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Statistical Methods Used to Test for Agreement of Medical Instruments Measuring Continuous Variables in Method Comparison Studies: A Systematic Review

2012-05-25T21:00:00Z

by Rafdzah Zaki, Awang Bulgiba, Roshidi Ismail, Noor Azina Ismail

Background

Accurate values are a must in medicine. An important parameter in determining the quality of a medical instrument is agreement with a gold standard. Various statistical methods have been used to test for agreement. Some of these methods have been shown to be inappropriate. This can result in misleading conclusions about the validity of an instrument. The Bland-Altman method is the most popular method judging by the many citations of the article proposing this method. However, the number of citations does not necessarily mean that this method has been applied in agreement research. No previous study has been conducted to look into this. This is the first systematic review to identify statistical methods used to test for agreement of medical instruments. The proportion of various statistical methods found in this review will also reflect the proportion of medical instruments that have been validated using those particular methods in current clinical practice.

Methodology/Findings

Five electronic databases were searched between 2007 and 2009 to look for agreement studies. A total of 3,260 titles were initially identified. Only 412 titles were potentially related, and finally 210 fitted the inclusion criteria. The Bland-Altman method is the most popular method with 178 (85%) studies having used this method, followed by the correlation coefficient (27%) and means comparison (18%). Some of the inappropriate methods highlighted by Altman and Bland since the 1980s are still in use.

Conclusions

This study finds that the Bland-Altman method is the most popular method used in agreement research. There are still inappropriate applications of statistical methods in some studies. It is important for a clinician or medical researcher to be aware of this issue because misleading conclusions from inappropriate analyses will jeopardize the quality of the evidence, which in turn will influence quality of care given to patients in the future.

Induction of Neuronal Death by Microglial AGE-Albumin: Implications for Alzheimer’s Disease

2012-05-25T21:00:00Z

by Kyunghee Byun, Enkhjaigal Bayarsaikhan, Daesik Kim, Chae Young Kim, Inhee Mook-Jung, Sun Ha Paek, Seung U. Kim, Tadashi Yamamoto, Moo-Ho Won, Byoung-Joon Song, Young Mok Park, Bonghee Lee

Advanced glycation end products (AGEs) have long been considered as potent molecules promoting neuronal cell death and contributing to neurodegenerative disorders such as Alzheimer’s disease (AD). In this study, we demonstrate that AGE-albumin, the most abundant AGE product in human AD brains, is synthesized in activated microglial cells and secreted into the extracellular space. The rate of AGE-albumin synthesis in human microglial cells is markedly increased by amyloid-β exposure and oxidative stress. Exogenous AGE-albumin upregulates the receptor protein for AGE (RAGE) and augments calcium influx, leading to apoptosis of human primary neurons. In animal experiments, soluble RAGE (sRAGE), pyridoxamine or ALT-711 prevented Aβ-induced neuronal death in rat brains. Collectively, these results provide evidence for a new mechanism by which microglial cells promote death of neuronal cells through synthesis and secretion of AGE-albumin, thereby likely contributing to neurodegenerative diseases such as AD.

H19 Antisense RNA Can Up-Regulate Igf2 Transcription by Activation of a Novel Promoter in Mouse Myoblasts

2012-05-25T21:00:00Z

by Van Giang Tran, Franck Court, Anne Duputié, Etienne Antoine, Nathalie Aptel, Laura Milligan, Françoise Carbonell, Marie-Noëlle Lelay-Taha, Jacques Piette, Michaël Weber, Didier Montarras, Christian Pinset, Luisa Dandolo, Thierry Forné, Guy Cathala

It was recently shown that a long non-coding RNA (lncRNA), that we named the 91H RNA (i.e. antisense H19 transcript), is overexpressed in human breast tumours and contributes in trans to the expression of the Insulin-like Growth Factor 2 (IGF2) gene on the paternal chromosome. Our preliminary experiments suggested that an H19 antisense transcript having a similar function may also be conserved in the mouse. In the present work, we further characterise the mouse 91H RNA and, using a genetic complementation approach in H19 KO myoblast cells, we show that ectopic expression of the mouse 91H RNA can up-regulate Igf2 expression in trans despite almost complete unmethylation of the Imprinting-Control Region (ICR). We then demonstrate that this activation occurs at the transcriptional level by activation of a previously unknown Igf2 promoter which displays, in mouse tissues, a preferential mesodermic expression (Pm promoter). Finally, our experiments indicate that a large excess of the H19 transcript can counteract 91H-mediated Igf2 activation. Our work contributes, in conjunction with other recent findings, to open new horizons to our understanding of Igf2 gene regulation and functions of the 91H/H19 RNAs in normal and pathological conditions.

Exposures to Conditioned Flavours with Different Hedonic Values Induce Contrasted Behavioural and Brain Responses in Pigs

2012-05-25T21:00:00Z

by Caroline Clouard, Mélanie Jouhanneau, Marie-Christine Meunier-Salaün, Charles-Henri Malbert, David Val-Laillet

This study investigated the behavioural and brain responses towards conditioned flavours with different hedonic values in juvenile pigs. Twelve 30-kg pigs were given four three-day conditioning sessions: they received three different flavoured meals paired with intraduodenal (i.d.) infusions of 15% glucose (F_Glu), lithium chloride (F_LiCl), or saline (control treatment, F_NaCl). One and five weeks later, the animals were subjected to three two-choice feeding tests without reinforcement to check the acquisition of a conditioned flavour preference or aversion. In between, the anaesthetised pigs were subjected to three ¹⁸FDG PET brain imaging coupled with an olfactogustatory stimulation with the conditioned flavours. During conditioning, the pigs spent more time lying inactive, and investigated their environment less after the F_LiCl than the F_NaCl or F_Glu meals. During the two-choice tests performed one and five weeks later, the F_NaCl and F_Glu foods were significantly preferred over the F_LICl food even in the absence of i.d. infusions. Surprisingly, the F_NaCl food was also preferred over the F_Glu food during the first test only, suggesting that, while LiCl i.d. infusions led to a strong flavour aversion, glucose infusions failed to induce flavour preference. As for brain imaging results, exposure to aversive or less preferred flavours triggered global deactivation of the prefrontal cortex, specific activation of the posterior cingulate cortex, as well as asymmetric brain responses in the basal nuclei and the temporal gyrus. In conclusion, postingestive visceral stimuli can modulate the flavour/food hedonism and further feeding choices. Exposure to flavours with different hedonic values induced metabolism differences in neural circuits known to be involved in humans in the characterization of food palatability, feeding motivation, reward expectation, and more generally in the regulation of food intake.

A Policy-into-Practice Intervention to Increase the Uptake of Evidence-Based Management of Low Back Pain in Primary Care: A Prospective Cohort Study

2012-05-25T21:00:00Z

by Helen Slater, Stephanie Joy Davies, Richard Parsons, John Louis Quintner, Stephan Alexander Schug

Background

Persistent non-specific low back pain (nsLBP) is poorly understood by the general community, by educators, researchers and health professionals, making effective care problematic. This study evaluated the effectiveness of a policy-into-practice intervention developed for primary care physicians (PCPs).

Methods

To encourage PCPs to adopt practical evidence-based approaches and facilitate time-efficient, integrated management of patients with nsLBP, we developed an interdisciplinary evidence-based, practical pain education program (gPEP) based on a contemporary biopsychosocial framework. One hundred and twenty six PCPs from primary care settings in Western Australia were recruited. PCPs participated in a 6.5-hour gPEP. Self-report measures recorded at baseline and at 2 months post-intervention included PCPs' attitudes, beliefs (modified Health Care Providers Pain and Impairment Relationship Scale (HC-PAIRS), evidence-based clinical practices (knowledge and skills regarding nsLBP management: 5-point Likert scale with 1 = nil and 5 = excellent) and practice behaviours (recommendations based on a patient vignette; 5-point Likert scale).

Results

Ninety one PCPs participated (attendance rate of 72%; post-intervention response rate 88%). PCP-responders adopted more positive, guideline-consistent beliefs, evidenced by clinically significant HC-PAIRS score differences (mean change = −5.6±8.2, p<0.0001; 95% confidence interval: −7.6 to −3.6) and significant positive shifts on all measures of clinical knowledge and skills (p<0.0001 for all questions). Self management strategies were recommended more frequently post-intervention. The majority of responders who were guideline-inconsistent for work and bed rest recommendations (82% and 62% respectively) at pre-intervention, gave guideline-consistent responses at post-intervention.

Conclusion

An interprofessional pain education program set within a framework that aligns health policy and practice, encourages PCPs to adopt more self-reported evidence-based attitudes, beliefs and clinical behaviours in their management of patients with nsLBP. However, further research is required to determine cost effectiveness of this approach when compared with other modes of educational delivery and to examine PCP behaviours in actual clinical practice.

Exome Sequencing and Genetic Testing for MODY

2012-05-25T21:00:00Z

by Stefan Johansson, Henrik Irgens, Kishan K. Chudasama, Janne Molnes, Jan Aerts, Francisco S. Roque, Inge Jonassen, Shawn Levy, Kari Lima, Per M. Knappskog, Graeme I. Bell, Anders Molven, Pål R. Njølstad

Context

Genetic testing for monogenic diabetes is important for patient care. Given the extensive genetic and clinical heterogeneity of diabetes, exome sequencing might provide additional diagnostic potential when standard Sanger sequencing-based diagnostics is inconclusive.

Objective

The aim of the study was to examine the performance of exome sequencing for a molecular diagnosis of MODY in patients who have undergone conventional diagnostic sequencing of candidate genes with negative results.

Research Design and Methods

We performed exome enrichment followed by high-throughput sequencing in nine patients with suspected MODY. They were Sanger sequencing-negative for mutations in the HNF1A, HNF4A, GCK, HNF1B and INS genes. We excluded common, non-coding and synonymous gene variants, and performed in-depth analysis on filtered sequence variants in a pre-defined set of 111 genes implicated in glucose metabolism.

Results

On average, we obtained 45 X median coverage of the entire targeted exome and found 199 rare coding variants per individual. We identified 0–4 rare non-synonymous and nonsense variants per individual in our a priori list of 111 candidate genes. Three of the variants were considered pathogenic (in ABCC8, HNF4A and PPARG, respectively), thus exome sequencing led to a genetic diagnosis in at least three of the nine patients. Approximately 91% of known heterozygous SNPs in the target exomes were detected, but we also found low coverage in some key diabetes genes using our current exome sequencing approach. Novel variants in the genes ARAP1, GLIS3, MADD, NOTCH2 and WFS1 need further investigation to reveal their possible role in diabetes.

Conclusion

Our results demonstrate that exome sequencing can improve molecular diagnostics of MODY when used as a complement to Sanger sequencing. However, improvements will be needed, especially concerning coverage, before the full potential of exome sequencing can be realized.

Cdc42-Dependent Activation of NADPH Oxidase Is Involved in Ethanol-Induced Neuronal Oxidative Stress

2012-05-25T21:00:00Z

by Xin Wang, Zunji Ke, Gang Chen, Mei Xu, Kimberly A. Bower, Jacqueline A. Frank, Zhuo Zhang, Xianglin Shi, Jia Luo

It has been suggested that excessive reactive oxygen species (ROS) and oxidative stress play an important role in ethanol-induced damage to both the developing and mature central nervous system (CNS). The mechanisms underlying ethanol-induced neuronal ROS, however, remain unclear. In this study, we investigated the role of NADPH oxidase (NOX) in ethanol-induced ROS generation. We demonstrated that ethanol activated NOX and inhibition of NOX reduced ethanol-promoted ROS generation. Ethanol significantly increased the expression of p47^phox and p67^phox, the essential subunits for NOX activation in cultured neuronal cells and the cerebral cortex of infant mice. Ethanol caused serine phosphorylation and membrane translocation of p47^phox and p67^phox, which were prerequisites for NOX assembly and activation. Knocking down p47^phox with the small interfering RNA was sufficient to attenuate ethanol-induced ROS production and ameliorate ethanol-mediated oxidative damage, which is indicated by a decrease in protein oxidation and lipid peroxidation. Ethanol activated cell division cycle 42 (Cdc42) and overexpression of a dominant negative (DN) Cdc42 abrogate ethanol-induced NOX activation and ROS generation. These results suggest that Cdc42-dependent NOX activation mediates ethanol-induced oxidative damages to neurons.

p53 Modulation as a Therapeutic Strategy in Gastrointestinal Stromal Tumors

2012-05-25T21:00:00Z

by Joern Henze, Thomas Mühlenberg, Susanne Simon, Florian Grabellus, Brian Rubin, Georg Taeger, Martin Schuler, Juergen Treckmann, Maria Debiec-Rychter, Takahiro Taguchi, Jonathan A. Fletcher, Sebastian Bauer

The KIT-inhibitor imatinib mesylate (IM) has greatly improved the treatment of metastatic gastrointestinal stromal tumors (GIST). IM exhibits strong antiproliferative effects but fails to induce sufficient levels of apoptosis resulting in low pathologic complete remission rates and a high rate of secondary progression in the metastatic setting. Upregulation of p53 by MDM2 inhibitors has been shown to induce apoptosis in p53 wildtype tumors. Analyzing a series of 62 mostly untreated, localized and metastatic GIST we detected a low rate (3%) of inactivating p53 mutations, thus providing a rationale for further exploration of p53-directed therapeutic strategies. To this end, we studied nutlin-3, an inhibitor of the p53 antagonist MDM2, and RITA, a putative p53 activator, in GIST cell lines. Nutlin-3 effectively induced p53 at therapeutically relevant levels, which resulted in moderate antiproliferative effects and cell cycle arrest in p53 wildtype GIST cell lines GIST430, GIST48 and GIST48B. P53 reactivation substantially improved the apoptotic response after effective KIT inhibition with sunitinib and 17-AAG in IM-resistant cell lines. The commonly used imatinib-sensitive cell lines GIST882 and GIST-T1 were shown to harbor defective p53 and therefore failed to respond to nutlin-3 treatment. RITA induced p53 in GIST48B, followed by antiproliferative effects and a strong induction of apoptosis. Surprisingly, GIST-T1 was also highly sensitive to RITA despite lacking functional p53. This suggested a more complex, p53-independent mechanism of action for the latter compound. No antagonistic effects from p53-activating drugs were seen with any drug combination. Our data provide first evidence that modulation of the MDM2/p53 pathway may be therapeutically useful to improve the apoptotic response of KIT-inhibitory drugs in the treatment of naïve GIST, with p53 mutation status being a predictive factor of response.

Hyaluronan Binding Motifs of USP17 and SDS3 Exhibit Anti-Tumor Activity

2012-05-25T21:00:00Z

by Suresh Ramakrishna, Bharathi Suresh, Su-Mi Bae, Woong-Shick Ahn, Key-Hwan Lim, Kwang-Hyun Baek

Background

We previously reported that the USP17 deubiquitinating enzyme having hyaluronan binding motifs (HABMs) interacts with human SDS3 (suppressor of defective silencing 3) and specifically deubiquitinates Lys-63 branched polyubiquitination of SDS3 resulting in negative regulation of histone deacetylase (HDAC) activity in cancer cells. Furthermore, USP17 and SDS3 mutually interact with each other to block cell proliferation in HeLa cells but the mechanism for this inhibition in cell proliferation is not known. We wished to investigate whether the HABMs of USP17 were responsible for tumor suppression activity.

Methodology/Principal Findings

Similarly to USP17, we have identified that SDS3 also has three consecutive HABMs and shows direct binding with hyaluronan (HA) using cetylpyridinium chloride (CPC) assay. Additionally, HA oligosaccharides (6-18 sugar units) competitively block binding of endogenous HA polymer to HA binding proteins. Thus, administration of HA oligosaccharides antagonizes the interaction between HA and USP17 or SDS3. Interestingly, HABMs deleted USP17 showed lesser interaction with SDS3 but retain its deubiquitinating activity towards SDS3. The deletion of HABMs of USP17 could not alter its functional regulation on SDS3-associated HDAC activity. Furthermore, to explore whether HABMs in USP17 and SDS3 are responsible for the inhibition of cell proliferation, we investigated the effect of USP17 and SDS3-lacking HABMs on cell proliferation by soft agar, apoptosis, cell migration and cell proliferation assays.

Conclusions

Our results have demonstrated that these HABMs in USP17 and its substrate SDS3 are mainly involved in the inhibition of anchorage-independent tumor growth.

Diurnal Rhythms in Neurexins Transcripts and Inhibitory/Excitatory Synapse Scaffold Proteins in the Biological Clock

2012-05-25T21:00:00Z

by Mika Shapiro-Reznik, Anje Jilg, Hadas Lerner, David J. Earnest, Nava Zisapel

The neurexin genes (NRXN1/2/3) encode two families (α and β) of highly polymorphic presynaptic proteins that are involved in excitatory/inhibitory synaptic balance. Recent studies indicate that neuronal activation and memory formation affect NRXN1/2/3α expression and alternative splicing at splice sites 3 and 4 (SS#3/SS#4). Neurons in the biological clock residing in the suprachiasmatic nuclei of the hypothalamus (SCN) act as self-sustained oscillators, generating rhythms in gene expression and electrical activity, to entrain circadian bodily rhythms to the 24 hours day/night cycles. Cell autonomous oscillations in NRXN1/2/3α expression and SS#3/SS#4 exons splicing and their links to rhythms in excitatory/inhibitory synaptic balance in the circadian clock were explored. NRXN1/2/3α expression and SS#3/SS#4 splicing, levels of neurexin-2α and the synaptic scaffolding proteins PSD-95 and gephyrin (representing excitatory and inhibitory synapses, respectively) were studied in mRNA and protein extracts obtained from SCN of C3H/J mice at different times of the 24 hours day/night cycle. Further studies explored the circadian oscillations in these components and causality relationships in immortalized rat SCN2.2 cells. Diurnal rhythms in mNRXN1α and mNRXN2α transcription, SS#3/SS#4 exon-inclusion and PSD-95 gephyrin and neurexin-2α levels were found in the SCN in vivo. No such rhythms were found with mNRXN3α. SCN2.2 cells also exhibited autonomous circadian rhythms in rNRXN1/2 expression SS#3/SS#4 exon inclusion and PSD-95, gephyrin and neurexin-2α levels. rNRXN3α and rNRXN1/2β were not expressed. Causal relationships were demonstrated, by use of specific siRNAs, between rNRXN2α SS#3 exon included transcripts and gephyrin levels in the SCN2.2 cells. These results show for the first time dynamic, cell autonomous, diurnal rhythms in expression and splicing of NRXN1/2 and subsequent effects on the expression of neurexin-2α and postsynaptic scaffolding proteins in SCN across the 24-h cycle. NRXNs gene transcripts may have a role in coupling the circadian clock to diurnal rhythms in excitatory/inhibitory synaptic balance.

Chronic Exposure of Corals to Fine Sediments: Lethal and Sub-Lethal Impacts

2012-05-25T21:00:00Z

by Florita Flores, Mia O. Hoogenboom, Luke D. Smith, Timothy F. Cooper, David Abrego, Andrew P. Negri

Understanding the sedimentation and turbidity thresholds for corals is critical in assessing the potential impacts of dredging projects in tropical marine systems. In this study, we exposed two species of coral sampled from offshore locations to six levels of total suspended solids (TSS) for 16 weeks in the laboratory, including a 4 week recovery period. Dose-response relationships were developed to quantify the lethal and sub-lethal thresholds of sedimentation and turbidity for the corals. The sediment treatments affected the horizontal foliaceous species (Montipora aequituberculata) more than the upright branching species (Acropora millepora). The lowest sediment treatments that caused full colony mortality were 30 mg l⁻¹ TSS (25 mg cm⁻² day⁻¹) for M. aequituberculata and 100 mg l⁻¹ TSS (83 mg cm⁻² day⁻¹) for A. millepora after 12 weeks. Coral mortality generally took longer than 4 weeks and was closely related to sediment accumulation on the surface of the corals. While measurements of damage to photosystem II in the symbionts and reductions in lipid content and growth indicated sub-lethal responses in surviving corals, the most reliable predictor of coral mortality in this experiment was long-term sediment accumulation on coral tissue.

Selection of Aptamers Specific for Adipose Tissue

2012-05-25T21:00:00Z

by Jun Liu, Huixia Liu, Kwame Sefah, Bo Liu, Ying Pu, Dimitri Van Simaeys, Weihong Tan

Background

Obesity has reached epidemic proportions, affecting more than one tenth of the world’s population. As such, adipose tissue is being increasingly recognized as an important therapeutic target for obesity and related metabolic disorders. While many potential targets of adipose tissue have been established and drugs developed, very few of those drugs specifically target adipose tissue without affecting other tissue. This results from a limited knowledge of both cell-surface markers and physicochemical traits specific to adipocytes that might otherwise be exploited by circulating drugs.

Methodology/Principal Findings

Here we report the use of cell-SELEX technology to select two aptamers that can specifically recognize mature adipocytes: adipo-1 and adipo-8. Adipo-8 shows high affinity for differentiated, mature 3T3-L1 adipocytes with a K_d value of 17.8±5.1 nM. The binding was sustained upon incubation at 37°C and insulin stimulation, but was lost upon trypsin treatment. The binding ability was also verified on frozen tissue slides with low background fluorescence and isolated adipocytes.

Conclusions/Significance

Aptamer adipo-8 selected from a random library appears to bind to mature differentiated adipocytes specifically. This aptamer holds great promise as a molecular recognition tool for adipocyte biomarker discovery or for targeted delivery of molecules to adipocytes.

Regulation of Adipocyte 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) by CCAAT/Enhancer-Binding Protein (C/EBP) β Isoforms, LIP and LAP

2012-05-25T21:00:00Z

by Cristina L. Esteves, Val Kelly, Valérie Bégay, Tak Y. Man, Nicholas M. Morton, Achim Leutz, Jonathan R. Seckl, Karen E. Chapman

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyses intracellular regeneration of active glucocorticoids, notably in liver and adipose tissue. 11β-HSD1 is increased selectively in adipose tissue in human obesity, a change implicated in the pathogenesis of metabolic syndrome. With high fat (HF)-feeding, adipose tissue 11β-HSD1 is down-regulated in mice, plausibly to counteract metabolic disease. Transcription of 11β-HSD1 is directly regulated by members of the CCAAT/enhancer binding protein (C/EBP) family. Here we show that while total C/EBPβ in adipose tissue is unaltered by HF diet, the ratio of the C/EBPβ isoforms liver-enriched inhibitor protein (LIP) and liver-enriched activator protein (LAP) (C/EBPβ-LIP:LAP) is increased in subcutaneous adipose. This may cause changes in 11β-HSD1 expression since genetically modified C/EBPβ^(+/L) mice, with increased C/EBPβ-LIP:LAP ratio, have decreased subcutaneous adipose 11β-HSD1 mRNA levels, whereas C/EBPβ^ΔuORF mice, with decreased C/EBPβ-LIP:LAP ratio, show increased subcutaneous adipose 11β-HSD1. C/EBPβ-LIP:LAP ratio is regulated by endoplasmic reticulum (ER) stress and mTOR signalling, both of which are altered in obesity. In 3T3-L1 adipocytes, 11β-HSD1 mRNA levels were down-regulated following induction of ER stress by tunicamycin but were up-regulated following inhibition of mTOR by rapamycin. These data point to a central role for C/EBPβ and its processing to LIP and LAP in transcriptional regulation of 11β-HSD1 in adipose tissue. Down-regulation of 11β-HSD1 by increased C/EBPβ-LIP:LAP in adipocytes may be part of a nutrient-sensing mechanism counteracting nutritional stress generated by HF diet.

Gas7-Deficient Mouse Reveals Roles in Motor Function and Muscle Fiber Composition during Aging

2012-05-25T21:00:00Z

by Bo-Tsang Huang, Pu-Yuan Chang, Ching-Hua Su, Chuck C.-K. Chao, Sue Lin-Chao

Background

Growth arrest-specific gene 7 (Gas7) has previously been shown to be involved in neurite outgrowth in vitro; however, its actual role has yet to be determined. To investigate the physiological function of Gas7 in vivo, here we generated a Gas7-deficient mouse strain with a labile Gas7 mutant protein whose functions are similar to wild-type Gas7.

Methodology/Principal Findings

Our data show that aged Gas7-deficient mice have motor activity defects due to decreases in the number of spinal motor neurons and in muscle strength, of which the latter may be caused by changes in muscle fiber composition as shown in the soleus. In cross sections of the soleus of Gas7-deficient mice, gross morphological features and levels of myosin heavy chain I (MHC I) and MHC II markers revealed significantly fewer fast fibers. In addition, we found that nerve terminal sprouting, which may be associated with slow and fast muscle fiber composition, was considerably reduced at neuromuscular junctions (NMJ) during aging.

Conclusions/Significance

These findings indicate that Gas7 is involved in motor neuron function associated with muscle strength maintenance.

Expression of Concern: Immune Protection Induced on Day 10 Following Administration of the 2009 A/H1N1 Pandemic Influenza Vaccine

The PLoS ONE Editors — 2012-05-25T21:00:00Z

by The PLoS ONE Editors

Identification of Gene Modules Associated with Drought Response in Rice by Network-Based Analysis

2012-05-25T21:00:00Z

by Lida Zhang, Shunwu Yu, Kaijing Zuo, Lijun Luo, Kexuan Tang

Understanding the molecular mechanisms that underlie plant responses to drought stress is challenging due to the complex interplay of numerous different genes. Here, we used network-based gene clustering to uncover the relationships between drought-responsive genes from large microarray datasets. We identified 2,607 rice genes that showed significant changes in gene expression under drought stress; 1,392 genes were highly intercorrelated to form 15 gene modules. These drought-responsive gene modules are biologically plausible, with enrichments for genes in common functional categories, stress response changes, tissue-specific expression and transcription factor binding sites. We observed that a gene module (referred to as module 4) consisting of 134 genes was significantly associated with drought response in both drought-tolerant and drought-sensitive rice varieties. This module is enriched for genes involved in controlling the response of the plant to water and embryonic development, including a heat shock transcription factor as the key regulator in the expression of ABRE-containing genes. These results suggest that module 4 is highly conserved in the ABA-mediated drought response pathway in different rice varieties. Moreover, our study showed that many hub genes clustered in rice chromosomes had significant associations with QTLs for drought stress tolerance. The relationship between hub gene clusters and drought tolerance QTLs may provide a key to understand the genetic basis of drought tolerance in rice.

Autoantibody Epitope Spreading in the Pre-Clinical Phase Predicts Progression to Rheumatoid Arthritis

2012-05-25T21:00:00Z

by Jeremy Sokolove, Reuven Bromberg, Kevin D. Deane, Lauren J. Lahey, Lezlie A. Derber, Piyanka E. Chandra, Jess D. Edison, William R. Gilliland, Robert J. Tibshirani, Jill M. Norris, V. Michael Holers, William H. Robinson

Rheumatoid arthritis (RA) is a prototypical autoimmune arthritis affecting nearly 1% of the world population and is a significant cause of worldwide disability. Though prior studies have demonstrated the appearance of RA-related autoantibodies years before the onset of clinical RA, the pattern of immunologic events preceding the development of RA remains unclear. To characterize the evolution of the autoantibody response in the preclinical phase of RA, we used a novel multiplex autoantigen array to evaluate development of the anti-citrullinated protein antibodies (ACPA) and to determine if epitope spread correlates with rise in serum cytokines and imminent onset of clinical RA. To do so, we utilized a cohort of 81 patients with clinical RA for whom stored serum was available from 1–12 years prior to disease onset. We evaluated the accumulation of ACPA subtypes over time and correlated this accumulation with elevations in serum cytokines. We then used logistic regression to identify a profile of biomarkers which predicts the imminent onset of clinical RA (defined as within 2 years of testing). We observed a time-dependent expansion of ACPA specificity with the number of ACPA subtypes. At the earliest timepoints, we found autoantibodies targeting several innate immune ligands including citrullinated histones, fibrinogen, and biglycan, thus providing insights into the earliest autoantigen targets and potential mechanisms underlying the onset and development of autoimmunity in RA. Additionally, expansion of the ACPA response strongly predicted elevations in many inflammatory cytokines including TNF-α, IL-6, IL-12p70, and IFN-γ. Thus, we observe that the preclinical phase of RA is characterized by an accumulation of multiple autoantibody specificities reflecting the process of epitope spread. Epitope expansion is closely correlated with the appearance of preclinical inflammation, and we identify a biomarker profile including autoantibodies and cytokines which predicts the imminent onset of clinical arthritis.

Fluctuation-Driven Flocking Movement in Three Dimensions and Scale-Free Correlation

2012-05-25T21:00:00Z

by Takayuki Niizato, Yukio-Pegio Gunji

Recent advances in the study of flocking behavior have permitted more sophisticated analyses than previously possible. The concepts of “topological distances” and “scale-free correlations” are important developments that have contributed to this improvement. These concepts require us to reconsider the notion of a neighborhood when applied to theoretical models. Previous work has assumed that individuals interact with neighbors within a certain radius (called the “metric distance”). However, other work has shown that, assuming topological interactions, starlings interact on average with the six or seven nearest neighbors within a flock. Accounting for this observation, we previously proposed a metric-topological interaction model in two dimensions. The goal of our model was to unite these two interaction components, the metric distance and the topological distance, into one rule. In our previous study, we demonstrated that the metric-topological interaction model could explain a real bird flocking phenomenon called scale-free correlation, which was first reported by Cavagna et al. In this study, we extended our model to three dimensions while also accounting for variations in speed. This three-dimensional metric-topological interaction model displayed scale-free correlation for velocity and orientation. Finally, we introduced an additional new feature of the model, namely, that a flock can store and release its fluctuations.

Central Role of SREBP-2 in the Pathogenesis of Osteoarthritis

2012-05-25T21:00:00Z

by Fotini Kostopoulou, Vasiliki Gkretsi, Konstantinos N. Malizos, Dimitrios Iliopoulos, Pagona Oikonomou, Lazaros Poultsides, Aspasia Tsezou

Background

Recent studies have implied that osteoarthritis (OA) is a metabolic disease linked to deregulation of genes involved in lipid metabolism and cholesterol efflux. Sterol Regulatory Element Binding Proteins (SREBPs) are transcription factors regulating lipid metabolism with so far no association with OA. Our aim was to test the hypothesis that SREBP-2, a gene that plays a key role in cholesterol homeostasis, is crucially involved in OA pathogenesis and to identify possible mechanisms of action.

Methodology/Principal Findings

We performed a genetic association analysis using a cohort of 1,410 Greek OA patients and healthy controls and found significant association between single nucleotide polymorphism (SNP) 1784G>C in SREBP-2 gene and OA development. Moreover, the above SNP was functionally active, as normal chondrocytes’ transfection with SREBP-2-G/C plasmid resulted in interleukin-1β and metalloproteinase-13 (MMP-13) upregulation. We also evaluated SREBP-2, its target gene 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR), phospho-phosphoinositide3-kinase (PI3K), phospho-Akt, integrin-alphaV (ITGAV) and transforming growth factor-β (TGF-β) mRNA and protein expression levels in osteoarthritic and normal chondrocytes and found that they were all significantly elevated in OA chondrocytes. To test whether TGF-β alone can induce SREBP-2, we treated normal chondrocytes with TGF-β and found significant upregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13. We also showed that TGF-β activated aggrecan (ACAN) in chondrocytes only through Smad3, which interacts with SREBP-2. Finally, we examined the effect of an integrin inhibitor, cyclo-RGDFV peptide, on osteoarthritic chondrocytes, and found that it resulted in significant upregulation of ACAN and downregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13 expression levels.

Conclusions/Significance

We demonstrated, for the first time, the association of SREBP-2 with OA pathogenesis and provided evidence on the molecular mechanism involved. We suggest that TGF-β induces SREBP-2 pathway activation through ITGAV and PI3K playing a key role in OA and that integrin blockage may be a potential molecular target for OA treatment.

Polymorphisms in Toll-Like Receptors 2, 4, and 9 Are Highly Associated with Hearing Loss in Survivors of Bacterial Meningitis

2012-05-25T21:00:00Z

by Gijs Th J. van Well, Marieke S. Sanders, Sander Ouburg, A. Marceline van Furth, Servaas A. Morré

Genetic variation in innate immune response genes contributes to inter-individual differences in disease manifestation and degree of complications upon infection. We recently described an association of single nucleotide polymorphisms (SNPs) in TLR9 with susceptibility to meningococcal meningitis (MM). In this study, we investigate the association of SNPs in multiple pathogen recognition and immune response genes with clinical features that determine severity and outcome (especially hearing loss) of childhood MM and pneumococcal meningitis (PM). Eleven SNPs in seven genes (TLR2, TLR4, TLR9, NOD1, NOD2, CASP1, and TRAIL) were genotyped in 393 survivors of childhood bacterial meningitis (BM) (327 MM patients and 66 PM patients). Genotype distributions of single SNPs and combination of SNPs were compared between thirteen clinical characteristics associated with severity of BM. After correction for multiple testing, TLR4+896 mutant alleles were highly associated with post-meningitis hearing loss, especially MM (p = 0.001, OR 4.0 for BM, p = 0.0004, OR 6.2 for MM). In a multigene analysis, combined carriership of the TLR2+2477 wild type (WT) with TLR4+896 mutant alleles increases the risk of hearing loss (p<0.0001, OR 5.7 in BM and p = 0.0001, OR 7.6 in MM). Carriage of one or both mutant alleles in TLR4+896 and TLR9 -1237 increases the risk for hearing loss (p = 0.0006, OR 4.1 in BM). SNPs in immune response genes contribute to differences in clinical severity and outcome of BM. The TLR system seems to play an important role in the immune response to BM and subsequent neuronal damage as well as in cochlear inflammation. Genetic markers may be used for identification of high-risk patients by creating prediction rules for post-meningitis hearing loss and other sequelae, and provide more insight in the complex immune response in the CNS possibly resulting in new therapeutic interventions.

Evaluation of a Screening Instrument for Autism Spectrum Disorders in Prisoners

2012-05-25T21:00:00Z

by Louise Robinson, Michael D. Spencer, Lindsay D. G. Thomson, Andrew C. Stanfield, David G. C. Owens, Jeremy Hall, Eve C. Johnstone

Aims

We aimed to evaluate this tool in Scottish prisoners by comparing scores with standard measures of autistic traits (Autism Quotient (AQ)), neurodevelopmental history (Asperger Syndrome (and High-Functioning Autism) Diagnostic Interview (ASDI)), and social cognition (Ekman 60 Faces test).

Methods

Prison officers across all 12 publicly-run closed prisons in Scotland assessed convicted prisoners using the screening tool. This sample included male and female prisoners and both adult and young offenders. Prisoners with high scores, along with an equal number of age and sex-matched controls, were invited to take part in interviews. Prisoners' relatives were contacted to complete a neurodevelopmental assessment.

Results

2458 prisoners were screened using the tool, and 4% scored above the cut-off. 126 prisoners were further assessed using standardised measures. 7 of those 126 assessed scored 32 or above (cut-off) on the AQ. 44 interviews were completed with prisoners' relatives, no prisoner reached the cut-off score on the ASDI. Scores on the screening tool correlated significantly with AQ and ASDI scores, and not with the Ekman 60 Faces Test or IQ. Sensitivity was 28.6% and specificity 75.6%; AUC was 59.6%.

Conclusions

Although this screening tool measures autistic traits in this population, sensitivity for scores of 32 or above on the AQ is poor. We consider that this limits its usefulness and do not recommend that the tool is routinely used to screen for ASDs in prisons.

Bisphenol A-Mediated Suppression of LPL Gene Expression Inhibits Triglyceride Accumulation during Adipogenic Differentiation of Human Adult Stem Cells

2012-05-25T21:00:00Z

by Chris Linehan, Sanjeev Gupta, Afshin Samali, Lynn O'Connor

The endocrine disrupting chemical, bisphenol A (BPA), has been shown to accelerate the rate of adipogenesis and increase the amount of triglyceride accumulation during differentiation of 3T3-L1 preadipocytes. The objective of this study was to investigate if that observation is mirrored in human primary cells. Here we investigated the effect of BPA on adipogenesis in cultured human primary adult stem cells. Continuous exposure to BPA throughout the 14 days of differentiation dramatically reduced triglyceride accumulation and suppressed gene transcription of the lipogenic enzyme, lipoprotein lipase (LPL). Results presented in the present study show for the first time that BPA can reduce triglyceride accumulation during adipogenesis by attenuating the expression of LPL gene transcription. Also, by employing image cytometric analysis rather than conventional Oil red O staining techniques we show that BPA regulates triglyceride accumulation in a manner which does not appear to effect adipogenesis per se.

Variable Carbon Catabolism among Salmonella enterica Serovar Typhi Isolates

2012-05-25T21:00:00Z

by Lay Ching Chai, Boon Hong Kong, Omar Ismail Elemfareji, Kwai Lin Thong

Background

Salmonella enterica serovar Typhi (S. Typhi) is strictly a human intracellular pathogen. It causes acute systemic (typhoid fever) and chronic infections that result in long-term asymptomatic human carriage. S. Typhi displays diverse disease manifestations in human infection and exhibits high clonality. The principal factors underlying the unique lifestyle of S. Typhi in its human host during acute and chronic infections remain largely unknown and are therefore the main objective of this study.

Methodology/Principal Findings

To obtain insight into the intracellular lifestyle of S. Typhi, a high-throughput phenotypic microarray was employed to characterise the catabolic capacity of 190 carbon sources in S. Typhi strains. The success of this study lies in the carefully selected library of S. Typhi strains, including strains from two geographically distinct areas oftyphoid endemicity, an asymptomatic human carrier, clinical stools and blood samples and sewage-contaminated rivers. An extremely low carbon catabolic capacity (27% of 190 carbon substrates) was observed among the strains. The carbon catabolic profiles appeared to suggest that S. Typhi strains survived well on carbon subtrates that are found abundantly in the human body but not in others. The strains could not utilise plant-associated carbon substrates. In addition, α-glycerolphosphate, glycerol, L-serine, pyruvate and lactate served as better carbon sources to monosaccharides in the S. Typhi strains tested.

Conclusion

The carbon catabolic profiles suggest that S. Typhi could survive and persist well in the nutrient depleted metabolic niches in the human host but not in the environment outside of the host. These findings serve as caveats for future studies to understand how carbon catabolism relates to the pathogenesis and transmission of this pathogen.

Impact of Sauropod Dinosaurs on Lagoonal Substrates in the Broome Sandstone (Lower Cretaceous), Western Australia

2012-05-25T21:00:00Z

by Tony Thulborn

Existing knowledge of the tracks left by sauropod dinosaurs (loosely ‘brontosaurs’) is essentially two-dimensional, derived mainly from footprints exposed on bedding planes, but examples in the Broome Sandstone (Early Cretaceous) of Western Australia provide a complementary three-dimensional picture showing the extent to which walking sauropods could deform the ground beneath their feet. The patterns of deformation created by sauropods traversing thinly-stratified lagoonal deposits of the Broome Sandstone are unprecedented in their extent and structural complexity. The stacks of transmitted reliefs (underprints or ghost prints) beneath individual footfalls are nested into a hierarchy of deeper and more inclusive basins and troughs which eventually attain the size of minor tectonic features. Ultimately the sauropod track-makers deformed the substrate to such an extent that they remodelled the topography of the landscape they inhabited. Such patterns of substrate deformation are revealed by investigating fragmentary and eroded footprints, not by the conventional search for pristine footprints on intact bedding planes. For that reason it is not known whether similar patterns of substrate deformation might occur at sauropod track-sites elsewhere in the world.

The Transmembrane Region Is Responsible for Targeting of Adaptor Protein LAX into “Heavy Rafts”

2012-05-25T21:00:00Z

by Matous Hrdinka, Pavel Otahal, Vaclav Horejsi

Background

The importance of membrane compartmentalization into specific membrane microdomains has been shown in many biological processes such as immunoreceptor signaling, membrane trafficking, pathogen infection, and tumor progression. Microdomains like lipid rafts, caveolae and tetraspanin enriched microdomains are relatively resistant to solubilization by some detergents. Large detergent-resistant membrane fragments (DRMs) resulting from such membrane solubilization can be conveniently isolated by density gradient ultracentrifugation or gel filtration. Recently, we described a novel type of raft-like membrane microdomains producing, upon detergent Brij98 solubilization, “heavy DRMs” and containing a number of functionally relevant proteins. Transmembrane adaptor protein LAX is a typical “heavy raft” protein. The present study was designed to identify the molecular determinants targeting LAX-derived constructs to heavy rafts.

Methodology/Principal Findings

We prepared several constructs encoding chimeric proteins containing various informative segments of the LAX sequence and evaluated their effects on targeting to heavy rafts. Replacement of the polybasic membrane-proximal part of LAX by CD3ε-derived membrane-proximal part had no effect on LAX solubilization. Similarly, the membrane-proximal part of LAX, when introduced into non-raft protein CD25 did not change CD25 detergent solubility. These results indicated that membrane-proximal part of LAX is not important for LAX targeting to heavy rafts. On the other hand, the replacement of transmembrane part of CD25 by the transmembrane part of LAX resulted in targeting into heavy rafts. We also show that LAX is not S-acylated, thus palmitoylation is not involved in LAX targeting to heavy rafts. Also, covalent dimerization was excluded as a cause of targeting into heavy rafts.

Conclusions/Significance

We identified the transmembrane domain of LAX as a first motif targeting transmembrane protein constructs to detergent-resistant heavy rafts, a novel type of membrane microdomains containing a number of physiologically important proteins.

Risk Factors for Breast Cancer and Expression of Insulin-Like Growth Factor-2 (IGF-2) in Women with Breast Cancer in Wuhan City, China

2012-05-25T21:00:00Z

by Jun Qiu, Rong Yang, Yanhua Rao, Yukai Du, Fatch W. Kalembo

Purpose

The purpose of this study was to explore the risk factors for breast cancer and establish the expression rate of IGF-2 in female patients.

Methods

A case control study with 500 people in case group and 500 people in control group. A self-administered questionnaire was used to investigate risk factors for breast cancer. All cases were interviewed during a household survey. Immune-histochemical method was used to inspect the expression of IGF-2 in different tissues (benign breast lesions, breast cancer and tumor-adjacent tissue).

Results

Multivariate adjusted odds ratios and 95% confidence intervals were calculated using unconditional logistic regression. High body mass index (OR = 1.012,95%CI = 1.008–1.016), working attributes (OR = 1.004, 95%CI = 1.002 = 1.006), long menstrual period (OR = 1.007, 95%CI = 1.005–1.009), high parity OR = 1.003, 95%CI = 1.001–1.005) , frequent artificial abortion (OR = 1.004, 95%CI = 1.001–1.005), family history of cancer (OR = 1.003, 95%CI = 1.000–1.005), period of night shift (OR = 1.003, 95%CI = 1.001–1.006), live in high risk environment (OR = 1.005, 95%CI = 1.002–1.008), and family problems (OR = 1.010, 95%CI = 1.005–1.014) were associated with increased risk for breast cancer. In this study, good sleeping status, positive coping strategies, subjective support, and utility degree of social support were associated with reduced risk for breast cancer (OR = 0.998, 0.997, 0.985, 0.998 respectively; 95%CI = 0.996–1.000, 0.994–1.000, 0.980–0.989, 0.996–1.000, respectively). In benign breast lesions, breast cancer and tumor-adjacent tissue, IGF-2 was mainly expressed in the cytoplasm, but its expression rate was different (p<0.05).

Conclusions

The incidence of breast cancer is a common result of multiple factors. IGF-2 is involved in the development of breast cancer, and its expression varies in different tissues (benign breast lesions, breast cancer and tumor-adjacent tissue).

Insecticide-Mediated Apparent Displacement between Two Invasive Species of Leafminer Fly

2012-05-25T21:00:00Z

by Yulin Gao, Stuart R. Reitz, Qingbo Wei, Wenyan Yu, Zhongren Lei

Background

Closely related invasive species may often displace one another, but it is often difficult to determine mechanisms because of the historical nature of these events. The leafmining flies Liriomyza sativae and Liriomyza trifolii have become serious invasive agricultural pests throughout the world. Where both species have invaded the same region, one predominates over the other. Although L. sativae invaded Hainan Island of China first, it recently has been displaced by the newly invasive L. trifolii. We hypothesized that differential susceptibilities to insecticides could be causing this demographic shift.

Methodology/Principal Findings

Avermectin and cyromazine are the most commonly used insecticides to manage leafminers, with laboratory bioassays demonstrating that L. trifolii is significantly less susceptible to these key insecticides than is L. sativae. In trials where similar numbers of larvae of both species infested plants, which subsequently were treated with the insecticides, the eclosing adults were predominately L. trifolii, yet similar numbers of adults of both species eclosed from control plants. The species composition was then surveyed in two regions where L. trifolii has just begun to invade and both species are still common. In field trials, both species occurred in similar proportions before insecticide treatments began. Following applications of avermectin and cyromazine, almost all eclosing adults were L. trifolii in those treatment plots. In control plots, similar numbers of adults of the two species eclosed, lending further credence to the hypothesis that differential insecticide susceptibilities could be driving the ongoing displacement of L. sativae by L. trifolii.

Conclusions/Significance

Our results show that differential insecticide susceptibility can lead to rapid shifts in the demographics of pest complexes. Thus, successful pest management requires the identification of pest species to understand the outcome of insecticide applications. These results further demonstrate the importance of considering anthropogenic factors in the outcome of interspecific interactions.

ATP Induced Brain-Derived Neurotrophic Factor Expression and Release from Osteoarthritis Synovial Fibroblasts Is Mediated by Purinergic Receptor P2X4

2012-05-25T21:00:00Z

by Kerstin Klein, André Aeschlimann, Suzana Jordan, Renate Gay, Steffen Gay, Haiko Sprott

Brain-derived neurotrophic factor (BDNF), a neuromodulator involved in nociceptive hypersensitivity in the central nervous system, is also expressed in synoviocytes of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. We investigated the role of P2 purinoreceptors in the induction of BDNF expression in synovial fibroblasts (SF) of OA and RA patients. Cultured SF from patients with symptomatic knee OA and RA were stimulated with purinoreceptor agonists ATP, ADP, or UTP. The expression of BDNF mRNA was measured by quantitative TaqMan PCR. BDNF release into cell culture supernatants was monitored by ELISA. P2X4 expression in synovial tissue was detected by immunohistochemistry. Endogenous P2X4 expression was decreased by siRNA transfection before ATP stimulation. Kinase pathways were blocked before ATP stimulation. BDNF mRNA expression levels in OASF were increased 2 h and 5 h after ATP stimulation. Mean BDNF levels in cell culture supernatants of unstimulated OASF and RASF were 19 (±9) and 67 (±49) pg/ml, respectively. BDNF levels in SF supernatants were only elevated 5 h after ATP stimulation. BDNF mRNA expression in OASF was induced both by P2X receptor agonists ATP and ADP, but not by UTP, an agonist of P2Y purinergic receptors. The ATP-induced BDNF mRNA expression in OASF was decreased by siRNA-mediated reduction of endogenous P2X4 levels compared to scrambled controls. Inhibition of p38, but not p44/42 signalling reduced the ATP-mediated BDNF mRNA induction. Here we show a functional role of the purinergic receptor P2X4 and p38 kinase in the ATP-induced expression and release of the neurotrophin BDNF in SF.

The Influence of Social Comparison on Visual Representation of One's Face

2012-05-25T21:00:00Z

by Ethan Zell, Emily Balcetis

Can the effects of social comparison extend beyond explicit evaluation to visual self-representation—a perceptual stimulus that is objectively verifiable, unambiguous, and frequently updated? We morphed images of participants' faces with attractive and unattractive references. With access to a mirror, participants selected the morphed image they perceived as depicting their face. Participants who engaged in upward comparison with relevant attractive targets selected a less attractive morph compared to participants exposed to control images (Study 1). After downward comparison with relevant unattractive targets compared to control images, participants selected a more attractive morph (Study 2). Biased representations were not the products of cognitive accessibility of beauty constructs; comparisons did not influence representations of strangers' faces (Study 3). We discuss implications for vision, social comparison, and body image.

In Silico Prediction and Analysis of Caenorhabditis EF-hand Containing Proteins

2012-05-25T21:00:00Z

by Manish Kumar, Shadab Ahmad, Ejaz Ahmad, Muheet Alam Saifi, Rizwan Hasan Khan

Calcium (Ca⁺²) is a ubiquitous messenger in eukaryotes including Caenorhabditis. Ca⁺²-mediated signalling processes are usually carried out through well characterized proteins like calmodulin (CaM) and other Ca⁺² binding proteins (CaBP). These proteins interact with different targets and activate it by bringing conformational changes. Majority of the EF-hand proteins in Caenorhabditis contain Ca⁺² binding motifs. Here, we have performed homology modelling of CaM-like proteins using the crystal structure of Drosophila melanogaster CaM as a template. Molecular docking was applied to explore the binding mechanism of CaM-like proteins and IQ1 motif which is a ∼25 residues and conform to the consensus sequence (I, L, V)QXXXRXXXX(R,K) to serve as a binding site for different EF hand proteins. We made an attempt to identify all the EF-hand (a helix-loop-helix structure characterized by a 12 residues loop sequence involved in metal coordination) containing proteins and their Ca⁺² binding affinity in Caenorhabditis by analysing the complete genome sequence. Docking studies revealed that F165, F169, L29, E33, F44, L57, M61, M96, M97, M108, G65, V115, F93, N104, E144 of CaM-like protein is involved in the interaction with IQ1 motif. A maximum of 170 EF-hand proteins and 39 non-EF-hand proteins with Ca⁺²/metal binding motif were identified. Diverse proteins including enzyme, transcription, translation and large number of unknown proteins have one or more putative EF-hands. Phylogenetic analysis revealed seven major classes/groups that contain some families of proteins. Various domains that we identified in the EF-hand proteins (uncharacterized) would help in elucidating their functions. It is the first report of its kind where calcium binding loop sequences of EF-hand proteins were analyzed to decipher their calcium affinities. Variation in Ca⁺²-binding affinity of EF-hand CaBP could be further used to study the behaviour of these proteins. Our analyses postulated that Ca⁺² is likely to be key player in Caenorhabditis cell signalling.