood 2004; 104: 4311–4318. | Article | PubMed | ISI | ChemPort |
42. Parker MH, Kuhr C, Tapscott SJ, Storb R. Hematopoietic cell transplantation provides an immune-tolerant platform for myoblast transplantation in dystrophic dogs. Mol Ther 2008; 16: 1340–1346. | Article | PubMed | ChemPort |
43. Aranguren XL, McCue JD, Hendrickx B, Zhu XH, Du F, Chen E et al. Multipotent adult progenitor cells sustain function of ischemic limbs in mice. J Clin Invest 2008; 118: 505–514. | PubMed | ChemPort |
44. Biressi S, Molinaro M, Cossu G. Cellular heterogeneity during vertebrate skeletal muscle development. Dev Biol 2007; 308: 281–293. | Article | PubMed | ChemPort |
45. Cossu G, Biressi S. Satellite cells, myoblasts and other occasional myogenic progenitors: possible origin, phenotypic features and role in muscle regeneration. Semin Cell Dev Biol 2005; 16: 623–631. | Article | PubMed | ISI | ChemPort |
46. Collins CA, Olsen I, Zammit PS, Heslop L, Petrie A, Partridge TA et al. Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 2005; 122: 289–301. | Article | PubMed | ISI | ChemPort |
47. Montarras D, Morgan J, Collins C, Relaix F, Zaffran S, Cumano A et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science 2005; 309: 2064–2067. | Article | PubMed | ISI | ChemPort |
48. Sacco A, Doyonnas R, Kraft P, Vitorovic S, Blau HM. Self-renewal and expansion of single transplanted muscle stem cells. Nature 2008; 456: 502–506. | Article | PubMed | ChemPort |
49. Cerletti M, Jurga S, Witczak CA, Hirshman MF, Shadrach JL, Goodyear LJ et al. Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles. Cell 2008; 134: 37–47. | Article | PubMed | ChemPort |
50. Zammit PS. All muscle satellite cells are equal, but are some more equal than others? J Cell Sci 2008; 121: 2975–2982. | Article | PubMed | ChemPort |
51. Kuang S, Rudnicki MA. The emerging biology of satellite cells and their therapeutic potential. Trends Mol Med 2008; 14: 82–91. | Article | PubMed | ChemPort |
52. Partridge T. Denominator problems in a muscle stem cell study? Cell 2008; 135: 997–998; author reply 998–999. | Article | PubMed | ChemPort |
53. Asakura A, Seale P, Girgis-Gabardo A, Rudnicki MA. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002; 159: 123–134. | Article | PubMed | ISI | ChemPort |
54. Bachrach E, Perez AL, Choi YH, Illigens BM, Jun SJ, del Nido P et al. Muscle engraftment of myogenic progenitor cells following intraarterial transplantation. Muscle Nerve 2006; 34: 44–52. | Article | PubMed
55. Tanaka KK, Hall JK, Troy AA, Cornelison DD, Majka SM, Olwin BB. Syndecan-4-expressing muscle progenitor cells in the SP engraft as satellite cells during muscle regeneration. Cell Stem Cell 2009; 4: 217–225. | Article | PubMed | ChemPort |
56. Minasi MG, Riminucci M, De Angelis L, Borello U, Berarducci B, Innocenzi A et al. The meso-angioblast: a multipotent, self-renewing cell that originates from the dorsal aorta and differentiates into most mesodermal tissues. Development 2002; 129: 2773–2783. | PubMed | ISI | ChemPort |
57. Sampaolesi M, Torrente Y, Innocenzi A, Tonlorenzi R, D'Antona G, Pellegrino MA et al. Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science 2003; 301: 487–492. | Article | PubMed | ISI | ChemPort |
58. Sampaolesi M, Blot S, D'Antona G, Granger N, Tonlorenzi R, Innocenzi A et al. Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature 2006; 444: 574–579. | Article | PubMed | ISI | ChemPort |
59. Dellavalle A, Sampaolesi M, Tonlorenzi R, Tagliafico E, Sacchetti B, Perani L et al. Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells. Nat Cell Biol 2007; 9: 255–267. | Article | PubMed | ChemPort |
60. Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008; 3: 301–313. | Article | PubMed | ChemPort |
61. Zheng B, Cao B, Crisan M, Sun B, Li G, Logar A et al. Prospective identification of myogenic endothelial cells in human skeletal muscle. Nat Biotechnol 2007; 25: 1025–1034. | Article | PubMed | ChemPort |
62. Dezawa M, Ishikawa H, Itokazu Y, Yoshihara T, Hoshino M, Takeda S et al. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science 2005; 309: 314–317. | Article | PubMed | ChemPort |
63. Brack AS, Conboy IM, Conboy MJ, Shen J, Rando TA. A temporal switch from notch to Wnt signaling in muscle stem cells is necessary for normal adult myogenesis. Cell Stem Cell 2008; 2: 50–59. | Article | PubMed | ChemPort |
64. Palumbo R, Sampaolesi M, De Marchis F, Tonlorenzi R, Colombetti S, Mondino A et al. Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation. J Cell Biol 2004; 164: 441–449. | Article | PubMed | ISI | ChemPort |
65. Galvez BG, Sampaolesi M, Brunelli S, Covarello D, Gavina M, Rossi B et al. Complete repair of dystrophic skeletal muscle by mesoangioblasts with enhanced migration ability. J Cell Biol 2006; 174: 231–243. | Article | PubMed | ChemPort |
66. Gargioli C, Coletta M, De Grandis F, Cannata SM, Cossu G. PlGF-MMP-9-expressing cells restore microcirculation and efficacy of cell therapy in aged dystrophic muscle. Nat Med 2008; 14: 973–978. | Article | PubMed | ChemPort |
67. Bachrach E, Li S, Perez AL, Schienda J, Liadaki K, Volinski J et al. Systemic delivery of human microdystrophin to regenerating mouse dystrophic muscle by muscle progenitor cells. Proc Natl Acad Sci USA 2004; 101: 3581–3586. | Article | PubMed | ChemPort |
68. Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell 2008; 2: 313–319. | Article | PubMed | ChemPort |

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Acknowledgements

We thank Paolo Luban for his support. We are particularly grateful to Guido Tettamanti and Giulio Cossu for critical reading of the paper and for helpful comments, and Shea Carter for the ms proofreading service. This work was supported by FWO Odysseus Program n. G.0907.08; Wicka Funds n. zkb8720, University of Minnesota US; the Italian Ministry of University and Scientific Research (grant n. 2005067555_003, COFIN 2006–08), the Muscular Dystrophy Association, Association Francoise contre les Myopathies, CARIPLO Funds 2007-5639 and 2008-2005.

In vitro and in vivo multidrug resistance reversal activity by a Betti-base derivative of tylosin

21september1978@gmail.com (Unknown) — Sat, 19 Mar 2011 09:58:00 +0000

N Gyémánt, H Engi, Z Schelz, I Szatmári, D Tóth, F Fülöp, J Molnár and P A M de Witte

Background:

The multidrug resistance (MDR) proteins are present in a majority of human tumours. Their activity is important to understand the chemotherapeutic failure. A search for MDR-reversing compounds was conducted among various Betti-base derivatives of tylosin.

Methods:

Here, we evaluate the in vitro and in vivo P-glycoprotein (P-gp)-modulating activity of the most promising compound N-tylosil-1-α-amino-(3-bromophenyl)-methyl-2-naphthol (TBN) using human MDR1 gene-transfected and parental L5178 mouse lymphoma cell lines.

Results:

In vitro experiments showed that TBN dramatically increased the P-gp-mediated cellular uptake of the fluorescent substrate rhodamine 123. Similarly, TBN was found to act as a very potent enhancer of the cytotoxicity of doxorubicin on the resistant cell line. We also provide in vivo evidence using DBA/2 mice in support for an increased tumoural accumulation of doxorubicin, without affecting its tissue distribution, resulting in an enhanced antitumoural effect.

Conclusion:

Our results suggest that TBN is a potent modulator of the P-gp membrane pump and that the compound could be of clinical relevance to improve the efficacy of chemotherapy in MDR cancers.

British Journal of Cancer 103, 178-185

Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

21september1978@gmail.com (Unknown) — Sat, 19 Mar 2011 09:57:00 +0000

Acta Pharmacologica Sinica (2010) 31: 821–830; doi: 10.1038/aps.2010.67; published online 28 June 2010

Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

Chen Zhang¹, Kun-zheng Wang¹, Hui Qiang¹, Yi-lun Tang¹, Qian Li², Miao Li² and Xiao-qian Dang¹

¹Department of Orthopedic Surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China
²Department of Ultrasound, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China

Correspondence: Xiao-qian Dang, E-mail dang_xiaoqian@sohu.com

Received 22 February 2010; Accepted 6 May 2010; Published online 28 June 2010.

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Abstract

Aim:

To investigate the therapeutic potential of adeno-associated virus (AAV)-mediated expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP).

Methods:

Results:

Conclusion:

AAV-mediated VEGF and BMP gene transfer stimulates angiogenesis and bone regeneration and may be a new therapeutic technique for the treatment of avascular necrosis of the femoral head (ANFH).

Keywords:

adeno-associated virus; vascular endothelial growth factor; bone morphogenetic protein (BMP); avascular necrosis of the femoral head (ANFH); gene therapy

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Introduction

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Materials and methods

Materials and reagents

Figure 1.

(A) Schematic representation of the structure of plasmids in AAV Helper Free System. (B) Conceptual diagram of construction of pAAV-hVEGF₁₆₅-IRES-hBMP-7. hVEGF₁₆₅ gene (600 bp) and hBMP-7 gene (1300 bp) were respectively inserted into upstream MCS and downstream MCS located on either side of IRES sequence (631 bp). The length of the bicistronic frame is 2.5 kb.
Full figure and legend (72K)

rAAV vector production

The construction of the rAAV-hVEGF₁₆₅-IRES-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF₁₆₅-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP) vectors was carried out as previously described¹⁰. The structure of the pAAV-hVEGF₁₆₅-IRES-hBMP-7 vector is shown in Figure 1B. IRES sequences were incorporated into the pAAV MCS to construct a bicistronic vector with two multiple cloning sites. Then, the hVEGF₁₆₅ (Pubmed NM-003376) and hBMP-7 (Pubmed NM-001719) genes were inserted into the upstream and downstream MCS, respectively. The length of the bicistronic frame is 2.5 kb, which is within the capacity of the vector. The AAV helper-free system was used to generate recombinant AAV. HEK-293 cells were cultured in H-DMEM supplemented with 10% fetal bovine serum containing 20 mg/mL penicillin-streptomycin and incubated with 5% CO₂ at 37 ^oC. The AAV vector was co-transfected with the pAAV-helper and pAAV-RC vectors into HEK 293 cells by a calcium phosphate method according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). A primary virus stock was collected 72 h after transfection and further concentrated and purified by chloroform/PEG8000 protocols¹¹. The recombinant adeno-associated virus had a titer of 5.5×10¹¹ vp/mL.

Rabbit bone marrow-derived mesenchymal stem cells (BMSC) culture and rAAV infection in vitro

Male New Zealand rabbits were used to obtain rabbit BMSCs. The cells were harvested by gently flushing the tibiae and femora with L-DMEM. Density gradient centrifugation and adherent screening methods were used to isolate BMSCs as previously described¹². The cells were cultured in L-DMEM supplemented with 10% fetal bovine serum containing 20 mg/mL penicillin-streptomycin and incubated with 5% CO₂ at 37 ^oC. Following the 3rd passage, BMSCs (5×10⁴ cells/well) were seeded onto 24-well plates 24 h before rAAV infection. By taking into account the cytopathogenic effect, infection efficiency, and cost of recombinant virus, we determined that the best multiplication of infection (MOI) for infecting rabbit BMSCs with rAAV was 5×10⁴ vp/cell. The four rAAV virus variants were introduced into BMSCs using this MOI. Cells were incubated as above and were swirled gently at 30-min intervals. One hour later, the medium was replaced with L-DMEM supplemented with 10% fetal bovine serum. Medium was then completely replaced every three days.

Rabbit hind limb ischemia model and rAAV infection in vivo

Male New Zealand rabbits were kept under specific pathogen free conditions and supplied with sterile food and acidified water. The hind limb ischemia model was developed as described previously¹³. Rabbits were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Under a surgical microscope, a vertical longitudinal incision was made in the right hind limb. The right femoral arteries were separated from the origin of the external iliac artery, ligated, and completely excised. Immediately after ligation of the femoral artery, the four rAAV virus variants were each injected into five different sites¹⁴ on the three major thigh muscles of each rabbit (5.5×10¹¹ vp/20 μL per site), including the adductor (two sites), the quadriceps (two sites), and the semimembranous (one site) muscles. Subsequently, the skin was sutured. After surgery, all animals were housed under standard conditions (temperature: 21±1 °C; humidity: 55%–60%) with food and water continuously available. The hind limbs were mobilized without any fixation. To prevent infection, animals received prophylactic injections of gentamicin (0.03 mg·kg⁻¹·d⁻¹, im) within 3 days after surgery.

Rabbits were sacrificed at various time points post-injection to characterize gene expression efficiency and the effects on angiopoiesis and bone regeneration in vivo. Each group contained 30 rabbits and was divided into four experimental subgroups: group A (n=6) was examined at week 2 for GFP expression (n=3) and immunoblotting (n=3), group B (n=6) at week 4 for GFP expression (n=3) and immunoblotting (n=3), group C (n=9) at week 6 for GFP expression (n=3), immunoblotting (n=3), and ELISA (n=3), and group D (n=9) at week 8 for GFP expression (n=3) and for blood flow measurement, X-ray radiography, and immunohistochemistry (n=6).

Reporter gene (GFP) expression in vitro and in vivo

Following 3, 7, 14, and 28 days of infection with AAV-GFP virus in vitro, the expression of GFP protein was observed by inverted fluorescence microscopy. At 2, 4, 6, and 8 weeks post-injection in vivo, the muscles injected with the AAV-GFP virus were sliced by the frozen section method and the expression of the GFP protein was observed as above. Each assay was performed in triplicate.

Preparation of culture medium and assessment of VEGF₁₆₅ and BMP-7 gene expression

Total cellular RNA was isolated at 1, 2, 3, 7, 14, 21, and 28 days following infection with the AAV-GFP, AAV-VEGF, AAV-BMP or AAV-VEGF/BMP viruses using TRIzol Reagent (Invitrogen). Extracted RNA was treated with DNase I (Takara, Tokyo, Japan) to eliminate DNA contamination, and first-strand cDNA was synthesized with random hexamer primers using the reverse first-strand cDNA synthesis kit from MBI Fermentas (Glen Burnie, MD, USA). PCR was performed to amplify humanVEGF₁₆₅ (forward primer 5′-CCATCGATATGAACTTTCTGCTGTCTTG-3′; reverse primer 5′-CGGAATTCTCACCGCCTCGGCTTGTC-3′) and BMP-7 (forward primer 5′-GGCCGGATCCATGCACGTGCGCTCACTGCG-3′; reverse primer 5′-GGCCGTCGACCTAGTGGCAGCCACAG-3′). β-actin (forward primer 5′-GAGGGAAATCGTGCGTGAC-3′; reverse primer 5′-TAGGAGCCAGGGCAGTAATCT-3′) was detected by RT-PCR as an internal control. PCR was performed using the following program: 94 °C for 3 min for one cycle and 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s. The PCR products were electrophoresed on ethidium bromide-stained 2.0% agarose gels. Each assay was performed in triplicate.

Muscle extract preparation and assessment of VEGF₁₆₅ and BMP-7 gene expression

At 2, 4, and 6 weeks following injection with the AAV-GFP, AAV-VEGF, AAV-BMP, or AAV-VEGF/BMP viruses, the frozen muscles were pulverized in liquid nitrogen and homogenized in 3 mL of ice-cold lysis buffer (1% Nonidet P-40; 50 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 200 U/mL aprotinin; 1 mmol/L phenylmethylsulfonyl fluoride, PMSF). The tissue lysates (50 mg of protein) were separated by 12% polyacrylamide gel electrophoresis and blotted onto polyvinylidene diﬂuoride membranes. Immunoblotting was performed with anti-human VEGF₁₆₅ and BMP-7 antibodies and the speciﬁc binding of the antibody was visualized with an ECL detection system. At 6 weeks post-injection, muscle extracts were measured with an enzyme-linked immunosorbent assay (ELISA) kit using the Biotrak ELISA system (R&D, Minneapolis, MN, USA) according to the manufacturer's instructions. Each assay was performed in triplicate.

Angiogenic and osteogenic in vitro assays

Tube formation assay

HUVECs were cultured as previously described¹⁵. Basement membrane matrigel matrix (BD, Bedford, MA, USA) was diluted by serum-free medium, added to a 24-well plate, and incubated at 37 °C for 30 min to allow solidification to occur. HUVECs (5×10⁴ cells/well) were seeded on the matrigel and fresh L-DMEM medium supplemented with 10% FBS was added. Next, 1 mL of culture supernatant was harvested from the AAV-GFP, AAV-VEGF, AAV-BMP, or AAV-VEGF/BMP groups 14 days post-infection and added to the 24-well plate. The plate was then incubated at 37 ^oC with 5% CO₂ for 12 h. The images of tube formation were captured under a light microscope from three random fields, and quantification of the tubes was analyzed by image processing software (Media Cybernetics, USA) to assess the biological activity of VEGF in vitro.

Mineralization assay

BMSCs were infected with the four virus groups above. The cells were then cultured in L-DMEM supplemented with 10% fetal bovine serum containing 20 mg/mL penicillin-streptomycin with 5% CO₂ at 37 °C (the culture medium did not contain osteogenic induction factors, such as ascorbic acid, β-glycerophosphate, or dexamethasone). Mineralization effects were detected by von Kossa and alizarin red (AZR) staining¹⁶ for calcium deposits 4 weeks post-infection and observed using an inverted phase contrast microscope. The images of mineral nodules were captured under a light microscope from three random fields, and quantification of the mineral nodules was analyzed by image processing software to assess the biological activity of BMP in vitro.

Blood flow measurement and orthotopic bone formation in vivo

Eight weeks after injection, rabbits in the four groups were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Blood flow in the anterior tibial artery of ischemic and normal hind limbs was measured at rest with an Aspen Advanced Doppler ultrasound device from Acuson (Siemens Medical Solutions, Mountain View, CA, USA) using a perivascular flow probe and calculated by the inlay automatic processing software. The data were expressed as a percentage of the contralateral limbs. Three separate measurements were performed for each rabbit at every time point and the results were averaged. In addition, rabbits in the four groups were subjected to X-ray radiography to assess orthotopic bone formation.

Histological assessment

Eight weeks after injection, thigh muscle tissue sections of ischemic limbs from the four groups were harvested and ﬁxed in 10% neutral-buffered formalin. To identify the proliferation of capillary endothelial cells, tissue sections were immunostained for CD34. The monoclonal antibody against CD34 was applied at a 1:500 dilution after blocking with 1% normal bovine serum. Subsequent incubation with biotinylated horse anti-mouse IgG and an ABC Elite kit (Santa Cruz) was performed. The number of CD34-positive vessels was counted at a magnification of 200×, and twenty fields from each typical slide were counted (mean number of capillaries per square millimeter). To assess orthotopic bone formation, the slides were stained by von Kossa staining to detect mineralization.

Statistical analysis

The results are reported as means±standard deviation. The normality of the data distribution was assessed with the Shapiro-Wilk (W) test. ANOVA followed by the Fisher's test was conducted to assess differences among treatment groups. Statistical significance was set at a P-value less than or equal to 0.05. The SPSS mathematical statistics software used for this analysis was purchased from SPSS Inc (version 8; SPSS Inc, Chicago, IL, USA).

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Results

Animal condition after rAAV infection

There were no symptoms of local or systemic toxicity after rAAV infection. In the region of the injection sites, no inflammatory reaction, such as rubeosis, engorgement, or abscessus, was observed. The activities of all animals were normal. There was no systemic toxicity, such as nutation, instability of gait, anhelation, retardation, cyanosis, or convulsion. No animals died before the end of the experiments.

GFP gene expression

In vitro: GFP protein expression could be detected on the third day post-infection. However, the efficiency and density of infection were unstable. The expression of GFP protein increased from the 7th day and could be detected at 28 days post-infection (Figures 2A, 2B). In vivo: With prolonged infection time, GFP protein expression increased from the 4th week and could be detected at 8 weeks post-infection (Figures 2C, 2D).

Figure 2.

Representative images of GFP protein expression. (A–B) on the 3rd, 7th, 14th, and 28th days after rAAV-IRES-GFP virus transfection in vitro. (A) Magnification×100; (B) Magnification×200; (C–D) on the 2nd, 4th, 6th, and 8th weeks after rAAV-IRES-GFP virus injection in vivo. (C) Magnification×100; (D) magnification×200.
Full figure and legend (161K)

Efficient genes expression of hVEGF₁₆₅ and hBMP-7

To confirm hVEGF₁₆₅ and hBMP-7 gene expression in vitro, RT-PCR assays were performed. As shown in Figure 3A–3D, the sizes of the PCR products for VEGF₁₆₅, BMP-7, and β-actin were 600 bp, 1300 bp, and 340 bp, respectively. With prolonged infection time, the intensity of the VEGF₁₆₅ and BMP-7 bands increased in the AAV-VEGF/BMP group. Together, these data demonstrate that VEGF₁₆₅ was expressed in the AAV-VEGF and AAV-VEGF/BMP groups but not in the AAV-BMP and AAV-GFP groups and that BMP-7 was expressed in the AAV-BMP and AAV-VEGF/BMP groups but not in the AAV-VEGF and AAV-GFP groups. Protein expression of 2, 4, and 6 weeks following injection with AAV-VEGF/BMPin vivo is shown in Figure 3E–3H. Expression of the VEGF₁₆₅ and BMP-7 proteins was visualized by Western blot analysis. Strong staining at the expected molecular weights of 23 kDa (hVEGF₁₆₅), 55 kDa (hBMP-7), and 43 kDa (β-actin) was observed. With prolonged infection time, the intensity of the VEGF₁₆₅ and BMP-7 bands increased. These data demonstrate that VEGF₁₆₅ was expressed in the AAV-VEGF and AAV-VEGF/BMP groups but not in the AAV-BMP and AAV-GFP groups and that BMP-7 was expressed in the AAV-BMP and AAV-VEGF/BMP groups but not in the AAV-VEGF and AAV-GFP groups. As shown in Figure 3I, 3J, the production of hVEGF₁₆₅ and hBMP-7 was quantified in relevant muscle extracts 6 weeks post-injection. The average amounts of hVEGF₁₆₅ protein in the AAV-VEGF/BMP and AAV-VEGF groups were significantly higher than those in the AAV-GFP and AAV-BMP groups (P<0.05, n=30). The average amounts of hBMP-7 protein in the AAV-VEGF/BMP and AAV-BMP groups were significantly higher than those in the AAV-GFP and AAV-VEGF groups (P<0.05, n=30).

Figure 3.

Expression of hVEGF₁₆₅ and hBMP-7. (A–D) Representative images of RT-PCR assay of AAV-GFP group (A), AAV-VEGF group (B), AAV-BMP group (C) and AAV-VEGF/BMP group (D). The size of the PCR products for hVEGF₁₆₅, hBMP-7, and β-actin were 600 bp, 1300 bp, and 340 bp, respectively. With prolonged infection time, the brightness of the VEGF₁₆₅ or BMP-7 bands increased in AAV-VEGF/BMP group. No hVEGF₁₆₅ or hBMP-7 band could be detected in AAV-GFP group. Track 1–7 stands for the 1st, 2nd, 3rd, 7th, 14th, 21st, and 28th days post-transfection. (E–H) Representative images of Western blotting assay of AAV-GFP group (E), AAV-VEGF group (F), AAV-BMP group (G) and AAV-VEGF/BMP group (H). The molecular weights of hVEGF₁₆₅, hBMP-7, and β-actin were 23 kDa, 55 kDa and 43 kDa respectively. Strong staining with the expected molecular weight was observed in AAV-VEGF/BMP group, and no hVEGF₁₆₅ or hBMP-7 band was observed in AAV-GFP group. (I) ELISA assay for VEGF protein expression. The data is expressed as the mean±SD from three independent experiments. ^bP<0.05 vs AAV-GFP group, ^eP<0.05 vs AAV-BMP group. (J) ELISA assay for BMP protein expression. The data is expressed as the mean±SD from three independent experiments. ^bP<0.05 vs AAV-GFP group. ^eP<0.05 vs AAV-VEGF group.
Full figure and legend (53K)

Biological activity of hVEGF₁₆₅ and hBMP-7 in vitro

As shown in Figure 4A, hVEGF₁₆₅ secreted from BMSCs in the AAV-VEGF/BMP group enhanced HUVEC migration, proliferation, and tube formation in comparison with the other three groups. The number of tubes in the AAV-VEGF/BMP group was significantly higher than that in the AAV-GFP and AAV-BMP groups. However, there was no statistical difference between the AAV-VEGF/BMP group and the AAV-VEGF group (Figure 4B). In addition, the mineralization effect of hBMP-7 was detected by von Kossa (Figure 5A) and alizarin red staining (Figure 5B). The AAV-VEGF/BMP group displayed stronger osteogenic activity than all the other groups. The number of mineralized nodules in the AAV-VEGF/BMP group was significantly higher than that in the AAV-GFP and AAV-VEGF groups. However, there was no statistical difference between the AAV-VEGF/BMP group and the AAV-BMP group (Figure 5C).

Insurance Income Protection Insurance - Comparison With Critical Illness Insurance Income Protection Insurance - Comparison With Critical Illness Insurance

21september1978@gmail.com (Unknown) — Tue, 01 Mar 2011 10:21:00 +0000

There are several companies out there, which provide insurance policies. A person can approach these companies directly or via internet. It is far better to consult these companies on the websites, since many of the companies possess online customer care services. An individual can read the portfolio of a company on the web, and can decide which one would be better for him.

If you are interested to gain the quotes, you are required to find out a company, which can provide you more benefits. A suitable company would be one, which does not charge high fee for providing insurance cover.

The good thing is that, the insurance is provided on monthly basis. Hence those who have lost the job, or could not find feasible to join the office due to sickness may acquire the compensated amount every month.

Insurance provides reassurance to an individual, he feels secured and safe. He come to the realization that the world will not go to an end, if he would not maintain his position in the company or developed any illness.

Quotes are much better as compared to the critical illness insurance. The can be provided in any type of illness, no matter if it is a minor or a critical one. On the contrary, the critical illness insurance is provided to an individual only at the time of critical illness.

Insurance furnishes the ease in the life of person. He does develop stress if in case; he acquires any type of illness. He does not care, whether the company will provide him wages or not, and he remain able to spend money on the treatment of his disease.

If you want to get income protection insurance, you should try to find out those companies which are the best in their dealing, and which can provide a lot of benefits to the individuals. Income Protection Quotes should be protected at any cost, and it is the right of every individual.

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Stem cell From Wikipedia, the free encyclopedia

21september1978@gmail.com (Unknown) — Sun, 27 Feb 2011 03:07:00 +0000

Stem cells are biological cells found in all multicellular organisms, that can divide through mitosis and differentiate into diverse specialized cell types. In mammals, there are two broad types of stem cells: embryonic stem cells that are isolated from the inner cell mass of blastocysts, and adult stem cells that are found in various tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenished in adult tissues. In a developing embryo, stem cells can differentiate into all the specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues.

Stem cells can now be artificially grown and transformed into specialized cell types with characteristics consistent with cells of various tissues such as muscles or nerves through cell culture. Highly plastic adult stem cells are routinely used in medical therapies. Stem cells can be taken from a variety of sources, including umbilical cord blood and bone marrow. Embryonic cell lines and autologous embryonic stem cells generated through therapeutic cloning have also been proposed as promising candidates for future therapies.[1] Research of stem cells grew out of findings by Ernest A. McCulloch and James E. Till at the University of Toronto in the 1960s.[2][3]

Properties

The classical definition of a stem cell requires that it possess two properties:

* Self-renewal - the ability to go through numerous cycles of cell division while maintaining the undifferentiated state.
* Potency - the capacity to differentiate into specialized cell types. In the strictest sense, this requires stem cells to be either totipotent or pluripotent - to be able to give rise to any mature cell type, although multipotent or unipotent progenitor cells are sometimes referred to as stem cells.

[edit] Self-renewal

Two mechanisms exist to ensure that the stem cell population is maintained:

1. Obligatory asymmetric replication - a stem cell divides into one father cell that is identical to the original stem cell, and another daughter cell that is differentiated
2. Stochastic differentiation - when one stem cell develops into two differentiated daughter cells, another stem cell undergoes mitosis and produces two stem cells identical to the original.

[edit] Potency definitions
Pluripotent, embryonic stem cells originate as inner mass cells within a blastocyst. The stem cells can become any tissue in the body, excluding a placenta. Only the morula's cells are totipotent, able to become all tissues and a placenta.
Human embryonic stem cells
A: Cell colonies that are not yet differentiated.
B: Nerve cell
Main article: Cell potency

Potency specifies the differentiation potential (the potential to differentiate into different cell types) of the stem cell.[4]

* Totipotent (a.k.a omnipotent) stem cells can differentiate into embryonic and extraembryonic cell types. Such cells can construct a complete, viable, organism.[4] These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.[5]
* Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all cells,[4] i.e. cells derived from any of the three germ layers.[6]
* Multipotent stem cells can differentiate into a number of cells, but only those of a closely related family of cells.[4]
* Oligopotent stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells.[4]
* Unipotent cells can produce only one cell type, their own,[4] but have the property of self-renewal which distinguishes them from non-stem cells (e.g. muscle stem cells).

[edit] Identification

The practical definition of a stem cell is the functional definition - a cell that has the potential to regenerate tissue over a lifetime. For example, the gold standard test for a bone marrow or hematopoietic stem cell (HSC) is the ability to transplant one cell and save an individual without HSCs. In this case, a stem cell must be able to produce new blood cells and immune cells over a long term, demonstrating potency. It should also be possible to isolate stem cells from the transplanted individual, which can themselves be transplanted into another individual without HSCs, demonstrating that the stem cell was able to self-renew.

Properties of stem cells can be illustrated in vitro, using methods such as clonogenic assays, where single cells are characterized by their ability to differentiate and self-renew.[7][8] As well, stem cells can be isolated based on a distinctive set of cell surface markers. However, in vitro culture conditions can alter the behavior of cells, making it unclear whether the cells will behave in a similar manner in vivo. Considerable debate exists whether some proposed adult cell populations are truly stem cells.
[edit] Embryonic
Main article: Embryonic stem cell

Embryonic stem cell lines (ES cell lines) are cultures of cells derived from the epiblast tissue of the inner cell mass (ICM) of a blastocyst or earlier morula stage embryos.[9] A blastocyst is an early stage embryo—approximately four to five days old in humans and consisting of 50–150 cells. ES cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta.

Nearly all research to date has taken place using mouse embryonic stem cells (mES) or human embryonic stem cells (hES). Both have the essential stem cell characteristics, yet they require very different environments in order to maintain an undifferentiated state. Mouse ES cells are grown on a layer of gelatin and require the presence of Leukemia Inhibitory Factor (LIF).[10] Human ES cells are grown on a feeder layer of mouse embryonic fibroblasts (MEFs) and require the presence of basic Fibroblast Growth Factor (bFGF or FGF-2).[11] Without optimal culture conditions or genetic manipulation,[12] embryonic stem cells will rapidly differentiate.

A human embryonic stem cell is also defined by the presence of several transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and Sox2 form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency.[13] The cell surface antigens most commonly used to identify hES cells are the glycolipids SSEA3 and SSEA4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81. The molecular definition of a stem cell includes many more proteins and continues to be a topic of research.[14]

After nearly ten years of research,[15] there are no approved treatments using embryonic stem cells. The first human trial was approved by the US Food & Drug Administration in January 2009.[16] However, as of August 2010, the first human trial had not yet been initiated. The first human medical trial for embryonic stem cells started in Atlanta on October 13, 2010 for spinal injury victims. ES cells, being pluripotent cells, require specific signals for correct differentiation - if injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma. Differentiating ES cells into usable cells while avoiding transplant rejection are just a few of the hurdles that embryonic stem cell researchers still face.[17] Many nations currently have moratoria on either ES cell research or the production of new ES cell lines. Because of their combined abilities of unlimited expansion and pluripotency, embryonic stem cells remain a theoretically potential source for regenerative medicine and tissue replacement after injury or disease.
[edit] Fetal

Fetal stem cells are primitive cell types found in the organs of fetuses.[18]
[edit] Adult
Main article: Adult stem cell
Stem cell division and differentiation. A - stem cell; B - progenitor cell; C - differentiated cell; 1 - symmetric stem cell division; 2 - asymmetric stem cell division; 3 - progenitor division; 4 - terminal differentiation

Also known as somatic (from Greek Σωματικóς, "of the body") stem cells and germline (giving rise to gametes) stem cells, they can be found in children, as well as adults.[19]

Pluripotent adult stem cells are rare and generally small in number but can be found in a number of tissues including umbilical cord blood.[20] A great deal of adult stem cell research has focused on clarifying their capacity to divide or self-renew indefinitely and their differentiation potential.[21] In mice, pluripotent stem cells are directly generated from adult fibroblast cultures. Unfortunately, many mice don't live long with stem cell organs.[22]

Most adult stem cells are lineage-restricted (multipotent) and are generally referred to by their tissue origin (mesenchymal stem cell, adipose-derived stem cell, endothelial stem cell, dental pulp stem cell, etc.).[23][24]

Adult stem cell treatments have been successfully used for many years to treat leukemia and related bone/blood cancers through bone marrow transplants.[25] Adult stem cells are also used in veterinary medicine to treat tendon and ligament injuries in horses.[26]

The use of adult stem cells in research and therapy is not as controversial as embryonic stem cells, because the production of adult stem cells does not require the destruction of an embryo. Additionally, because in some instances adult stem cells can be obtained from the intended recipient, (an autograft) the risk of rejection is essentially non-existent in these situations. Consequently, more US government funding is being provided for adult stem cell research.[27]

An extremely rich source for adult mesenchymal stem cells is the developing tooth bud of the mandibular third molar. While considered multipotent they may prove to be pluripotent. The stem cells eventually form enamel (ectodrm), dentin,periodontal ligament, blood vessels, dental pulp, nervous tissues, including a minimum of 29 different unique end organs. Because of extreme ease in collection at 8–10 years of age before calcification and minimal to no morbidity will probably constitute a major source for personal banking, research and multiple therapies. These stem cells have been shown capable of producing hepatocytes.[citation needed]
[edit] Amniotic

Multipotent stem cells are also found in amniotic fluid. These stem cells are very active, expand extensively without feeders and are not tumorigenic. Amniotic stem cells are multipotent and can differentiate in cells of adipogenic, osteogenic, myogenic, endothelial, hepatic and also neuronal lines.[28] All over the world, universities and research institutes are studying amniotic fluid to discover all the qualities of amniotic stem cells, and scientists such as Anthony Atala[29][30] and Giuseppe Simoni [31][32][33] have discovered important results.

From an ethical point of view, stem cells from amniotic fluid can solve a lot of problems, because it's possible to catch amniotic stem cells without destroying embryos. For example, the Vatican newspaper "Osservatore Romano" called amniotic stem cell "the future of medicine".[34]

It's possible to collect amniotic stem cells for donors or for autologuous use: the first US amniotic stem cells bank [35][36] opened in 2009 in Medford, MA, by Biocell Center Corporation [37][38][39] and collaborates with various hospitals and universities all over the world.[40]
[edit] Induced pluripotent
Main article: Induced pluripotent stem cell

These are not adult stem cells, but rather reprogrammed cells (e.g. epithelial cells) given pluripotent capabilities. Using genetic reprogramming with protein transcription factors, pluripotent stem cells equivalent to embryonic stem cells have been derived from human adult skin tissue.[41][42][43] Shinya Yamanaka and his colleagues at Kyoto University used the transcription factors Oct3/4, Sox2, c-Myc, and Klf4[41] in their experiments on cells from human faces. Junying Yu, James Thomson, and their colleagues at the University of Wisconsin–Madison used a different set of factors, Oct4, Sox2, Nanog and Lin28,[41] and carried out their experiments using cells from human foreskin.

As a result of the success of these experiments, Ian Wilmut, who helped create the first cloned animal Dolly the Sheep, has announced that he will abandon nuclear transfer as an avenue of research.[44]

Frozen blood samples can be used as a source of induced pluripotent stem cells, opening a new avenue for obtaining the valued cells.[45]
[edit] Lineage
Main article: Stem cell line

To ensure self-renewal, stem cells undergo two types of cell division (see Stem cell division and differentiation diagram). Symmetric division gives rise to two identical daughter cells both endowed with stem cell properties. Asymmetric division, on the other hand, produces only one stem cell and a progenitor cell with limited self-renewal potential. Progenitors can go through several rounds of cell division before terminally differentiating into a mature cell. It is possible that the molecular distinction between symmetric and asymmetric divisions lies in differential segregation of cell membrane proteins (such as receptors) between the daughter cells.[46]

An alternative theory is that stem cells remain undifferentiated due to environmental cues in their particular niche. Stem cells differentiate when they leave that niche or no longer receive those signals. Studies in Drosophila germarium have identified the signals dpp and adherens junctions that prevent germarium stem cells from differentiating.[47][48]
Main article: Induced Pluripotent Stem Cell

The signals that lead to reprogramming of cells to an embryonic-like state are also being investigated. These signal pathways include several transcription factors including the oncogene c-Myc. Initial studies indicate that transformation of mice cells with a combination of these anti-differentiation signals can reverse differentiation and may allow adult cells to become pluripotent.[49] However, the need to transform these cells with an oncogene may prevent the use of this approach in therapy.

Challenging the terminal nature of cellular differentiation and the integrity of lineage commitment, it was recently determined that the somatic expression of combined transcription factors can directly induce other defined somatic cell fates; researchers identified three neural-lineage-specific transcription factors that could directly convert mouse fibroblasts (skin cells) into fully functional neurons. This "induced neurons" (iN) cell research inspires the researchers to induce other cell types implies that all cells are totipotent: with the proper tools, all cells may form all kinds of tissue.[50]
[edit] Treatments
Main article: Stem cell treatments
Diseases and conditions where stem cell treatment is promising or emerging.[51] Bone marrow transplantation is, as of 2009, the only established use of stem cells.

Medical researchers believe that stem cell therapy has the potential to dramatically change the treatment of human disease. A number of adult stem cell therapies already exist, particularly bone marrow transplants that are used to treat leukemia.[52] In the future, medical researchers anticipate being able to use technologies derived from stem cell research to treat a wider variety of diseases including cancer, Parkinson's disease, spinal cord injuries, Amyotrophic lateral sclerosis, multiple sclerosis, and muscle damage, amongst a number of other impairments and conditions.[53][54] However, there still exists a great deal of social and scientific uncertainty surrounding stem cell research, which could possibly be overcome through public debate and future research, and further education of the public.

One concern of treatment is the possible risk that transplanted stem cells could form tumors and have the possibility of becoming cancerous if cell division continues uncontrollably.[55]

Stem cells, however, are already studied extensively. While some scientists are hesitant to associate the therapeutic potential of stem cells as the first goal of the research, they find the investigation of stem cells as a goal worthy in itself.[56]

Contrarily, supporters of embryonic stem cell research argue that such research should be pursued because the resultant treatments could have significant medical potential. It is also noted that excess embryos created for in vitro fertilization could be donated with consent and used for the research.

The recent development of iPS cells has been called a bypass of the legal controversy. Laws limiting the destruction of human embryos have been credited for being the reason for development of iPS cells, but it is still not completely clear whether hiPS cells are equivalent to hES cells. Recent work demonstrates hotspots of aberrant epigenomic reprogramming in hiPS cells (Lister, R., et al., 2011).
[edit] Research patents

The patents covering a lot of work on human embryonic stem cells are owned by the Wisconsin Alumni Research Foundation (WARF). WARF does not charge academics to study human stem cells but does charge commercial users. WARF sold Geron Corp. exclusive rights to work on human stem cells but later sued Geron Corp. to recover some of the previously sold rights. The two sides agreed that Geron Corp. would keep the rights to only three cell types. In 2001, WARF came under public pressure to widen access to human stem-cell technology.[57]

A request for reviewing the WARF patents 5,843,780; 6,200,806; 7,029,913 US Patent and Trademark Office were filed by non-profit patent-watchdogs The Foundation for Taxpayer & Consumer Rights, and the Public Patent Foundation as well as molecular biologist Jeanne Loring of the Burnham Institute. According to them, two of the patents granted to WARF are invalid because they cover a technique published in 1993 for which a patent had already been granted to an Australian researcher. Another part of the challenge states that these techniques, developed by James A. Thomson, are rendered obvious by a 1990 paper and two textbooks. Based on this challenge, patent 7,029,913 has been rejected in 2010. The two remaining hES WARF patents are due to expire in 2015.

The outcome of this legal challenge is particularly relevant to the Geron Corp. as it can only license patents that are upheld.[58]
[edit] Key research events

* 1908 - The term "stem cell" was proposed for scientific use by the Russian histologist Alexander Maksimov (1874–1928) at congress of hematologic society in Berlin. It postulated existence of haematopoietic stem cells.
* 1960s - Joseph Altman and Gopal Das present scientific evidence of adult neurogenesis, ongoing stem cell activity in the brain; like André Gernez, their reports contradict Cajal's "no new neurons" dogma and are largely ignored.
* 1963 - McCulloch and Till illustrate the presence of self-renewing cells in mouse bone marrow.
* 1968 - Bone marrow transplant between two siblings successfully treats SCID.
* 1978 - Haematopoietic stem cells are discovered in human cord blood.
* 1981 - Mouse embryonic stem cells are derived from the inner cell mass by scientists Martin Evans, Matthew Kaufman, and Gail R. Martin. Gail Martin is attributed for coining the term "Embryonic Stem Cell".
* 1992 - Neural stem cells are cultured in vitro as neurospheres.
* 1997 - Leukemia is shown to originate from a haematopoietic stem cell, the first direct evidence for cancer stem cells.
* 1998 - James Thomson and coworkers derive the first human embryonic stem cell line at the University of Wisconsin–Madison.[59]
* 1998 - John Gearhart (Johns Hopkins University) extracted germ cells from fetal gonadal tissue (primordial germ cells) before developing pluripotent stem cell lines from the original extract.
* 2000s - Several reports of adult stem cell plasticity are published.
* 2001 - Scientists at Advanced Cell Technology clone first early (four- to six-cell stage) human embryos for the purpose of generating embryonic stem cells.[60]
* 2003 - Dr. Songtao Shi of NIH discovers new source of adult stem cells in children's primary teeth.[61]
* 2004–2005 - Korean researcher Hwang Woo-Suk claims to have created several human embryonic stem cell lines from unfertilised human oocytes. The lines were later shown to be fabricated.
* 2005 - Researchers at Kingston University in England claim to have discovered a third category of stem cell, dubbed cord-blood-derived embryonic-like stem cells (CBEs), derived from umbilical cord blood. The group claims these cells are able to differentiate into more types of tissue than adult stem cells.
* 2005 - Researchers at UC Irvine's Reeve-Irvine Research Center are able to partially restore the ability of mice with paralyzed spines to walk through the injection of human neural stem cells.
* August 2006 - Mouse Induced pluripotent stem cells: the journal Cell publishes Kazutoshi Takahashi and Shinya Yamanaka.[62]
* October 2006 - Scientists at Newcastle University in England create the first ever artificial liver cells using umbilical cord blood stem cells.[63][64]
* January 2007 - Scientists at Wake Forest University led by Dr. Anthony Atala and Harvard University report discovery of a new type of stem cell in amniotic fluid.[65] This may potentially provide an alternative to embryonic stem cells for use in research and therapy.[66]
* June 2007 - Research reported by three different groups shows that normal skin cells can be reprogrammed to an embryonic state in mice.[67] In the same month, scientist Shoukhrat Mitalipov reports the first successful creation of a primate stem cell line through somatic cell nuclear transfer[68]
* October 2007 - Mario Capecchi, Martin Evans, and Oliver Smithies win the 2007 Nobel Prize for Physiology or Medicine for their work on embryonic stem cells from mice using gene targeting strategies producing genetically engineered mice (known as knockout mice) for gene research.[69]
* November 2007 - Human induced pluripotent stem cells: Two similar papers released by their respective journals prior to formal publication: in Cell by Kazutoshi Takahashi and Shinya Yamanaka, "Induction of pluripotent stem cells from adult human fibroblasts by defined factors",[70] and in Science by Junying Yu, et al., from the research group of James Thomson, "Induced pluripotent stem cell lines derived from human somatic cells":[71] pluripotent stem cells generated from mature human fibroblasts. It is possible now to produce a stem cell from almost any other human cell instead of using embryos as needed previously, albeit the risk of tumorigenesis due to c-myc and retroviral gene transfer remains to be determined.
* January 2008 - Robert Lanza and colleagues at Advanced Cell Technology and UCSF create the first human embryonic stem cells without destruction of the embryo[72]
* January 2008 - Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts[73]
* February 2008 - Generation of pluripotent stem cells from adult mouse liver and stomach: these iPS cells seem to be more similar to embryonic stem cells than the previously developed iPS cells and not tumorigenic, moreover genes that are required for iPS cells do not need to be inserted into specific sites, which encourages the development of non-viral reprogramming techniques.[74]
* March 2008-The first published study of successful cartilage regeneration in the human knee using autologous adult mesenchymal stem cells is published by clinicians from Regenerative Sciences[75]
* October 2008 - Sabine Conrad and colleagues at Tübingen, Germany generate pluripotent stem cells from spermatogonial cells of adult human testis by culturing the cells in vitro under leukemia inhibitory factor (LIF) supplementation.[76]
* 30 October 2008 - Embryonic-like stem cells from a single human hair.[77]
* 1 March 2009 - Andras Nagy, Keisuke Kaji, et al. discover a way to produce embryonic-like stem cells from normal adult cells by using a novel "wrapping" procedure to deliver specific genes to adult cells to reprogram them into stem cells without the risks of using a virus to make the change.[78][79][80] The use of electroporation is said to allow for the temporary insertion of genes into the cell.[81][82][83][84]
* 28 May 2009 Kim et al. announced that they had devised a way to manipulate skin cells to create patient specific "induced pluripotent stem cells" (iPS), claiming it to be the 'ultimate stem cell solution'.[85]
* 11 October 2010 First trial of embryonic stem cells in humans.[86]
* 25 October 2010 - Ishikawa et al. write in the Journal of Experimental Medicine that research shows that transplanted cells which contain their new host's nuclear DNA could still be rejected by the invidual's immune system due to foreign mitochondrial DNA. Tissues made from a person's stem cells could therefore be rejected, because mitochondrial genomes tend to accumulate mutations.[87]

biotechnology from wikipedia

21september1978@gmail.com (Unknown) — Mon, 31 Jan 2011 06:38:00 +0000

iotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Modern use of similar terms includes genetic engineering as well as cell- and tissue culture technologies. The concept encompasses a wide range of procedures (and history) for modifying living organisms according to human purposes - going back to domestication of animals, cultivation of plants, and "improvements" to these through breeding programs that employ artificial selection and hybridization. By comparison to biotechnology, bioengineering is generally thought of as a related field with its emphasis more on higher systems approaches (not necessarily altering or using biological materials directly) for interfacing with and utilizing living things. The United Nations Convention on Biological Diversity defines biotechnology as:[1]

"Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use."

Biotechnology draws on the pure biological sciences (genetics, microbiology, animal cell culture, molecular biology, biochemistry, embryology, cell biology) and in many instances is also dependent on knowledge and methods from outside the sphere of biology (chemical engineering, bioprocess engineering, information technology, biorobotics). Conversely, modern biological sciences (including even concepts such as molecular ecology) are intimately entwined and dependent on the methods developed through biotechnology and what is commonly thought of as the life sciences industry.

biotechnology information media,news

21september1978@gmail.com (Unknown) — Mon, 31 Jan 2011 06:35:00 +0000

biotechnology information media,news

ISAAA RILIS KAMPANYE “SEJUTA TANGAN PENYEMBUHAN UNTUK MEMBANTU SEMILIAR ORANG KELAPARAN”

Tanggal : 15 October 2010

Pada peringatan Hari Pangan Sedunia, International Service for the Acquisition of Agri-biotech Applications (ISAAA) merilis suatu Kampanye Pengetahuan mengenai bioteknologi yang bertajuk “A million healing hands to help a billion hungry”. Disertai satu dekade berbagi pengetahuan, ISAAA merilis profil tinggi Kampanye Pengetahuan guna memenuhi rasa haus akan pengetahuan dalam bidang teknologi tanaman, pangan dan pertanian baik di negara berkembang maupun industri demi memerangi kemiskinan dan kelaparan – suatu tujuan moral yang mulia. Kampanye tersebut bertujuan untuk mengedukasi, menginformasikan dan memobilisasi masyarakat untuk berpartisipasi dan menjadi ujung tombak pengetahuan, mengeliminasi celah pengetahuan serta membangun sebuah jembatan berkesinambungan antara sains dan masyarakat. Pusat Kampanye Pengetahuan ISAAA adalah “orang-orang” – mobilisasi massa dengan sejuta pasang tangan penyembuhan, menawarkan pengetahuan mengenai bioteknologi tanaman, guna membantu 1 miliar orang yang sedang mendertia kemiskinan dan kelaparan. Dalam konteks ini, tema kampanye Pengetahuan ISAAA adalah “A million healing hands to help a billion hungry".

Sesuai dengan semangat Sasaran Pembangunan Milenium PBB untuk memangkas setengah angka kemiskinan dan kelaparan antara tahun 1990 sampai 2015, ISAAA menegaskan kembali komitmennya bagi bioteknologi tanaman dan pengembangan pertanian sebagai kunci dalam mencapai sasaran ini. Diperkirakan bahwa para petani dan buruh di Asia, Afrika dan Amerika Latin mewakili 70% dari miliaran orang miskin di dunia. Dengan demikian, ISAAA menegaskan kembali keyakinan kuatnya atas peran penting pengetahuan berbasis sains tentang bioteknologi tanaman, sebagai kontribusi penting guna menekan kemiskinan dan kelaparan, yang secara moral tidak dapat diterima dalam suatu masyarakat yang adil.

Kampanye Pengetahuan ISAAA mengenai bioteknologi tanaman tersebut dipersembahkan kepada Dr. Norman Borlaug – peraih Nobel perdamaian di tahun 1970 yang merupakan patron pertama ISAAA. Dengan dukungan dan inisiatif penuhnya, ISAAA mendirikan Global Knowledge Center on Crop Biotechnology di tahun 2000 di Filipina dengan node aktif yang disebut Biotechnology Information Centers (BIC) di 24 negara di dunia. Pada tahun 2010, ISAAA dan keluarga global BICnya merayakan satu dekade keberhasilan dalam memimpin berbagi pengetahuan dan pengembangan kemampuan mengenai bioteknologi tanaman guna membantu mengurangi kemiskinan di negara-negara berkembang.

ISAAA telah melembagakan berbagi pengetahuan mengenai bioteknologi tanaman dengan menciptakan dan mendistribusikan newsletter email mingguan - Crop Biotech Update (CBU) – yang membagi pengetahuan mengenai bioteknologi tanaman secara gratis, penting, menghormati hak-hak orang lain untuk membuat keputusan berbasis sains dan pengetahuan. CBU meringkas perkembangan-perkembangan terkini dunia dalam bidang pertanian, pangan dan bioteknologi tanaman yang relevan bagi negara-negara berkembang. CBU kini disebarkan secara mingguan ke lebih dari 850,000 pelanggan di 200 negara - tujuan Kampanye Pengetahuan ISAAA tentang tanaman bioteknologi adalah untuk meningkatkan jumlah pelanggan menjadi 1 juta pada 31 Desember 2010.

Dengan menegaskan kembali misinya bagi pengetahuan, teknologi dan pengentasan kemiskinan, Kampanye Pengetahuan ISAAA tentang bioteknologi tanaman mengharapkan partisipasi publik hanya dengan mendaftar, tanpa biaya atau kewajiban, 1 sampai 5 alamat email, atau lebih sahabat atau rekannya termasuk siswa, untuk undian yang dilakukan setiap minggunya setelah rilis kampanye tersebut pada peringatan Hari Pangan Sedunia, 16 Oktober 2010. Tiga pemenang akan dianugerahi sebuah duplikat perunggu dari Medali Emas Kongresional Dr. Norman E. Borlaug tiap minggunya dan disoroti dalam CBU mingguan. Undian terakhir akan dilakukan pada Jumat, 31 Desember 2010 dengan hadiah sebuah netbook untuk mengenali peranan penting elektronik dalam komunikasi, yang akan dimenangi oleh pemenang medali Borlaug dari negara-negara berkembang. ISAAA bertujuan untuk menerangi 1 juta atau lebih pelanggan agar menjadi pembawa obor bagi sains dan teknologi mengenai bioteknologi tanaman dan kontribusi pentingnya untuk mengentaskan kemiskinan, yang disimbolkan dalam moto sederhana dan menarik “A million healing hands to help a billion hungry”.

bio technology today

21september1978@gmail.com (Unknown) — Mon, 24 Jan 2011 02:04:00 +0000

bio technology today,bio technology today news

HARGA KAKAO TURUN TERUS, PETANI PUSING

21september1978@gmail.com (Unknown) — Fri, 21 Jan 2011 17:12:00 +0000

MAMUJU, KOMPAS.com - Sebagian besar petani tanaman Kakao di Kabupaten Mamuju, Sulawesi Barat (Sulbar) mengeluh, menyusul harga komoditi unggulan bagi petani di daerah ini sejak beberapa pekan terakhir mulai turun.

Suparman, (42) petani kakao di Mamuju, Minggu (4/7/2010) mengatakan, para petani Kakao di sejumlah kecamatan di Kabupaten Mamuju mulai tidak bersemangat karena terus anjloknya harga coklat di sejumlah pasar di daerah ini.

"Sudah beberapa pekan terakhir ini, harga komoditi Kakao anjlok dari harga normal," katanya.

Harga kakao saat ini hanya sekitar Rp 21.000/kilogram, padahal sebelumnya, harga kakao cukup menguntungkan para petani di daerah ini dengan kisaran sebesar Rp 28.000/kilogram hingga Rp 29.000/kilogram.

"Turunnya harga kakao di pasar tradisional di Mamuju membuat petani Kakao sedikit pusing karena biaya yang dikeluarkan untuk pembelian pupuk cukup tinggi," katanya.

Dia mengatakan, selain harga komoditi itu jatuh di pasaran, tingkat produksi hasil panen juga cenderung berkurang, karena buah saat memasuki musim panen banyak yang rusak akibat terserang hama.

"Jika berjalan normal maka tingkat produksi hasil panen raya untuk komoditi kakao dapat mencapai ratusan ton, namun, kali ini produksinya turun dua kali lipat akibat terserang penyakit atau hama," katanya.

Ia berharap, pemerintah daerah yang mencanangkan program gerakan nasional peningkatan mutu dan produksi kakao (Gernas-pro Kakao) tidak hanya dijadikan simbol, namun, diharapkan program ini dapat diimplementasikan ke petani yang ada di Sulbar.

"Selama ini banyak membantu petani melalui program gernas, namun, program ini belum menyentuh secara merata terhadap petani," katanya.

Mestinya, pihak Dinas Perkebunan (Disbun) yang mewadahi petani dapat melakukan pendataan petani kakao, sehingga bantuan itu tersalurkan secara merata kesemua petani yang ada.

"Jangan ada pengecualian, karena petani sangat mengharapkan bantuan itu untuk memenuhi program peningkatan mutu dan produksi kakao," katanya.

Ia juga berharap pemerintah daerah segera menurunkan petugas penyuluh pertanian untuk mendampingi petani kakao yang mayoritas penduduknya bertumpu di bidang pertanian.

"Sekitar 60 persen dari penduduk Sulbar bertumpu pada sektor pertanian tanaman Kakao, sehingga peran pemerintah itu sangat diharapkan dapat membantu secara optimal kepada petani di Sulbar," katanya.

Konferensi Internasional ke-14 mengenai Riset Komputasional Biologi Molekular

21september1978@gmail.com (Unknown) — Fri, 21 Jan 2011 17:11:00 +0000

Konferensi Internasional ke-14 mengenai Riset Komputasional Biologi Molekular akan diadakan di Lisbon, Portugal dari 12 sampai 15 Agustus 2010. Acara tersebut merupakan seri keempat belas dalam menjembatani bidang komputasi, ilmu matematika dan biologi. Konferensi itu akan menampilkan pembicara utama oleh para ilmuwan yang unggul dalam ilmu kehidupan, bersama-sama dengan presentasi makalah-makalah penelitian di bidang komputasi biologi.

Untuk informasi lebih lanjut, silahkan kunjungi: http://kdbio.inesc-id.pt/recomb2010/home.html.