Biomedicine blog

4.5.2. Cell reproduction II

noreply@blogger.com (David Funes) — Sun, 25 Sep 2016 14:41:00 +0000

The cell cycle is a set of ordered events that involve the cell growth and division to produce two new daughters cells.

Prior to cell division, cells go through a series of stages of growth, DNA replication and division, carefully regulated, with the objective of creating two identical cells.

The two main phases of the cell cycle are the interphase and the mitotic phase. During the first stage, the cell grows and replicates its DNA. While, in the mitotic phase, the replicated DNA and the cytoplasmatic content come apart and the cell divides.

Interphase

The interphase is the longest period of the cell cycle, it occurs between two mitosis and we can distinguish the following phases within it:

▣ G1: the cell grows, reaching its critical size. RNA and proteins are synthesised and the centrosome is duplicated. If conditions are appropiate, the cell passes the restriction point (a G1 phase checkpoint) and the next step begins.
Differentiated cells that do not divide (e.g., neurons or cells from the cardiac muscle) leave the cell cycle at this point and start the G0 phase^[1].

▣ S: DNA is replicated. In this way, two daughter cells inherit the same genome from the mother cell.

▣ G2: repair phase of DNA and preparation for mitosis. The cell doubles its size compared to G1 phase.

Mitotic phase

The mitotic phase is a multistep process in which the duplicated chromosomes align, come apart and move to the two new daughters created.
The first stage of the mitotic phase is called karyokinesis, mitosis or nuclear division and the second step is known as cytokinesis, which is the physical separation of the cytoplasmatic components into the two daughter cells.

Mitosis, where the genetic material is divided equally, comprises the following stages:

▣ Prophase: chromatin is condensed, the mitotic spindle^[2] is formed and the nucleolus disappears.

▣ Prometaphase: the nuclear envelope is removed and the chromosomes join the microtubules through the kinetochores^[3].

▣ Metaphase: the chromosomes align randomly in a plane called metaphase plate, or equatorial plane, between the two poles of the cell.

▣ Anaphase: the microtubules of the spindle become shorter, which causes the separation of the two sister chromatids.

▣ Telophase: the chromosomes decondense and the nuclear envelope, along with the nucleoulus is formed. Lastly, the mitotic spindle disappears.

During the last step of the mitotic phase, cytokinesis, a contractil ring of actin filaments is produced which divides the cytoplasm into the two daughter cells.

Control of the cell cycle

The duration of the cell cycle is higly variable, even in cells of the same organism. In human beings, it ranges from just a few hours in the embryonic cells, to two - five days for epithelial cells, to a whole human life for specialised cells, like cardiac muscle cells or cortical neurons.

The time that a cell spends in each phase of the cycle varies too. Thus, for example, those human cells with a 24-hour cycle spend approximately nine hours in the G1 phase, ten hours in the S phase, around four and a half hours in the G2 phase and about 30 minutes in the M phase.

Each step of the cell cycle is controlled by mechanisms both internal and external to the cell.

Regulation of the cell cycle by external mechanisms

Both the start and the termination of the cell replication cycle are activated by events external to the cell.
For instance, events such as the death of a near cell, the release of hormones that favour growth or the cell reaching a specific size can start cell division.
Regardless of the type of externally received message, a series of internal effects then lead it to the interphase.
From this starting point, each required parameter in each phase must be satisfied so that the cell cycle can continue.

Regulation at internal checkpoints

The checkpoints of the cell cycle prevent mistakes in the duplication or in the distribution of chromosomes (which can cause mutations).

We can distinguish three checkpoints:

▣ The G1 checkpoint: also named the ‘rectriction point’ in mammals. It acts at the end of G1 phase and determines if all the necessary requirements are fulfilled to proceed the cell division.
Si no se cumplen, la célula puede parar el ciclo hasta que las condiciones adversas se solucionen o puede pasar a la fase G0 y esperar señales que indiquen que las condiciones han mejorado.

▣ The G2 checkpoint: carries out its role during the transition between G2 to M phases. Its most important function is to make sure that the chromosomes have been replicated and that replication has been accomplished without DNA damage. If damage is detected, the cycle stops and the cell tries to complete the DNA replication or repair the damaged one.

▣ The M checkpoint: is also known as the spindle checkpoint. It acts near the end of the metaphase stage of karyokinesis. It determines whether the sister chromatids are firmly and correctly bound to the spindle microtubules by the kinetochores.

The control system of the cell cycle could be compared to the program of a washing machine, where the washing machine only carries out its functions when the signals from its sensors (lock door, levels of water and detergent, power supply) indicate that it is ok to move forward during the diverse stages of the washing cycle.

Regulator molecules of the cell cycle

In addition to the checkpoints, there are some intracellular molecules that regulate the cell cycle and can act individually or affect the activity or production of other regulator molecules.

Regulator molecules are classified into two groups:

▣ Those molecules that promote the progress of a cell to the next phase (positive regulation). They are, in turn, divided into two types: cyclins^[4] and cyclin-dependent kinases (CDKs)^[5].

▣ The second group are negative regulators, which are in charge of halting the cell cycle. The best-known negative regulators are retinoblastoma protein (Rb), p53 and p21.
Retinoblastoma protein is a suppressor protein for tumours that is altered in many kinds of cancer (prostate, breast), but it is retina cancer from where it takes its name.
P21 inhibits the cell cycle, with its levels controlled, in turn, by p53, which regulates cell growth and controls DNA damage. This protein can halt the cycle if needed, and is able to cause apoptosis too.

[1] Permanent or temporary quiescence (inactivity) phase in which the cell cycle stops.
[2] Set of microtubules whose function is to enable the migration and correct separation of the chromosomes during mitosis.
[3] Protein structures whose function it is to initiate, control and supervise the movement of the chromosomes during cell division.
[4] Proteins synthesised during the interphase and destroyed at the end of the mitosis. They regulate the enzymatic activity of CDKs.
[5] Enzymes that activate or inhibit other proteins by phosphorylating them (adding phosphate groups).

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
  http://www.biocancer.com/journal/1219/41-regulacion-positiva-del-ciclo-celular
  http://www.pucmmsti.edu.do/websise/estudiante/materias/201120122/ST-BIO-112-T-003/04.%20Regulacion%20del%20ciclo%
20celular%20Apoptosis%20Cancer%20%20.pdf
  http://bio3400.nicerweb.com/Locked/media/ch02/interphase.html
  http://www.slideshare.net/obessionforu/cell-cycle-and-molecular-basis-of-cancer
  http://biologicalexceptions.blogspot.com.es/2013_01_01_archive.html
  http://gleesonbiology.pbworks.com/w/page/7537859/C9
  http://www.bioscirep.org/content/30/4/243

4.5.1. Cell reproduction I

noreply@blogger.com (David Funes) — Sat, 03 Sep 2016 15:24:00 +0000

Human beings, like other organisms that reproduce sexually, start life from a zygote or fertilised egg.
Later, trillions of cell divisions take place in a controlled way to lead to a complex multicellular human being, or in other words, a single cell is the common ancestor of the rest of the organism’s cells.
Even when a living being has developed completely, cell division is still necessary for the development, maintenance and repair of cells and tissues. This division is strictly regulated to avoid consequences that might be fatal for that organism.

Cell division

Cell reproduction via the cell cycle constitutes the foundation that enables the continuity of life from one cell to another. This cycle, strongly regulated, includes the diverse stages of a cell life from the division of a single parent cell to the reproduction of two new daughter cells.

Before going on with the cell reproduction process, it is necessary to understand the function and structure of the genetic information of a cell.
The number of chromosomes^[1] of eukaryotic cells varies depending on the species.
In human beings, body cells (somatic cells) have 46 chromosomes, while sex cells or gametes (eggs and sperm) have 23 each.
Somatic cells have two sets of chromosomes (2n^[2]) in a configuration called diploid.
Gametes, that have a single set of chromosomes, are designated as 1n and they are known as haploid cells.

Genes (functional units of the chromosome) determine particular features by coding specific proteins, to represent variations in specific features. For instance, eye colour is a characteristic whose traits can be brown, blue or green.
These traits in an individual are established by the inherited genes from each parent.

If the DNA of those 46 chromosomes in a human cell unfolded from one end to the other, it would measure around 2 metres and its diameter just 2 nm. Taking into account that the typical size of a cell is about 10 μm, DNA has to condense itself to fit into the cell nucleus and must be quickly accesible so that genes can be expressed.

During several phases of the cell cycle, the long chains of DNA are highly condensed in the chromosome. There are a number mechanisms by which chromosomes are compacted, in all of them various classes of proteins help to organise and pack the chromosomal DNA.

In the first level of compaction, DNA is strongly packed around a core of eight structural proteins (histones) in regular intervals along the chromosome, forming the structure known as chromatin.
The fundamental unit of organisation for chromatin in eukaryotic cells is the nucleosome, composed of a DNA fragments around 200 base pairs (bp) long and a histone octamer (group of eight). This structure compacts seven times the DNA molecule which under a microscope looks like a ‘necklace of beads’ with a 10 nm diameter.

In the second level of compaction, the nucleosome fibres and the linker DNA^[3] roll into 30 nm fibres (DNA molecule is now 50 times packed).

The third level of compaction uses fibrous proteins to compact chromatin, creating radial loops of 250-400 nm long. These proteins make sure that each chromosome in the non-dividing phase occupies a certain area in the nucleus avoiding overlap.

In higher levels of organization we find chromatids (700 nm - 1μm long). When two of these chromatids join, through a region called the centromere, they form metaphasic chromosomes^[4].

[1] Highly organized structures comprising DNA and proteins, which contain most of the genetic information of an individual.
[2] n indicates the number of chromosome sets.
[3] DNA regions that are not transcribed and are located between genes.
[4] Metaphase: second phase of mitosis (cell division) in which the nuclear membrane is removed and the chromosomes are placed in the equator of the cell.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
  https://www.quora.com/What-are-the-differences-between-chromosomes-chromatids-and-chromatin
  https://burningscience.wordpress.com/biology/dna-structure-and-replication/
http://27.109.7.67:1111/econtent/cell-communication/principles-part-3.php
  https://www.quora.com/What-are-the-differences-between-chromosomes-chromatids-and-chromatin

1.6. Personalised medicine

noreply@blogger.com (David Funes) — Sun, 31 Jul 2016 15:03:00 +0000

In recent years, the definition of a succesful drug has changed. The time when a drug would become a blockbuster is over, since there are no longer illnesses that can be treated with just a drug. At the same time, patients and markets demand more effective medicines.

But this demand turns out to be harder to fulfil because our knowledge about diseases is getting more and more complex. Besides, there are numerous external pressures (from governments, financial institutions, regulatory agencies and even scientists) to achieve the best clinical results.
Then, how is it possible to create new drugs when their development costs increase year after year and it is more difficult to generate them with a clinical benefit for patients?

To overcome this challenge, science has provided us with new tools, such as the ability to map the human genome (at a lower and lower cost), which allows us to understand the expression of different proteins in response to diverse illnesses.
Consequently, personalised medicine, which uses these new kinds of tools, is expected to take the lead in the discovery of new drug development processes and of specific medicines that are more effective and even safer.

But, how is a personalised drug defined? Personalised medicine is that medical treatment tailored to the features of each and every patient.
It is expected that personalised drugs will be prescribed only to those for whom there will be a known and personal benefit.
In this way, not only the capacity to treat diseases improves, but also, the associated savings in prescribing in a targeted way.

Within personalised medicine, we find three categories:

▣ Diagnostics: these products can diagnose the risk of suffering from a specific disease, the real existence of a disease in an individual, or even guide the treatment for those people who have already been diagnosed.

▣ Companion diagnostics: : these products work in tandem with pharmaceutical treatments, indicating to the physician the type of drugs that a particular patient should receive.

▣ Screening tools: they are able to detect diseases in their early stages, which allows quick interventions with the hope of diminishing the effects caused by illnesses.

Ideally, it would be interesting to find a way of evaluating a person’s vulnerability to a disease or their response to a certain treatment even before administering it.
With the help of personalised medicine, we could know beforehand which people would respond positively to a specific drug and those that would not; which people would suffer grave side effects and those that would not; which people would need that medicine at an early stage and those who would need it at a later stage. All with the goal of accomplishing the best possible results in each patient.

Personalised medicine has experienced spectacular growth in the last few years, from 13 main products in the market in 2006 to 72 products in the year 2011 and sales of 30 billion dollars in the USA.
Presently, 94% of biopharmaceutical companies are researching personalised medicine. They are aware that it can be used both as a research tool as well as to improve a drug’s potential.

Among the personalised medicine products that physicians use currently to treat their patients are the following:

▣ BRACAnalysis tests that identify potential mutations in the genes BRCA1 and BRCA2 responsible for most ovary and breast inherited cancers. Depending upon the presence or not of these mutations, the patient’s likelihood of developing these types of cancer in the future is determined. According to that, other relatives are guided and advised about what steps and decisions to make.

▣ Oncotype DX is a 21gene test that predicts a patient’s likelihood of benefiting from chemotherapy and the risk of a tumour recurrence in the following ten years.

▣ PreDX is a blood test that examines the levels of seven protein markers associated with diabetes. These markers are related to fat tissue quality, inflammatory conditions of the pancreas and glucose metabolism.

Some of the current benefits from personalised medicine include:

▣ A 34% reduction of chemotherapy in those women who suffer from breast cancer and prior to these treatments they have been undergone genetic testing.

▣ Up to 17,000 strokes can be prevented every year in those patients who are prone to cerebrovascular disorder by giving the proper dose of warfarin^[1] provided that a genetic test is carried out.

▣ Over 600 million dollar savings for the health care system in the USA if patients with metastatic colorectal cancer undergo the KRAS gene^[2] test before receiving their treatments.

▣ Development of new diagnostic tools that can help in the drug development process. Thus, if pharmaceutical companies know in advance what patients will respond better to a clinical trial, better results will be obtained in that study.

We are still in the early stages of personalised medicine, which will provide us with a better understanding about disease conditions, minimise side effects, and identify new drug targets.
By incorporating it into our experiments and research methods, we will also identify new innovations related to the drug discovery process.

[1] An anticoagulant (blood thinner) used in the prevention of thrombosis and thromboembolism.
[2] Gene that produces KRAS protein, which takes part in many cell signalling patways, cell growth and apoptosis.

Sources: UTAustinX: UT.4.01x Take Your Medicine - The Impact of Drug Development.
http://www.patia.com.mx/braca.html
https://breast-cancer.oncotypedx.com/es-MX/Professional-Invasive/WhatIsTheOncotypeDXBreastCancerTest.aspx
http://www.tuorigen.cl/index.php/preguntas-frecuentes
http://www.cancer.gov/espanol/publicaciones/diccionario?cdrid=652256
http://www.lampadia.com/analisis/tecnologia/medicina-del-futuro-despliegue-de-un-nuevo-paradigma/

4.4. Cell communication

noreply@blogger.com (David Funes) — Sat, 23 Jul 2016 15:56:00 +0000

In the same way that social organisations require communication between individuals and their environment to maintain societal cohesion, cells must also be able to interact with their environment.

In order to respond to external stimuli, cells have developed complex communication systems that allow them to receive messages, transfer information across the plasma membrane and produce changes within the cell in response to those messages.

Cells of multicellular organisms constantly send and receive messages to coordinate the actions of cells, tissues and distant organs. The capacity to send messages in an effective and quick way allows cells to coordinate and adjust their functions.
This capacity to communicate via chemical signals, initially developed by individual cells, was a vital attribute for the evolution of multicellular organisms.

Signalling molecules and cellular receptors

Cells are in contact via intercellular signaling (communication between cells) and intracellular signaling (communication within the cell).
Signalling cells are responsible for secreting ligands^[1] or they bind to target cells, triggering a event chain within them.

In multicellular organisms, chemical signalling is classified into four categories: endocrine signalling, autocrine signalling, paracrine signalling and direct signalling by gap junctions (juxtacrine signalling).
Endocrine signalling is performed by hormones across long distances through the bloodstream.
In autocrine signalling, signals are received by the same cell that sends them or other close cells of the same type.
Paracrine signalling also acts at short distances under the action of ligands that travel through the liquid medium of the extracellular matrix.
Lastly, gap junctions enable signalling molecules to flow between neighbouring cells.

Types of receptors

Receptors are proteins located within, or on the surface of target cells that bind to ligands. Receptors are thus divided into two different classes: internal receptors and cell-surface receptors.

Internal receptors are located in the cell cytoplasm attached to the ligand molecules that pass through the cell membrane. These receptor-ligand complexes move to the nucleus and there they interact directly with the cellular DNA.

Cell-surface receptors convey a signal from outside the cell to the cytoplasm. They comprise three components: a region outside the cell to which the ligand binds (also called extracellular domain), a hydrophobic central region in the membrane and the intracellular domain within the cell.

They are divided into three categories:

▣ Ion channel-linked receptors^[2]. When they bind to their specific ligands they form a channel across the plasma membrane through which certain ions can pass.

▣ G-protein-linked receptors. They interact with G-proteins^[3] on the cytoplasmatic side of the cell membrane.
Once the G-protein binds to the receptor, the resultant compound activates the G-protein, which releases GDP^[4] and pick up GTP^[5], interacting with other enzymes or ionic channels to transmit the signal.

▣ Enzyme-linked receptors. They carry a signal of membrane-bound enzymes from outside the cell to the intracellular domain. The union with the ligand causes the enzyme activation.

Small hydrophobic ligands (e.g. steroids) are able to penetrate the cell membrane and stick to internal receptors.
On the other hand, hydrophilic receptors (soluble in water) are unable to pass through the membrane; consequently they link with cell-surface receptors, which convey signals inside the cell.

Propagation of the signal

The binding of a ligand to a receptor enables signal transduction^[6] throughout the cell.
The chain of events that creates signal transmission is called the ‘signalling cascade’ or ‘signalling pathway’. This pathway can be very complex as a consequence of the interaction among different proteins.
One of the most important events in this process is the phosphorylation of molecules by way of specific enzymes known as kinases^[7].
Phosphorylation adds a phosphate group to residues (R-groups) of the following amino acids: serine, threonine and tyrosine, activating and deactivating the protein to these amino acids belong to or changing its shape. Also, small molecules such as nucleotides can be phosphorylated.

Once a receptor has been activated, the signal propagates throughout the cell via the cytoplasm by modifying the behaviour of certain cellular proteins. These signals are released by small non-protein molecules, called second messengers. Among them are: cyclic AMP (cAMP), calcium ions (Ca²⁺), inositol triphosphate (IP₃) and diacylglycerol.

Response to the signal

The initiation of signalling routes is triggered in response to external stimuli. The effects of these responses are quite diverse, depending on the type of cell involved, as well as internal and external conditions.
Among these effects are cell growth, protein synthesis, changes in the cell metabolism and even cell death.

But these signalling routes have their most significant impact on the cell by initiating gene expression^[8].
Thus, some routes activate enzymes that interact with transcription factors of RNA and others regulate protein transduction from mRNA.
Other routes again, however, work on the cellular metabolism, e.g. they enable muscle cells to communicate their functional energy requirements (these requirements are in the form of glucose).

Signalling routes play a very significant role in cellular growth too, which is stimulated by external signals, called ‘growth factors’ (types of ligands that bind to cell-surface receptors).
Uncontrolled cell growth lead to cancer. Cancer’s origin is often due to mutations presented by those genes responsible for encoding the proteins that are part of signalling pathways.

When a cell is damaged, is unnecessary or is potentially dangerous, the organism must initiate the process that triggers programmed cell death or apoptosis. Apoptosis allows a cell to die in a controlled way, avoiding the release of potentially harmful molecules, in contrast to uncontrolled death or necrosis.

Cell signalling controls the process of apoptosis so that the dismantling of cells is carried out in an organised way, as well as the efficiently recycling the different components of dead cells.

The end of the signalling cascade is very important in ensuring that the signal response is appropriate both in time and intensity.
Two of the most common ways of ending signalling within cells is with the degradation of signalling molecules and dephosphorylation of intermediate products of the pathway (previously phosphorylated) via phosphatase^[9] enzyme.

Some of the abnormal signals originated in tumour cells are good evidence that termination of cellular cascades at the appropriate time is as important as the timing of initiation.

[1] Small volatile or soluble molecules that bind to other specific molecules (receptors) releasing a signal in the process.
[2] Cell-surface receptors in the channels of plasma membranes that open when a ligand sticks to its extracellular domain.
[3] Signal transducers that transmit information from the receptor to effector proteins.
[4] Guanosine diphosphate: dephosphorylation product of GTP.
[5] Guanosine triphosphate is a purine nucleoside triphosphate used in the cell metabolism.
[6] Transmission of a signal from outside the cell to its inside.
[7] Enzymes that catalyse the transfer of a phosphate group from ATP to another molecule.
[8] Process by which cells transform the encoding information of amino acids into necessary proteins for their development and functioning.
[9] Enzyme in charge of removing the phosphate group of a molecule that has been phosphorylated.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
http://mol-biol4masters.masters.grkraj.org/html/Cellular_Signal_Transduction1-Cells_And_Signals.htm
http://27.109.7.67:1111/econtent/cell-communication/principles-part-3.php
http://www.scq.ubc.ca/protein-phosphorylation-a-global-regulator-of-cellular-activity/
http://medicinembbs.blogspot.com.es/2011/03/programmed-cell-death-apoptosis.html

1.5. Quality control in pharmaceutical industry

noreply@blogger.com (David Funes) — Sat, 09 Jul 2016 15:02:00 +0000

Quality control is used in each and every stage of the drug development process. It ensures that the drug product fulfils requirements that include safety, quality, stability and efficacy.
The word ‘quality’ refers to a pharmaceutical drug’s features from both a quantitative and qualitative point of view. It refers to both the quality of the manufacturing process and the product itself.
The word ‘control’ implies a procedure by which production is carried out, according to a specific plan fulfilling the standards previously established.

The maintance of drug quality depends on each and every person and the equipment that is used in its development. There is strict supervision in each stage of the process so that the final product achieves its highest quality. The quality control department in a pharmaceutical company is in charge of monitoring records, procedures, systems, facilities, staff and analyses that are made in development and production of a drug product.

Quality variety can occur throughout the process, from the receipt of raw materials to the packaging of the final product. The risk of this happening is increased as the manufacturing method gets more and more complex. Possible causes of diminished quality are:

▣ Methods:

Negligence in the process
Wrong process
Inadequate process

▣ Materials:

Variations amongst the suppliers of the same substance
Variations amongst batches of the same supplier
Variations in a batch

▣ Staff:

Fatigue and carelessness
Lack of interest and attention
Inappropriate training
Unsuitable work conditions

Errors can be controlled, reduced or eliminated by the monitoring of material, its packaging and with good manufacturing practices (GMPs).

Material control starts just after its delivery. Active substances, excipients, packaging and printed materials are stored properly and classified alphabetically or depending on their physical nature.
In the case of active principles the following are examined: their adulteration, percentage purity, expiry date and batch number.
For printed and packaging materials, their weight, colour and grammage are checked too.

Manufacturing practices control

To achieve the highest quality, the following elements are controlled:
- Personnel
- Equipment and facilities
- Production process
- Records

▣ Staff

Staff have to be appropriately educated and trained to work in the pharmaceutical industry.
They have to receive continous training suitable for their role.
They have to be warned about the risks and responsibilities in their role.
The work performed by workers has to be monitored by highly-educated senior managers with broad and deep expertise.
Senior managers always have to be available in case of any potential incident.

▣ Equipment and buildings

Equipment and buildings must have the right design, size and construction requirements to store, process, examine and package drug products.
Equipment surfaces must be non-absorptive, non-additive and non-reactive.
Equipment must be built and assembled in such a way that it is easy to replace, wash and operate.
Buildings must be free of any kind of contamination.

▣ Record control

The most important record controls are related to drug formulation and lot production.

The drug formulation record must contain:
- Product name and dosage form.
- Quality by weight or volume of each ingredient.
- Control and manufacturing instructions, specifications and precautions.
- Complete list of ingredients used, including excipients.
- Standards or specifications of each ingredient.
- Complete description of packaging, containers and labelling materials.

The batch production record must contain the following information:
- Lot number.
- Code number.
- Manufacturing date.
- Expiry date.

▣ Production process control

Manufacturing processes are carried out from the delivery of the material through to the final product commercialisation in accordance with a set of rules previously established.
The Master Formula has a full list of all drug components alongside their quantities, procedures, equipment and precautions that must be taken during its manufacturing.

This Master Formula is delivered to the Production Department where all ingredients are re-examined and tested in the laboratory.
Some of these analyses are performed during the production process itself, this is called ‘In Process Quality Control’ (IPQC), which is under supervision of the Quality Control Department.
These tests vary depending on the drug dosage form (syrups, injectables, tablets, semi-solid forms…).

Packaging control

The final product is packaged in its recommended containers avoiding mistakes in labelling or lot number.
Packaging materials are chosen according to the distribution and nature of product.

Distribution control

The responsibilities of the Quality Control Department do not finish when the pharmaceutical product is distributed to the market.
The deparment registers samples of each batch and these are stored for years with the purpose of being examined in case of need or demand.

Organisation of quality control

Generally, there are five different departments in Quality Control:

▣ Analytical department

The quality of the final product largely depends on the quality of the raw materials used in the manufacturing process. This department analyses these raw materials and ensures that the drug fulfils a set of specifications to keep its quality. Among these specifications we find:
Solubility, viscosity, surface tension, crystal shape…

▣ Chemical testing laboratory

The chemical and physical properties of each lot of raw materials and final products are tested in this laboratory.
This laboratory must be located in an isolated area away from noise and vibration and it must have suitable equipment to carry out a wide range of chemical tests.

▣ Biological testing laboratory

Biological examinations analyse biologic drug products and a broad variety of products such as parenteral products which require pyrogen^[1] and sterility tests before their commercialisation.

▣ Central release office

This office evaluates the many records generated throughout manufacturing and packaging. These records report on the characteristics of each lot produced and distributed in the market, which facilitates the later investigation of potential product quality claims submitted by customers.

▣ Inspection and checking

Finally, Quality Control Inspectors are responsible for selecting random samples of raw materials received and final products for their inspection.

[1] Agents that cause fever.

Sources: http://thepharmacistpharma.blogspot.com.es/2009/03/quality-control-procedure-in.html
http://www.ancalmo.com/moderno-laboratorio-control-de-calidad/
http://controldecalidadifv.blogspot.com.es/
http://www.aemps.gob.es/industria/inspeccionNCF/guiaNCF/docs/reqBasicosMed/04_capitulo-1b.pdf

4.3.2. Cell respiration II: Oxidative phosphorylation (Electron transport chain). Metabolism without oxygen

noreply@blogger.com (David Funes) — Sun, 12 Jun 2016 15:15:00 +0000

Most of the ATP originated during the aerobic catabolism of glucose does not come from the two previous routes (Glycolysis and Krebs cycle), but rather from the movement of electrons through a series of electron carriers that undergo redox reactions, which causes the accumulation of hydrogen ions in the mitochondrial matrix.

As a consequence, a concentration gradient is formed in which hydrogen ions diffuse out of the mitochondrial matrix by passing through it with the help of the complex called ATP synthase^[1]. The current of hydrogen ions promotes the catalytic action of ATP synthase, which phosphorylates ADP, producing ATP.

Electron transport chain

The electron transport chain is the last component of aerobic respiration and the last stage of glucose metabolism that uses atmospheric oxygen.
This oxygen is used as a final receptor for the electrons that have been removed from the intermediate compounds in glucose catabolism.

The electron transport chain consists of four large multiprotein complexes (called complexes I-IV) embedded in the inner mitochondrial membrane and two small electron transporters that move the electrons between them.

Electrons participate in a series of redox reactions in which free energy is used to shuttle hydrogen ions across the mitochondrial membrane using the ATP synthase enzyme. This process contributes to the gradient used in chemiosmosis (a mechanism based on the diffusion of ions across a membrane) that produces 90% of ATP created during aerobic glucose metabolism.

The electrons that take part in the transport chain lose their energy gradually, so to complete it, the contribution of high-energy electrons from the NADH or FADH₂ compounds is necessary (generated in the previous process (Krebs cycle).

At the end of this metabolic pathway, electrons reduce oxygen molecules to oxygen ions and the extra electrons of these ions attract hydrogen ions (protons) from the surrounding environment, resulting in the release of water molecules and ATP as the final products of the electron transport chain.

The number of molecules created varies depending on a number of factors such as the number of hydrogen ions that the complexes of the electron chain pump across the mitochondrial membrane, the transport of electrons or the use of intermediate compounds produced in these routes for other purposes.

Metabolism without oxygen

In aerobic respiration, the final receptor of electrons is the oxygen molecule (O₂) and ATP is produced with the assistance of high-energy electrons transported to the transport chain by the molecules NADH or FADH₂.

If aerobic respiration does not take place, the NADH compound must be re-oxidased to NAD⁺ so that it can be reused as an electron carrier, and in this way the glycolytic pathway continues.
Living organisms employ two different mechanisms to achieve this:

▣ They can use an organic molecule as the final acceptor of electrons to regenerate NAD⁺ from NADH in a process called fermentation.
One of the most well known fermentation processes is lactic acid fermentation, used by red blood cells in skeletal muscles of mammals when they lack enough oxygen to continue aerobic respiration.

▣ The second option is to use an inorganic molecule instead.

In both, organisms transform energy to use it in the absence of oxygen and they are known as anaerobic cellular respiration.

Regulation of cellular respiration

Cellular respiration must be regulated in order to supply the required amounts of energy at any time in the form of ATP.
The cell must regulate, therefore, its metabolism and for that have a broad variety of mechanisms.
For instance, glucose entering the cell via the plasma membrane is controlled by transport proteins (GLUT proteins). But most of the control of the respiratory process is performed by specific enzymes that act on each route.

These enzymes react to the available levels of the nucleosides ATP, ADP, AMP, NAD⁺ and FAD, which, in turn, increase or decrease enzyme activity on the routes where they participate.

We have seen that glucose metabolism is responsible for providing energy to living cells. Yet, living beings consume a broad range of nutrients other than glucose in their diets. Hence, how do these foods become ATP in our cells?
Eventually the catabolic pathways of lipids, proteins and carbohydrates are connected with glycolysis and citric acid cycle pathways.
These pathways are not closed cycles, but many of their substrates, intermediate and final products are used in other routes.

[1] It is a transmembrane protein complex (enzyme) that catalyses ATP synthesis by the supplied energy from a proton flow (H+) and by adding a phosphate group to ADP.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
https://chelseaharripersad.wordpress.com/2013/04/13/the-electron-transport-chain/
http://healthylifemed.com/aerobic-vs-anaerobic-respiration/
https://adapaproject.org/bbk_temp/tiki-index.php?page=Leaf%3A+Why+do+cells+need+fermentation+to+continue+glycolysis%3F
http://163.178.103.176/Fisiologia/general/dinamica/FG05_17a.jpg

4.3.1. Cell respiration I: Glycolysis. Krebs cycle

noreply@blogger.com (David Funes) — Sat, 04 Jun 2016 15:12:00 +0000

The energy that enters into our organisms as food is transformed so that cells can use it and perform their functions.
Thus, through a set of metabolic pathways (collectively called cell respiration), the energy of glucose bonds is extracted and turned into an energy form that all living organisms can use.

Energy in living beings

A living cell does not collect excessive amounts of free energy because it could damage and subsequently destroy it. Hence, cells must store that energy in a safe way and free it only when the need arises, which is achieved by the adenosine triphosphate compound (ATP), which is considered the ‘energy currency’ of cells.

ATP allows the cell to store energy for a short time and to transport it within the cell to facilitate endergonic chemical reactions.
ATP’s structure is made up of an adenosine monophosphate molecule (AMP^[1]), which consists of an adenine molecule attached to a ribose molecule and one phosphate group. The addition of a second phosphate group produces adenosine diphosphate (ADP) and the addition of a third phosphate group forms finally adenosine triphosphate (ATP).

Life to accomplish its processes and obtain energy, it breaks down ATP into ADP^[2] constantly, through the hydrolysis ATP reaction, producing, at the same time, an inorganic phosphate ion:

ATP + H₂O ⇔ ADP + inorganic phosphate (Pi)

The water molecule that takes part in this reaction is split into a hydrogen atom and a hydroxyl group. This water is regenerated when a third phosphate is added to the ADP molecule, which reconstitutes, in turn, ATP. This mechanism works, therefore, like a rechargeable battery.

In practically all organisms, the required energy comes from glucose metabolism. This way, ATP is directly connected to the set of exergonic pathways of the catabolism of glucose and the large number of endergonic pathways that supply energy to cells.

The two processes of ATP regeneration used in combination with glucose catabolism are substrate phosphorylation^[3] and oxidative phosphorylation.
In the first mechanism, ATP is produced from ADP and a phosphate group from a reactive.
But most of the generated ATP (90%) during the glucose catabolism is derived from a much more complex process, which ocurrs in the mitochondrion: chemiosmosis. During chemiosmosis in which a proton gradient is involved across the mitochondrial membrane.
The production of ATP using this mechanism is called oxidative phosphorylation because of the involvement of oxygen in it.

Glycolysis

Glycolysis was probably one of the first metabolic pathways used during the evolution of living beings and it is used by nearly all of them.
Glycolysis is the first point of glucose breakdown for the extraction of energy during the cellular metabolism. It does not require oxygen; it is thus an anaerobic mechanism.

This process is composed of two steps:

▣ The six carbon atom ring of glucose molecule is divided into two sugar molecules (called pyruvate) with three carbon atoms each one. ATP is needed to cause this separation.

▣ ATP and high energy electrons are extracted from hydrogen atoms, and are attached to NAD⁺ compounds (oxidised form of the molecule NAD^[4]).

The first stage invests two ATP molecules, while the second stage produces four ATP molecules through the substrate phosphorylation mechanism.
The final outcome is a net gain of two ATP molecules and two NADH^[5] for the cell.
In the event that the cell cannot catalyse the pyruvate molecules, it only gets two ATP molecules from a glucose molecule.

Oxidation of pyruvate and the Citric Acid Cycle (Krebs cycle)

If there is oxygen available, aerobic respiration then takes place following the glycolysis process.
Pyruvate molecules originated at the end of that metabolic pathway are transported into mitochondria where cell respiration takes place. There, pyruvate is transformed into an acetyl group, which is collected and activated by a carrier called coenzyme A (CoA)..
The resultant compound, named acetyl coenzime A, is made up of vitamin B5 (pantothenic acid). This compound is used in a wide range of ways by cells, but its principal function is to distribute the acetyl group that comes from the pyruvate to the following stage in glucose metabolism (the citric acid cycle).

The conversion of pyruvate to an acetyl group removes a CO₂ molecule and two high energy electrons. This step occurs twice, so that the CO₂ molecule (2CO₂) holds two of the six carbon atoms from the initial molecule of glucose. While the NAD+ molecule picks up the electrons forming NADH that conducts those electrons to later pathways in the production of ATP.

At this point, the glucose molecule that entered the cellular respiration mechanism has been completely oxidised and its potential energy has been transferred to electron transporters or has been used to synthesise a few ATP molecules.

Next, the citric acid cycle also begins in the mitochondrial matrix.
Unlike glycolysis, the cytric acid cycle is a closed cycle, in which the last step regenerates the compound used in the first stage.
The cytric acid cycle is an eight step cycle that comprises a series of oxidation-reduction (redox), dehydration, hydration and decarboxylation reactions that produce: two CO₂ molecules, one GTP^[6]/ATP molecule and the reduced forms of three NADH molecules and one FADH₂^[7] molecule.

This cycle is considered an aerobic route because these two last compounds must transfer their electrons to the next pathway of the system, which will use oxygen and will generate ATP.

Some of the intermediate compounds in this cycle can be used to synthesise non-essential amino acids, lipids and sugars that can be energy sources for the metabolic pathways of glucose. Hence, the Krebs cycle is an amphibolic cycle (both anabolic and catabolic).

[1] AMP is one of the nucleotides used in RNA.
[2] Adenosine diphosphate: organic compound made up of adenosine and two phosphate groups.
[3] Addition of a phosphate group to a compound, usually a metabolic intermediate, a protein or ADP.
[4] Nicotinamida adenina dinucleótido: coenzima encontrada en las células vivas, cuya función principal es el intercambio de electrones y protones en las reacciones de producción de energía.
[5] Reduced form of the NAD molecule that therefore, it accepts electrons.
[6] Guanosine triphosphate is another nucleotide required for RNA synthesis and involved in cellular metabolism. Its nitrogenous base is purine guanine.
[7] Reduced form of FAD (flavin adenine dinucleotide: coenzyme that intervenes in metabolic reactions of oxidation-reduction) that accepts two hydrogen atoms.

Fuentes: OpenStax College, Biology. OpenStax College. 30 May 2013.
http://table4eversquishycells.pbworks.com/w/page/9947625/How%20Cells%20Get%20Energy
http://bio100.class.uic.edu/lectures/atp_energy.jpg
https://apbionotebook.wordpress.com/chapter-09-cellular-respiration-fermentation/
http://msdoranbiology.weebly.com/notes---cp.html

1.4.3. Clinical trials and drug approval in the US

noreply@blogger.com (David Funes) — Fri, 27 May 2016 15:32:00 +0000

In the post titled: ‘Safety and efficacy of drugs’, we saw how the regulatory agency in the USA, the FDA (Food and Drug Administration), started the drug review process by submiting an Investigational New Drug (IND) application to request the start of clinical trials in humans.

Later, there is a review at two levels:

▣ A review at national level, where the FDA inspects all the pre-clinic work performed in the laboratory and the data set that led to the submission of that IND.

▣ A second review at local level (cities, towns, health systems) that is carried out by local entities named Institutional Review Boards (IRBs) regulated by the FDA. Their functions are to evaluate the number of patients that take part in the study, the study safety and its design.

After these assessments, if it is concluded that the benefits outweigh the risks the FDA and IRBs approve human trials.

Clinical trials in humans are divided into three different phases:

▣ Phase I: they are small tests that involve from 10 to 20 healthy human volunteers, who do not have the disease of interest.
In this phase, the drug is, for the first time, administered to human subjects.
The objective of these studies is to find the tolerability and safety of the new product, as well as its pharmacokinetics[1].

▣ Phase II: once the phase I has been completed without issue, the researchers proceed to the phase II where they determine, by comparison, if the new drug works better than an inactive product (placebo) or, at least, as well as another current drug.
To achieve this, half of the patients of the study are administered with the new drug and the other half with the inactive product or the drug already on the market.
Patients receive a wide range of doses of the drug with the purpose of finding out which one provides fewer side effects along with the best efficacy.
This phase involves between 100 and 500 test subjects.

▣ Phase III: if everything goes according to plan, we reach phase III where the new drug is given to thousands of patients. Once again, it is compared to a placebo or a medicine that already works to discover whether the new product is safe and efficient, this time at a larger scale.
In this phase, the tests occur in many centres not only in the US but also all over the world.
Another characteristic feature of phase III is that the drug is administered by healthcare providers or practising clinicians, which gives the FDA new data about the safety and effectiveness of the new medicine in the real environment of health care.

In phases I, II, III and even subsequently, the safety of the new compound is always evaluated and is the main concern in these studies.

Biopharmaceutical companies do not conduct these clinical research studies alone, but working very closely with the FDA, in such a way that the FDA can halt a study at any moment, even before beginning, when it suspects that the patient safety could be at risk.
Equally, the FDA can finish a trial ahead of schedule when the drug is so effective that expectations are exceeded, so that the new medicine starts to be commercialised for those people who need it.

At the end of these clinical trials, a new application, named a New Drug Application (NDA), is drawn up and submitted to the FDA where it is examined by several members with different backgrounds: scientists, statisticians, clinicians… in order to give recommendations (as long as the drug fulfils the safety and effectiveness criteria).

The FDA also examines the facilities where the new drug will be manufactured in a standardised and robust way and confirms that they adhere to good manufacturing practices (GMP).

The FDA also reviews the set of information, called product or package labelling, distributed with the product, which helps patients and health providers to determine if a specific medicine is the most appropriate to treat a certain illness.
This leaflet includes information about the clinical testing done, the most suitable dosage, warnings about its safety (if any) and side effects.

Lastly, the FDA decides whether the new medication is approved or not. If the result is negative, it can ask for further studies from the manufacturer or deny its use in the US permanently in the event that there is enough evidence about the drug’s ineffectiveness or doubts about its safety.

[1] Measurement of drug levels in the organism and how the drug is excreted from it.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
  http://www.ice-epilepsy.org/us-drug-approval-process.html
  http://mscreations.org/clinical-trial-phases/
  https://www.ciscrp.org/education-center/charts-and-statistics/during-participation/

4.2. Cellular metabolism

noreply@blogger.com (David Funes) — Thu, 19 May 2016 17:13:00 +0000

Practically all tasks performed by living organisms require energy. Energy is not only needed to perform exercise or demanding tasks, but also when we think, and even when we sleep; its use is, therefore, constant.

Energy is used so that immune system cells can absorb, ingest and break down viruses and pathogenic bacteria. It is also fundamental to eliminate toxins and waste, to transport neurotransmitters and hormones and in the synthesis and chemical breakdown of molecules.

Nutrients and other molecules are imported, metabolised (broken down) and synthesised in other molecules, modified in some cases, transported to other cells and probably distributed throughout the body by using energy.

All cellular processes, such as the building and degradation of complex molecules occur through a series of staged chemical reactions.
These chemical reactions within cells, including those that release and use energy, is called cell metabolism.

Metabolic pathways

A metabolic pathway is a series of interconnected chemical reactions that transform one or several substrate molecules gradually, via a group of metabolic intermediates, into a final product or products.

In the case of sugar metabolism, glucose (a simple sugar) is synthesised from smaller molecules:

6CO₂ + 6H₂ O + energy → C₆ H₁₂ O₆ + 6O₂

While in another different metabolic pathway, glucose is broken down into smaller molecules:

C₆H₁₂O₆ + 6O₂ → 6CO₂ + 6H₂ O + energy

Hence, metabolism consists of building or anabolism (first reaction) and breakdown or catabolism (second reaction).

These metabolic pathways do not ocurr spontaneously, but each reaction is enabled or catalysed by specific proteins called enzymes, which catalyse both the reactions that release energy and the reactions that require its use.

Potential, free and activation energy

When the bonds in molecules are broken down, they release energy. This is known as potential energy. To carry out their function, cells depend on the extraction of that potential energy.

Free energy is a concept that measures the available energy to perform a task. This free energy is modified during chemical reactions (where energy transfers occur) and this change is denominated as Gibbs free energy (ΔG).

The value of ΔG can be negative (whereby energy is released). These reactions are called exergonic or spontaneous. On the contrary, if the value of ΔG is positive then the reaction consumes energy. These reactions are called endergonic or non-spontaneous.

If a reaction is spontaneous, this indicates that its products have less energy than its reactives, and vice versa for non-spontaneous reactions.
Regardless of the sort of reaction, all of them however require an initial contribution of energy to reach the transition state^[1]. This contribution is known as activation energy.

ATP: adenosine triphosphate

ATP is the molecule that provides energy to living cells. It is composed of a nucleotide, a pentose (a five carbon atom monosaccharide) and three phosphate groups. The bonds of these three phosphate groups have a high energy content that when are broken, promote a series of reactions and cell processes.
The energy that is released in the ATP hydrolysis reaction:

ATP +H₂O → ADP + Pi^[2] + energía libre (reacción reversible)

Cells use ATP by coupling the exergonic reaction of ATP hydrolysis with endergonic reactions. ATP gives its phosphate group to another molecule during a process known as phosphorylation. This phosphorylated molecule is less stable than its unphosphorylated form, but the added energy after the addition of the phosphate group enables it to perform the necessary endergonic reactions so that the cell can carry out its different functions.

Enzymes

Enzymes (usually one or more polypeptide chain proteins) speed up chemical reactions by diminishing their activation energy requirement.
Enzymes bind to substrates^[3] to catalyse reactions in four different ways:

▣ Taking part directly in chemical reactions by forming transient covalent bonds with substrates.

▣ Uniting substrates in an optimal orientation.

▣ Providing optimal conditions so that the reaction can take place.

▣ Acting on bond structures so that they can be broken more easily.

Enzymes are regulated by cell conditions like pH, temperature and their location within the cell (some of them are compartmentalised).
But the most common method by which cells regulate enzymes in metabolic pathways is via feedback inhibition. In this way the products of a metabolic pathway act as inhibitors (normally allosteric^[4]) of one or more enzymes involved in the pathway where these products are originated.

[1] Very high energy and short duration state that is produced when the reaction reactives approach and experiment a deformation.
[2] Inorganic phosphate group.
[3] Specific chemical reactives on which a specific enzyme performs.
[4] They decrease the enzyme activity by causing structural changes on them, enzyme receptors change and adopt their inactive conformation.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
  http://cienciasdejoseleg.blogspot.com.es/2013/03/plicacion-de-las-variables.html
  http://teenbiotechchallenge.ucdavis.edu/2010_TBC/Peter%20Wang,%20Clara%20Fannjiang,%20William%20Liu/Chemistry.html
  https://mollycools.wordpress.com/2014/03/10/enzymes/
https://commons.wikimedia.org/wiki/File:Allosteric_competitive_inhibition_3.svg

4.1.3. Plasma membrane. Cellular transport

noreply@blogger.com (David Funes) — Mon, 09 May 2016 16:24:00 +0000

Among the different functions of the plasma or cell membrane, the most basic is to establish the cell’s limits and keep it functional.
The membrane is selectively permeable. Consequently some elements can enter and exit the cell freely, whereas others need a specialised structure or even energy to accomplish this.

The most significant transport routes that the cell uses through its plasma membrane are:

Passive transport

Passive transport is a natural phenomenon where the cell does not need to use any sort of energy to move substances across the plasma membrane.

In passive transport, the substances pass from a high concentration region to a low concentration region.
The various mechanisms of passive transport are:

▣ Diffusion: a single substance moves from a high concentration area to a lower one until the concentration is equalised on both sides.
A variation of diffusion is the filtration process where materials move through the membrane according to their concentration gradient^[1]. At times, the diffusion rate is increased by pressure causing materials to be filtered much more quickly. This is the process that takes place in kidneys where the blood pressure forces big amounts of water and substances dissolved in it (solutes) from the bloodstream to the renal tubules.

▣ Facilitated transport: diffusion is carried out by the membrane with the help of its embedded proteins, which act like shields for these substances (polar molecules, ions) against the membrane’s repellent forces, facilitating their diffusion inside the cell.

▣ Osmosis: is a special case of diffusion in which water, not material, is carried across the membrane. Therefore, the membrane limits the diffusion of solutes in water. This process is mediated by a set of specific proteins called aquaporins.

Depending on the relationship between the cell osmolarity^[2] and the osmolarity of the surrounding extracellular fluid, we can identify three solution states:

▣ Hypotonic solutions: the osmolarity of the extracellular fluid is lower than the osmolarity of the fluid inside the cell, therefore water penetrates the cell.

▣ Hypertonic solutions: the osmolarity of the extracellular fluid is higher than the cytoplasm osmolarity, hence water leaves the cell.

▣ Isotonic solutions: both the cell and the extracellular fluid have the same osmolarity. As a consequence, there is a constant exchange of water between both regions.

Active transport

Active transport, also known as pumps, requires the use of energy in the form of adenosine triphosphate (ATP). This energetic requirement is necessary because the substance is moving against a concentration gradient (i.e., the substance concentration in the cell is higher than in the extracellular fluid or vice versa).
By this mechanism a wide range of different sized molecules can be carried.

In living organisms, there are not only simple concentration gradients but also electrical gradients that indicate a charge difference across the cell membrane.

The cell interior is negative electrically in comparison to the extracellular fluid, which has a higher concentration of K+ (potassium ions) and a lower concentration of Na+ (sodium ions) than the extracellular fluid.
Consequently, the electrical and concentration gradients of Na+ tend to lead these ions into the cell.
The K+ ion situation is more complex; while its electrical gradient also tends to drive these ions towards the interior of the cell, its concentration gradient leads out of the cell.

These combined gradients of electrical charge and concentration that affect an ion are named electrochemical gradient.

A notable adaptation of the cell membrane for active transport is the presence of specific transport proteins (pumps) that enable the movement of materials on both sides of the membrane. These proteins or transporters are classified into three types:

- Uniporters: they carry a specific molecule or ion in just one direction.
- Symporters: they transport two different ions or molecules in the same direction.
- Antiporters: they also transport two different molecules or ions but in opposite directions.

Primary active transport

This type of transport works with the active transport of K and Na and enables secondary active transport to occur at later stage.

One of the most important mechanisms of active transport in animal cells is the Na+ -K+ pump that maintains the electrochemical gradient and the appropiate concentrations of Na+ and K+ in cells.
During this process for every three Na+ ions that are expelled from the cell, two K+ ions penetrate into the cell, therefore the interior of the cell is slightly more negative than its exterior. This charge difference is vital to create the necessary conditions for the second process.

The Na-K pump belongs to the electrogenic pump category, which is a kind of ionic pump that generates a potential electrical charge difference on both sides of the cell membrane.

Secondary active transport (co-transport)

Via this process Na+ ions and other compounds penetrate into the cell taking advantage of the electrochemical gradient created in the previous step.
In this class of active transport a solute that moves against the concentration gradient is co-transported with other solutes that move in favour of its concentration gradient.
Thanks to this method, many diverse molecules, like amino acids or glucose molecules, can enter the cell.
This process is also used to store hydrogen ions of high energy in mitochondria to produce ATP.

Bulk transport

In addition to small ions and molecules, cells must also assimilate and eliminate larger size molecules and particles and in order to achieve it they need a supply of energy. However, these molecules are so large that not even with the supply of energy can they pass through the plasma membrane.
Hence, the cell uses the following mechanisms:

Endocytosis

Endocytosis is a type of active transport where particles, such as large molecules, parts of cells and even entire cells are enclosed by invagination of the membrane forming a vesicle whose content is carried from the outside to the inside of the cytoplasm.

Listed are the various endocytosis:

▣ Phagocytosis: is the process in which cells, or large parts are ‘consumed’ by a cell. This is the method used by a particular type of white cells called neutrophils. These immune system cells surround, cover and destroy the microrganisms that invade our body.

▣ Pinocytosis: the cell captures the molecules that are needed from the extracellular fluid. The cell membrane invaginates that unwelcome cell, creating a vesicle around the liquid of the external fluid, which is ‘consumed’ and released in the cytoplasm.
For example, the maturation egg process in the uterus is carried out by pynocytosis when the ovule takes in the nutrients that its supply cells release.

▣ Receptor-mediated endocytosis: uses receptor proteins from the cell membrane that have a specific affinity to binding to particular substances.

Some human diseases are caused by the malfunction of this mechanism. It is the case of familial hypercholesterolemia, in which the receptors of bad cholesterol are faulty or completely absent.

Exocytosis

Exocytosis is the opposite to endocytosis in which the cell expels material from its interior to outside.
This waste material is covered with a membranous sac that fuses to the plasma membrane and then opens to release its content into the extracellular space.
The neurotransmitter secretion by vesicles in the synaptic gap of neurons follows this process.

[1] Different concentration of molecules between two regions.
[2] It describes the overall concentration of solute in a solution.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
  http://www.educ.ar/sitios/educar/recursos/ver?id=14378
  http://sevendaysperweek.blogspot.com.es/2015/09/spm-biology-3-movement-of-substances_20.html
  http://cbc.arizona.edu/classes/bioc462/462a/NOTES/LIPIDS/transport.html
Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000
  http://es.slideshare.net/exitoinevitable/clulas-31270712
  https://micro.magnet.fsu.edu/cells/endosomes/endosomes.html

1.4.2. Good Clinical Practice (GCP)

noreply@blogger.com (David Funes) — Sat, 30 Apr 2016 11:04:00 +0000

According to the European regulations, all clinical trials must be performed in accordance with the rules of good clinical practice (GCP) that constitute a guarantee for patient protection and for test results.

That GCP guidelines of the International Conference on Harmonisation (ICH)^[1] have been followed since 1996 in Europe; they have also been adopted by the USA and Japan. They are a set of scientific and ethical quality requirements recognised internationally that must be fulfilled by the planning, execution, record and communication of clinical trials where human beings take part.
These rules must guarantee the rights, safety and well-being of trial subjects and the reliability of the results.

GCP arises from the need to reconciling the subject’s interests (who receives the treatment) and society’s interests in increasing its knowledge about drugs.

The main actors that participate in a clinical trial are the following:

▣ Promoter: legal entity or person interested in performing a clinical test and responsible for the same. It is usually a pharmaceutical multinational.

▣ Ethical committees: they belong to the centre where the research takes place.

▣ Regulatory agencies: they are usually QUANGOs with government oversight.
These two last participants must approve the protocol and supervise the experimental development. In addition, agencies evaluate the research results to authorise subsequent drug commercialisation.

▣ Researchers: carry out or manage the test research. They are normally physicians or health professionals that examine the compound response that is subject of study.

▣ Monitor: responsible for the direct follow-up of the clinical trial execution. Monitor is the intermediary between promoter and researcher and must guarantee the tracking of everything that occurs throughout the experiment.

▣ Patient: subjects who take part in the research and generally suffer from the disease of interest.

On occasion, some companies called CROs (contract research organisations) provide the trial management and monitoring.

After the II World War, the Nuremberg Code was promulgated to establish clinical research principles in human beings, which are as follows:

▣ Autonomy: respect the principle for subjects that they are treated as autonomous people to decide freely if they want to take part in the clinical test.
The informed consent^[2] signed by the patient is part of the fulfilment of this principle.

▣ Well-being: the physician is obligated to do good and to attend to the welfare of the patients, as well as their highest benefit.

▣ Non-malefience: to avoid harmful clinical tests for patients.

▣ Justice: obtained positive results must be shared with the people who suffer from the malady which is the focus of the trial (whether knowledge or new medication). This principle would not be fulfilled, for example, if a country from the third world, where the trial takes place, would not benefit from the treatment because of its high cost.

Phase IV clinical trials

Once a drug has been introduced to the market, studies continue to increase the knowledge about its safety and effectiveness, identify new risks and non-detected adverse response in previous research.
The group of controlled and randomised trials and studies that are performed at this given point is called phase IV of clinical trials.

The most common epidemiological (observational) studies after the drug authorisation are case-control and cohort studies.

Finally, the Yellow Card Scheme is the system for collecting suspected adverse reactions to drugs. This system is used especially when a medicine has just been marketed and it is mandatory for physicians (and current patients can also fill it out).

These cards consist of several sections among which we find the following:

▣ Patient details: first name or initials, age, sex, weight, height…

▣ Data about the suspected medicine causing adverse reactions.

▣ Description of those reactions.

▣ Reporter/clinician details: name, professional address, specialty…

[1] It matches the different regional requirements for the register of pharmaceutical products.
[2] The subject who participates in the clinical trial is informed by this document about the details, advantages and disadvantages of the test with an accessible language, being able to leave it freely at any time.

Sources: CEU Universidad San Pablo: Farmacología Básica, 2013.
https://www.gov.uk/guidance/the-yellow-card-scheme-guidance-for-healthcare-professionals
https://yellowcard.mhra.gov.uk/the-yellow-card-scheme/

http://www.ich.org/home.html

4.1.2. The endomembrane system. The cytoskeleton

noreply@blogger.com (David Funes) — Sun, 17 Apr 2016 16:13:00 +0000

The endomembrane system is a group of organelles and membranes that work together to modify, pack and transport lipids and proteins. This system includes the nuclear envelope, lysosomes and vesicles that we have already mentioned in the previous post: “Prokaryotic and eukaryotic cells. Main components” , the Golgi apparatus and the endoplasmic reticulum.

The endoplasmic reticulum (ER)

The endoplasmic reticulum is made up of a set of membranous sacs and interconnected tubules that function colectively to modify proteins and synthesise lipids. These two functions are carried out in two different areas: the rough endoplasmic reticulum (RER) and the smooth endoplasmic reticulum (SER).

Rough endoplasmic reticulum (RER)

The rough endoplasmic reticulum receives this name because of the large number of ribosomes that are stuck to its cytoplasmic surface.
The ribosomes transfer the proteins that they synthesise to the lumen of the RER where they undergo modifications and then they are incorporated into the plasma membrane or they are secreted outside the cell through vesicles that bud from the RER membrane.
Enzymes, hormones and even some phospholipids generated by the RER undergo these processes to become part of the cell membranes.
The rough endoplasmic reticulum is mainly found in cells that secrete a large amount of proteins, like hepatic cells.

Smooth endoplasmic reticulum (SER)

The smooth endoplasmic reticulum is next to the RER but, unlike this one, it has few or no ribosomes stuck to its surface.
Among its functions we can find the synthesis of lipids, proteins, steroid hormones, detoxification of poisons and drugs and the storage of calcium ions.
In the muscle cells, the smooth endoplasmic reticulum is called the sarcoplasmic reticulum (SR) and the SR is in charge of accumulating the necessary calcium ions for muscle contraction.

The Golgi apparatus

Before reaching their destination, the proteins and lipids that are carried by the vesicles of the ER must be tagged, classified, packed and distributed. The Golgi apparatus, which consists of a series of flattened membranes, performs these functions.
The receiving side of the Golgi apparatus is called the cis face, whereas the opposite side is called the trans face.

As lipids and proteins travel through the Golgi apparatus they undergo a set of modifications that enable classification; the most common modification is the addition of short chains of sugar molecules.

Afterwards, they are tagged with phosphate groups or other small molecules so that they can be delivered to their appropiate destinations.
Finally, they are packed into secretory vesicles that emerge from the trans face of the Golgi apparatus. Some of these lipids and proteins are placed in other parts of the cell, whereas others are fuse with the plasma membrane and they release their content outside the cell.

Immune system cells that secrete antibodies are characterised by having a large amount of Golgi.

Lysosomes

In addition to their digestive role and recycling of organelles, lysosomes are an important component of the endomembrane system too.
Lysosomes use their hydrolytic enzymes to destroy those pathogens that penetrate into the cell.
Lysosomes are used by a special type of white cell called macrophage. These cells via phagocytosis or endocytosis invaginate (fold) their plasma membranes to surround and enclose the pathogen. Later, these pathogens are destroyed under the action of the hydrolitic enzymes of lysosomes.

THE CYTOSKELETON

The cytoskeleton is the group of protein fibres that maintain the cell shape, attach the organelles in their appropiate positions, enable the vesicles to move in the cell and allow the cells of multicellular organisms to move.

Microfilaments

Of the three kinds of protein fibres of the cytoskeleton, microfilaments are the narrowest with a diameter around 7 nm.
They are two intertwined fibres of the actin protein, consequently they are also known as actin filaments.
They participate in processes that require movement such as the cell division in animal cells. They provide rigidity and shape to the cell.

Intermediate filaments

They purely have a structural function; they bear strain and fix the nucleus and other organelles in the necessary locations.
Their diameter is between 8 and 10 nm and they are made of several twisted protein fibres.
Within this category, keratin filaments are the best known; they strengthen nails, skin epidermis and hair.

Microtubules

Among the most important roles performed by microtubules, we find the movement of the replicated chromosomes to opposite ends of the cell during the cell division. They also provide a path for vesicles to move inside the cell and help the cell to maintain compression.
They are the widest components of the cytoskeleton with a diameter of around 25 nm.

Flagella and cilia

Flagella are moving appendages that extend from the cell membrane making possible the movement of those cells that have them (e.g., sperm).

On the contrary, cilia are short and are found widespread along the surface of the plasma membrane.
Like flagella, they enable the movement of cells (paramecia) or substances over the cell surface, like for example the cilia of the Fallopian tubes that move the ovule towards the uterus.

CELL CONNECTIONS

As you can guess, if cells have to work together they must communicate with each other. Let’s see what methods they use to achieve it.

Extracellular matrix of animal cells

The major role of the extracellular matrix is to hold cells together to form a tissue and to allow cell communication within that tissue.
The extracellular matrix is primarily made up of a sort of protein called collagen, intertwined with other types of proteins that contain carbohydrates (proteoglycans).

Overall, the cell communication is performed as follows:
Cells have quite a few receptors on the surface of their cell membranes.
When a molecule within the matrix joins the receptor, it modifies the molecular structure of the receptor. The receptor, in turn, changes the arrangement of the microfilaments located within the membrane. These changes produce chemical signals that reach the nucleus and they activate and deactivate the transcription of specific sections of DNA, which influences the creation of the related proteins.

Cells can also communicate through direct contact via intercellular joints, which are formed by diverse kinds of proteins. In animal cells, there are three categories of these joints:

▣ Tight junctions: they seal the plasma membranes of adjacent cells creating a waterproof barrier between them. Their main components are occludin and claudin proteins. These tight junctions are found, for instance, linking the epithelial cells of the urinary bladder.

▣ Desmosomes: they work like spot welds between the epithelial cells of organs and tissues that undergo contraction such as the skin, the heart and muscle cells. Desmosomes are composed of short proteins named cadherins.

▣ Gap junctions: they act like pores and channels that enable the transport of ions and nutrients. They play a significant role in the cardiac muscle, where they allow the movement of the electrical signal that contracts this muscle.
They are made up of a group of six proteins called connexins, which are arranged in the cell membrane in a donut-like configuration known as connexon.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
  http://www.oncoursesystems.com/images/user/9341/10845583/rough%20er.bmp
  http://apocketmerlin.tumblr.com/post/14923100822/a-summary-of-the-functions-of-major-eukaryotic
  http://iesicaria.xtec.cat/~SBG/BiologiaCurtis/Seccion%201/1%20-%20Capitulo%205.htm
  https://ohhaitrish.wordpress.com/2012/02/12/unit-one-compilation/

4.1.1. Prokaryotic and eukaryotic cells. Main components

noreply@blogger.com (David Funes) — Sun, 10 Apr 2016 18:32:00 +0000

We have already studied the different biological macromolecules, which comprise the main components of cell structure. So, we are now in position to deal with a new topic within this section: cell biology.

In the same way that bricks are the basic building blocks of a wall, cells are the building blocks of living organisms.

There is a huge range of cells, each one specialised in a particular function: growth, development and daily maintenance of the organism, but all of them share some fundamental features.
Among the diverse types of cells we find, for example, immune system cells that protect us against bacterial infections, blood cells that carry oxygen and nutrients throughout our body or epithelial cells that protect the body surface and cover organs and body cavities.

Cells are considered the smallest unit of a living being. When several cells of the same class interconnect with each other sharing a common function they become a tissue. Several tissues form an organ and several organs make up a system (nervous, circulatory, digestive systems…). Lastly, the union of several systems working together and in concert comprises an organism like the human being.

Cells are classified into two major groups: prokaryots and eukaryots. But all of them have four basic components: the cell membrane that protects it from outside, the cytoplasm that is composed of the cytosol with a jelly consistency and floating on it other cell components, the DNA that contains the genetic information of the cell and the ribosomes that synthesise proteins..

In prokaryotic cells, the organism (most of the time unicellular) lacks a nucleus with what its DNA located in the cell centre, or ‘nucleoid’. Bacteria and archaea^[1] belong to this category.

On the contrary, protist^[2], fungi, plants and animals cells are eukaryotic cells, which differ from the prokaryotic cells by having a nucleus that encloses its genetic material (DNA) surrounded by a membrane, a large variety of organelles^[3] limited by membranes and several rod-shaped chromosomes.

Due to the purpose of this blog, I will only focus on the components and functions of eukaryotic cells and particularly on animal cells.

The plasma membrane

We start the study of the diverse cell components with the plasma membrane or cell membrane, which separates the interior content of the cell from the external environment.

It consists of a lipid bilayer with many embedded proteins in it, which controls the movement of water, ions, oxygen, organic molecules and waste disposal (carbon dioxide and ammonia) between the inside and outside of a cell.

Microvilli located on the wall of the small intestine are an example of plasma membranes specialised in the absorption task. They are in charge of absorbing the nutrients from the digested food.
In the case of celiacs, the consumption of gluten (a cereal protein) causes an autoimmune respond that attacks these cells causing malnutrition, abdominal pain and diarrhoea.

The cytoplasm

The cytoplasm is the region between the plasma membrane and the nuclear membrane.
It has a jelly-like and semisolid consistency called cytosol, on which the organelles are suspended.

Also the cytoskeleton and a number of biochemical substances, such as amino acids, nucleic and fatty acids, polysaccharides, simple sugars and sodium, potassium and calcium ions form part of the cytoplasm.
In the cytoplasm a large range of metabolic reactions, like the synthesis of proteins, take place.

The nucleus

The nucleus is the most significant organelle in the cell. It contains the DNA and leads the synthesis of proteins and ribosomes.

The nuclear envelope

The nuclear envelope is a double-membrane structure, in which both the inner and the outer membranes are formed by phospholipid bilayers (to revisit what a phospholipid bilayer see the post: “Lipids”).
This structure has a set of pores on its surface to facilitate the movement of ions, molecules and RNA between the nucleoplasm and the cytoplasm.
The nucleoplasm is a semisolid fluid ubicated inside the nuclear membrane where the chromatin and the nucleolus are.

Chromosomes and chromatin

In eukaryotic cells, chromosomes are structures inside the nucleus made up of DNA and therefore, the hereditary genetic material.
Each eukaryotic species has a particular number of chromosomes in its cell nuclei, which in the case of human beings is 46 (23 pairs).

Chromosomes are only distinguishable when the cell is about to initiate the cell division process. When the cell is in the other phases of the cell cycle, the chromosomes join certain proteins (histones) forming a complex known as chromatin, which looks like a tangled skein.
Chromatin characterises the substance that forms the chromosomes both in decondensed and condensed states.

The nucleolus

The nucleolus is the part of the nucleus that aggregates the ribosomal RNA (rRNA) with specific proteins to assemble the ribosomal subunits. These subunits are then transported to the cytoplasm where they are put together. This is the way in which the nucleus performs one of its principal functions, the synthesis of ribosomes.

Ribosomes

Ribosomes are the organelles responsible for protein synthesis. They are made up of two subunits: large and small subunits.
They can be found in groups, or individually next to the cytoplasmic side of the plasma membrane, the endoplasmic reticulum or stuck to the outer membrane of the nuclear membrane.

Ribosomes receive instructions from the nucleus to produce proteins by means of DNA, this DNA is transcripted to mRNA and this one gets to the ribosome. Once there, the code provided by the nitrogenous bases of the mRNA is translated in a set of amino acids with a specific order, giving rise to the required protein.

Ribosomes are abundant in those cells that synthesise large amounts of protein, like pancreatic cells, which produce multiple digestive enzymes.

Mitochondria

Mitochondria are usually known as the energetic centre of cells, they take charge the production of the most crucial energetic molecule for cells: ATP (adenosine triphosphate). This molecule is generated using glucose and other nutrients during cellular respiration. During this process, mitochondria use oxygen and produce carbon dioxide as waste, which is expelled from the organism when we exhale.

Mitochondria are oval-shaped with a double membrane (phospholipid bilayer with embedded proteins) and have their own ribosomes and DNA.
Their inner layer has a set of folds called mitochondrial cristae, surrounded by the mitochondrial matrix. Both develop different roles in the cellular respiration.

Peroxisomes

Peroxisomes are small, spherical organelles surrounded by a monolayer membrane.
They carry out oxidation reactions that break down amino acids and fatty acids, as well detoxifying poisons that enter our body.
For instance, the peroxisomes of hepatic cells must detoxify the alcohol we consume.

Vesicles and vacuoles

These organelles are sac-shaped and their functions are transport and storage. The main difference between them is their size (vacuoles are larger) and the fact that vesicle membranes can fuse with the plasma membrane or with membranes of other components inside the cell.

The centrosome

The centrosome is a microtubule-organising centre (MTOC) located near animal cell nuclei. Centrosomes are made up of two structures that are perpendicular to each other named centrioles. These centrioles are cylinders of nine triplets of microtubules each.

The centrosome replicates before starting cell division and the centrioles pull the duplicated chromosomes to the opposite poles of the cell during its division stage.

Lysosomes

Lysosomes are dumps of cells. Their pH is more acidic than cytoplasm´s, which favours the fact that their enzymes are more active and assist the degradation of nucleic acids, lipids, polysaccharides and even the recycling of organelles that are no longer necessary for the cell by enclosing and digesting them.

[1] Unicellular microorganisms similar to bacteria.
[2] Unicellular organisms that form colonies at times.
[3] Small organs specialised in cell functions, in a similar way to our body organs.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
  http://michelleburden.weebly.com/cell-types-prokaryotes-v-eukaryotes-plant-v-animal.html
  http://cosbiology.pbworks.com/w/page/11556247/Lesson%204-02%20Prokaryotes%20and%20Eukaryotes
  http://ernsstev.com/bilayer-pattern-in-cell-membrane/
  http://www.biologyexams4u.com/2012/11/difference-between-chromatin-and.html#.Vu1BANI7uSp
  http://jptregularbio.pbworks.com/w/page/79985510/Cells
  https://oggisioggino.wordpress.com/2013/11/01/las-celulas-procariotas-y-eucariotas/
  http://www.studyrankers.com/2015/06/cell-the-unit-of-life-class-11th-ncert-solutions.html

1.4.1. Clinical research: Overview

noreply@blogger.com (David Funes) — Thu, 31 Mar 2016 15:09:00 +0000

Those compounds that have passed the preclinical phase are tested in human beings (clinical research).
This kind of research has significant associated issues of a scientific, ethical, social and economic nature.

In all developed societies it is mandatory to prove the efficacy and safety of drugs before commercialising them, a task that is fulfilled by clinical trials.

Clinical trials are defined as research studies in human beings to determine or confirm clinical, pharmacological, pharmacodynamic effects and/or detect adverse reactions and/or study the absorption, distribution, metabolism and excretion of a drug in order to establish its safety and efficacy.

Clinical tests are based on documentation, both national and international regulation and a series of ethical codes, declarations and international conventions.

Over the last fifty years hundreds of thousands of compounds have been produced, but only 10% of them have passed clinical evaluation, and even within this group some of them were withdrawn after being put on the market due to safety and efficacy reevaluations.

The methodology of clinical research can be carried out via one of the following procedures:

▣ Experimental: In experimental tests, researchers design the study, select the patients, establish the treatments and variables to study.

▣ Observational: Researchers are mere observers, without altering or intervening the habitual clinical practice.

Within the experimental tests, we find:

▣ Randomised trials. In these trials, patients are allocated to one treatment or another (often a placebo) in a random way. This is the procedure demanded by regulatory agencies to avoid bias in data interpretation before giving the authorisation to market the drug.

▣ Non-randomised trials. Here, patients are not allocated randomly.

Among the observational studies to develop the complete efficacy and safety profile of a drug after its commercialisation, we have:

▣ Case-control studies. Participants are chosen according to they have or not a certain disease and it is investigated if they were exposed or not to a factor of interest. The exposed group and the control group are compared.

▣ Cohort studies. The frequency of a sickness is compared between two populations, one exposed to a risk factor and the other population are not.

▣ Cross-sectional studies. They examine the relationship between an illness and a set of variables in a certain population at a given point in time.

Clinical trials chronologically go through three phases until achieving marketing authorisation for the drug. Once the drug is commercialised, it begins its phase IV, which lasts the whole lifespan of the drug. In Phase IV the drug can be removed from the market if undesired effects, not detected in the three previous stages, are identified.
These new side effects can be discovered when the drug is distributed among thousands of patients.

The measured efficacy in these real conditions is called effectiveness to distinguish it from the efficacy measured prior to drug distribution on the market.

Sources: CEU Universidad San Pablo: Farmacología Básica, 2013.
http://cmidd.northwestern.edu/about/drug-discovery-and-development-process/
http://indacea.org/desarrollo-de-medicamentos-1/

3.5. Nucleic acids

noreply@blogger.com (David Funes) — Wed, 23 Mar 2016 17:42:00 +0000

DNA and RNA are the most important biological macromolecules for the continuity of life.
DNA molecules contain the genetic material of all living organisms, from bacteria to multicellular mammals.
In prokaryotic cells DNA is not enclosed inside a membranous envelope, but is in eukaryotic cells where it is located inside the nucleus and organelles.

In eukaryotic cells, DNA along with some proteins, called histones, form chromatin (the material from which eukaryotic chromosomes are made), which can contain tens of thousands of genes.
DNA is in charge of controlling the activity of cells by activating and deactivating genes.

The other kind of nucleic acid, RNA, primarily conducts protein synthesis and its regulation through the action of three different sorts of RNA (rRNA or ribosomal RNA, tRNA or transfer RNA and miRNA or microRNA). The fourth type of RNA (mRNA or messenger RNA) acts as communication intermediary between DNA and the rest of the cell.

Nucleotides are nucleic acid monomers, which are combined to generate their polynucleotides (DNA and RNA). Each nucleotide is composed of three components: one nitrogenous base attached to a pentose sugar molecule, which, in turn, is attached to a phosphate group.

Nitrogenous bases are organic molecules made up of carbon (C) and nitrogen (N) that are divided into five kinds: adenine (A) and guanine (G) are known as purines, they have two fused rings of C-N. Cytosine (C), thymine (T) and uracil (U) are known as pyrimidines, they only have a fused ring of C-N.
Both purines and pyrimidines have different functional groups attached to these rings.

The DNA molecule comprises A, T, G and C, while the RNA molecule comprises A, U, G and C, therefore, tymine is substituted by uracil.

Another difference between DNA and RNA is that DNA sugar molecule is a deoxyribose (they have a hydrogen atom attached to the second carbon atom of the molecule) whereas RNA sugar molecule is a ribose (they have a hydroxyl group attached to that second carbon atom instead).

Carbon atoms of the sugar molecule are numbered as 1´, 2´, 3´, 4´ and 5´. The phosphate residue joins the hydroxyl group of the 5´carbon and the 3´ carbon of the sugar molecule of the next nucleotide forming a phosphodiester linkage between them.

DNA double-helix structure

In the DNA double-helix structure, the sugar molecule and phosphate group are located outside this structure, whereas the nitrogenous bases lie in the interior like the rungs of a ladder linked by hydrogen bonds, in such a way that A can only pair with T and G can only pair with C. This rule is known as the rule of complementary base pairing.
Each of these pairs is separated from the next one by 0.34 nm; both strands of the double-helix run in opposite directions in such a way that the 5' carbon of one strand faces the 3' carbon of the complementary strand.

During DNA replication, each of these strands are separated and they are used to synthesise a new complementary strand, in such a way that each daughter DNA double helix contains a new synthesised strand and one parental DNA strand.

RNA

Normally, RNA comprises a single ribonucleotide strand held together by phosphodiester bonds. These ribonucleotides contain, in turn, a phosphate group, a ribose molecule (pentose sugar) and one of the four nitrogenous bases (A, U, C and G).

The DNA that controls the activity of the cell uses the mRNA (messenger RNA) to carry its message
Thus, if a cell requires a certain protein, the gene for this product is activated and the nucleus synthesises the mRNA. This mRNA has a complementary sequence to the coding sequence of the DNA has been copied from.
The mRNA crosses the nuclear membrane, gets to the cytoplasm and there it interacts with the ribosomes and other cellular machinery.
To produce the required protein, the mRNA is read in groups of three bases called codons. Each codon codifies a single amino acid.

The second kind of RNA, rRNA, is responsible for the suitable alignment between the mRNA and ribosomes. It also has an enzymatic activity and catalyses the formation of peptide bonds between two aligned amino acids.

The tRNA is in charge of transporting the correct amino acid to the site where the protein synthesis takes place. The base pairing between the tRNA and the mRNA allows for the appropriate amino acid to be inserted in the polypeptide chain.

Lastly, the major function of the microRNA (the smallest of all types of RNA) is the regulation of gene expression by interfering with the expression of certain messages of the mRNA.

Hence, in an organism the information goes from DNA to RNA and from this one to proteins. During the first stage (transcription), the DNA dictates the structure of mRNA and during the second (translation), RNA rules the structure of a specific protein.
This process is known as the central dogma of molecular biology, which is valid for all living organisms except for some viral infections.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
  https://www.windowssearch-exp.com/images/search?q=Antiparallel+DNA+Replication&view=detailv2&&id=CE5B36CC30
3DB6E9F7C902454DF516328A0882B8&selectedIndex=1&ccid=uEIdkd5u&simid=608029733518446573&thid=OIP.Mb84
21d91de6e5ee9db88b338c5b77e9aH0&ajaxhist=0
  http://www.scienceprofonline.com/genetics/ribonucleic-acid-rna-structure-and-function.html
  http://guia.bio.br/tag/purinas/
  http://www.discoveryandinnovation.com/BIOL202/notes/lecture12.html
  http://www.ib.bioninja.com.au/standard-level/topic-3-chemicals-of-life/35-transcription-and-transl.html
  http://thelessonlocker.com/kvhs/biology/biology.html

2.2. Atomic force microscope (AFM)

noreply@blogger.com (David Funes) — Tue, 08 Mar 2016 16:16:00 +0000

In the post titled “Top-down and bottom-up approach” I mentioned that, in order to examine and handle objects at the nanoscale it is necessary to develop devices that enable observation of samples at the atomic scale. These instruments are called scanning probe microscopes (SPMs), from which 3D images are obtained at a very high resolution without damaging the analysed sample (an improvement on electron microscopes (TEMs)).

The scanning probe microscopes are divided, basically, into two types: scanning tunneling microscope (STMs), which we studied in the previous entry within this section, and the atomic force microscopes (AFMs), which we address below.

Atomic force microscopes are based on the following operating principle:
The sample topography is scanned by a microcantilever or strip around a few hundreds of microns (micrometres) long, built in silicon or silicon nitride, whose end has an extremely sharp tip and a curvature radius on the order of nanometers.

When this tip approximates the sample surface, the forces between them produce the cantilever deflection according to Hooke’s law^[1].
Depending on the type of tip-sample interaction (whether there is contact or not), the forces measured by AFMs can be: electrostatic, magnetic, capillary forces or Van der Waals interactions...
The deflection that the cantilever experiences is measured by a laser beam focused on the upper surface of this cantilever and reflected in an array of photodiodes that register the laser spot shift.

In contrast with STMs, AFMs can be used in the study of non-conductive samples, and they can perform two types of measurement depending on the application: image (which, in turn, it is divided into contact mode and dynamic mode) and force.

Contact mode

In this operating mode, the bending of the cantilever is kept constant during the sample scan, hence this cantilever is correspondingly moved up and down to keep invariable the pressure force of the tip on the sample. This up and down movement is registered and a specimen image is obtained.
A relatively fast scan speed and the opportunity to scan rough surfaces are its main advantages. Its principal drawback is that it may damage biological samples (soft and delicate), therefore these samples must be strongly adhered to the surface.

Dynamic mode

In the dynamic mode, the microcantilever tip does not come into contact with the sample surface. Instead the strip oscillates at its resonant frequency or just above by a piezoelectric actuator^[2] that determines the tip height over the sample.

The tip-sample interaction modifies the resonant frequency and amplitude. Depending on which of these two properties keeps constant during the surface scan, we have:

▣ Non-contact mode where the resonant frequency is stable. This mode is specially indicated for the study of soft biological samples and thin organic films. Because there is no contact, the samples can be subjected to countless analyses without being damaged.
Among its disadvantages, we find its low image resolution and samples must be examined in ultra-high vacuum environments, to avoid the presence of pollutants on the specimen surface that could interfere with imaging.

▣ Tapping mode where the resonant amplitude does not vary. This method provides high-resolution images of those samples that are susceptible to be easily damaged or are weakly adhered to the analysis surface. The major disadvantage is that the scanning has to be done at a slower speed.

Force mode

In the force operating mode, the tip oscillates vertically while the cantilever deflection is registered.
This method has as its main applications the examination of specific interactions between molecules (antigen-antibody, the complementary strands of DNA), structural interactions (protein folding) or the study of polymer elasticity.

[1] This law states that the lenghtening of materials is directly proportional to the applied force on the same.
[2] Mechanical devices whose function is provide force to move another mechanical device.

Sources: http://www.dme-spm.com/funktion.html
  https://es.wikipedia.org/wiki/Microscopio_de_fuerza_at%C3%B3mica
  http://www.phy.mtu.edu/nue/images/atomicforce/Nanoscope2.jpg
http://slideplayer.com/slide/6856881/
  http://www.biozentrum.unibas.ch/research/groups-platforms/overview/unit/lim/

1.3.6. Repurposing drugs

noreply@blogger.com (David Funes) — Mon, 29 Feb 2016 17:02:00 +0000

So far, we have studied the basics concerning the drug discovery process.
We have seen how these discoveries were made, at the beginning, by serendipity to give way later to the methodogical method: the target-based approach.
We still do not know how the new drugs will be discovered in the future, but biologics, genetics and phenotypic^[1] screening in drug discovery will increasingly play a critical role.

There is another area of increasing interest within the drug development pipeline that, although it does not belong to the discovery process, should be remarked upon. It is usually named drug repurposing, and it is based on giving a known compound a new use.
This practice consists of choosing a compound from the catalogue of shelved drugs that have failed in their clinical trials, or even have been commercialised, for the purpose of treating a different disease.

The main advantage that pharmaceutical developers find in reexamining these known compounds is that their safety profile and chemical properties are already well studied and understood, which considerably reduces the chances of failure in later stages during the development process.

Existing drugs can be repurposed in two fundamental ways:

The first one is to use them for a new or different therapeutic target. Sometimes drugs are shelved because they are considered too “dirty”, i.e. they bind too many targets and give various undesirable side effects. It may be that a different dosage or route of administration can reach a new target, and hence, be used to treat other pathologies.

The second method is based on taking a known target and a known drug for treating a different disease. We can find an illustrative example of this in the medication called finasteride, which originally was developed against prostate enlargement, and later it was discovered that it could stop or even reverse male pattern alopecia.
In spite of the fact that this medication performs on the same target for both pathologies, its approval to treat male baldness was eight years later than the authorisation for prostate enlargement. This was due to the fact that finasteride had to pass successfully a series of clinical trials before receiving its permission to treat the second indication.

A medication that fulfills both types of repurposing is chlorpromazine, which was originally produced as a antihistamine^[2] and later was used as an antipsychotic on account of its sedative effect. While, these are very different illnesses, the drug acts on the same target for both.
Currently, chlorpromazine is used in combination with chemotherapy since its anticancer potential was discovered some years later. This property was learnt thanks to its ability to bind a previously unknown target.

But the medicine that is many times seen as model of repurposing drugs is thalidomide. In principle, this medication was developed to treat morning sickness^[3] in pregnant women.

When this drug was approved, the safety regulations were not as rigorous as they are today, and some years after it was found out that thalidomide was responsible for serious birth defects; 20,000 people were affected worldwide.
As a consequence, this drug was removed from the market for 50 years until the company Celgene studied it again, in order to use it against leprosy and multiple melanoma. After this new research, this medication was authorised again, warning strictly against its use by potentially pregnant women.

[1] Phenotype: set of an organism's visible traits such as eye and hair color, height...It is the interaction between a genotype and the environment.
[2] Drug used to reduce or eliminate allergy effects.
[3] Nausea in pregnancy, usually in the first few months.

Sources: UTAustinX: UT.4.01x Take Your Medicine - The Impact of Drug Development.
  http://www.imujer.com/salud/4707/tratamientos-naturales-para-la-enfermedad-de-la-manana
http://www.academica.mx/observatorio/noticias/desarrolla-la-unam-f%C3%A1rmaco-controlar-algunos-tipos-c%C3%A1ncer
  http://www.elsevier.es/es-revista-educacion-quimica-78-articulo-avances-el-diseno-farmacos-asistido-90434983
  http://www.dddmag.com/articles/2007/09/innovative-strategies-drug-repurposing
  https://embryology.med.unsw.edu.au/embryology/index.php/Abnormal_Development_-_Thalidomide

2.2.3. Nanobiosensors

noreply@blogger.com (David Funes) — Tue, 23 Feb 2016 20:42:00 +0000

The great potential that nanotechnology offers enables the progress of new devices in the nanodiagnostic field, mainly in nanobiosensors.
Their properties are characterised by the nanoscale at which they are produced. Unlike biosensors , which we studied in the post: "Introduction to biosensors", where their sensory receptors are distanced by hundreds of micrometers, here they are distanced by a few nanometers.

Thus, even sensors constituted by single molecules bound to their surface have been designed. Consequently the process of diagnosis is performed with a much smaller size device, improving portability and the potential for use anywhere.
Also, only extremely small sample quantities (micro or nanolitres) are required to carry out the analysis, so the sample extraction methods are less traumatic and invasive for patients.

These instruments also facilitate the use of samples without fluorescent or radioactive markers, which are used in biological and clinical analysis. This property is essential, since it has been proven that when a sample is not marked or altered before examining, the detection sensitivity significantly increases.

Within nanobiosensors we can distinguish the following categories:

Nanophotonic biosensors

This class of nanobiosensors, also known as evanescent wave biosensors, is based on how the light is transmitted by multiple internal reflections through the optical waveguide, which is a component of the biosensor.

When light is propagated in a medium and passes into a lower refractive index^[1] medium, it is not totally reflected. As a consequence, a light component (called evanescent wave), in each of these reflections, is propagated in the lower refractive index that covers the waveguide. This propagation is really short, a few hundreds of nanometers, but it permits interaction between the light and the detection surface of the sensor, demonstrating which specific bioreceptors anchored to that surface interact with the analysed sample.

With these devices, only a few microliters of sample are necessary to determine protein concentrations or the variations of just one DNA base.

Also, it is possible to evaluate the metabolic state of just a single cell because some of these nanobiosensors have sharp fibre optic (30-50 nm) filaments, which can be introduced into cells through the plasma membrane without harming the cell.
This method facilitates the study of in vivo cell functions (apoptosis^[2], cell division, biological nanomachines…) and it can detect pathological changes in a single cell.

Nanoplasmonic biosensors

In the Surface Plasmon Resonance (SPR) biosensor, a thin metal layer (normally a 50- nm-thick layer of gold) is placed on a dielectric material (a crystal), and its functioning is based on the detection of changes in the refractive index around the separation area between both elements.
By exciting the interface between these two layers (under conditions of total internal reflection) a plasmonic resonance^[3] is activated with a certain angle of incidence.. This angle, which produces a plasmonic wave (with an evanescent character) is very sensitive to the changes from molecular interactions that happen on the metal surface. Therefore, these interactions between the analyte^[4] of the sample and the sensor surface of the biosensor are shown as variations of the resonance angle.

Currently, new biosensors are being developed, which rely on the plasmon resonance phenomenon in nanoparticles. While their detection range would be very similar to the SPRs, their functioning system can be made simpler by measuring light transmission instead of light reflection, in addition to the advantage of the device miniaturisation.

Due to the tiny size of nanoparticles, the electron oscillations are more localised than in the previous case (SPR), in specific areas of nanoparticles. This phenomenon is called ‘Localized Surface Plasmon Resonance’ (LSPR). The colour (wavelength: λ) changes that nanoparticles experience by adsorbing^[5] the (bio)molecules of the examined sample are used to analyse this sample.

An alternative, within this kind of device,, is DNA sensors detecting colour changes produced by the aggregation of gold nanoparticles marked with stranded DNA complementary to the target DNA.

Nanoplasmonic biosensors are characterised by their real-time detection, specificity, high sensitivity and recognisition rate.
Their main applications are found in veterinarian, biomedical and environmental fields as well as in the food industry.

Nanomechanical biosensors

These sorts of nanobiosensors use the deflection or the resonance frequency variation of a microcantilever (as a transducing^[6] method) when they interact with the studied sample. This position variation (Δx) is just a few nanometers derived from the biomolecular identification of the analysed sample.

Because these microcantilevers can be produced in mass at a low cost by standar microelectronic technology, thousands of them can be manufactured to identify thousands of analytes in a single sample.

The sensor area of these microcantilevers is around 1,000 μm², which provides them to test sample volume lower than femtomole (10^-15 moles).

Both photonic and nanomechanical biosensors could help us to obtain unlimited genetic and proteomic information that would offer the discovery of pathogens, new drugs, vaccines and undiscovered mutations of certain diseases in a faster way than the current technology.

Microfluidic devices or lab-on-a-chip (LOC)

This class of instruments uses an electric field to move liquids, particles, molecules or cells through microcapillaries designed on chips of different materials like silicon, crystal, quartz or plastic.

This kind of chip is configured by a large number of integrated microchannels and microchambers, where the examined sample is subjected to complex chemical and biochemical reactions.
The required volume of the sample fluids is very small, therefore the analysis can be carried out very quickly.
It is a portable and disposable detection method with a high grade of automation.

The ultimate objective for all these biocompatible nanodevices is to finally be implanted in vivo inside our organism, where they would perform a similar function to ‘sentinels’ in the presence of the first diseased cells. Some advances have already been made to that effect at the microscale (pills with built-in image cameras), but, without a doubt, this will become one the major research fields in nanomedicine in the near future.

[1] It describes how light propagates through a certain medium. It is defined as n = c/v, where c is the speed of light in vaccum and v the speed of light in that medium.
[2] Process of programmed cell death.
[3] Excitation or oscillation of conduction electrons at the interface between two materials, in our case a metal layer on a dielectric substrate.
[4] Substance (ion, compound or element) of interest in an analysed sample.
[5] Adhesion of atoms, ions or molecules to a surface.
[6] Process by one signal or energy is converted to another.

Sources: Nanomedicina: aplicación de la nanotecnología en la salud. Laura M. Lechuga. Grupo de Nanobiosensores y Aplicaciones Bioanalíticas
Centro de Investigación en Nanociencia y Nanotecnología (CIN2). CSIC
  http://www.kennislink.nl/publicaties/de-opmars-van-de-twentse-lab-on-a-chip
  http://images.slideplayer.es/16/5040279/slides/slide_47.jpg
  http://www.mdpi.com/sensors/sensors-10-09630/article_deploy/html/images/sensors-10-09630f1-1024.png
  https://www.ifm.liu.se/applphys/molphys/research/biosensing_using_nanopart/
  http://www.tcd.ie/Physics/people/Martin.Hegner/ReviewNSST-The_impact_of_STM_and_AFM.html?ntherodt_01.pdf
  Fritz, J., Baller, M.K., Lang, H.P., Rothuizen, H., Vettiger, P., Meyer, E., Guntherodt, H.-J., Gerber, CH. and Gimzewski, J.K.,
Science 288 (2000) 316.
  https://www.theengineer.co.uk/lab-on-chip-device-promises-hiv-diagnosis-in-10-minutes/

3.4. Proteins

noreply@blogger.com (David Funes) — Sat, 06 Feb 2016 16:12:00 +0000

Proteins are the biological macromolecules with the largest variety in structure and functions. We can find thousands of them in a single cell, each performing a unique and specific role. In this way, we find proteins carrying out transport or storage functions, protective, contractile and structural proteins, enzymes or toxins.
All of them have in common that they are made up of structural polymers of amino acids arranged in lineal chains.

Types and functions of proteins

▣ Enzymes: they are known as complex or conjugated proteins, whose essential function is to act as an organic catalyst in biochemical reactions, where they assist to synthesise, reorder or break down different elements on which they perform.
An example are digestive enzymes, like salivary amylase, hydrolyses amylose (starch component).

▣ Hormones: they control and regulate physiological processes such as metabolism, growth or reproduction. They are chemical-signalling molecules, usually stereoids or small proteins, released by endocrine cells.
Examples include insulin, which is a protein hormone in charge of regulating glucose levels in blood.

Proteins present a wide range of molecular weights and shapes, where shapes are vital to their activities. But these functions can be modified by the exposure to chemicals and changes in pH or temperature, which lead to shape variations, and as a consequence the loss of their functions in a process named denaturation.

Amino acids

Amino acids are the monomers that constitute proteins. Each one has a structure formed by a central carbon atom, known as α carbon, to which an amino group (NH₂), a hydrogen atom and a carboxyl group (COOH) are attached. The name ‘amino acid’ comes from the presence of these two groups in the amino acid basic structure.

Each amino acid also have another atom or group of atoms joined to the central atom, called R group or side chain which is different for each kind of amino acid. The chemical nature of this chain establishes the type of aminoacid (whether it is polar, nonpolar, basic, acidic…). Thus, we can find basic aminoacids, like arginine and lysine, with a positive charge. Others such as cysteine or serine are polar due to their hydrophilic side chains, or if this chain is hydrophobic we have nonpolar amino acids like alanine or valine.

Out of the twenty different amino acids that form proteins, ten of them are considered essentials, because they are the necessary blocks for the protein construction. However, the human body cannot sinthesise them and must be obtained from the diet.

Amino acids are represented by a single upper case letter or three-letter abbreviation, e.g.: cysteine is symbolised by the letter C or the three letters Cys.

The number and sequence of amino acids ultimately set up the shape, function and size of proteins.

Amino acids are linked by covalent bonds, called a peptide bond, in which the carboxyl group of one amino acid is bonded to the amino group of the adjacent amino acid, releasing a water molecule during the process (dehydration reaction).
These bond products are called peptides and their joining give rise to a polypeptide chain. Each one has a free amino group at the end of the chain, named N terminal or amino terminal, having a C terminal or carboxyl terminal at the other end.

Often, the terms polypeptide and protein are used interchangeably, although technically a polypeptide is an amino acid polymer, while a protein is a polypeptide or combined polypeptides, each one with distinct shape and unique functions.

Protein structure

As I previously mentioned, protein shape is vital for the role they perform. In order to understand how a protein adopts its shape or final conformation, it is necessary to study the four structural levels in which they are organised: primary, secondary, tertiary and quaternary structures.

Primary structure

The primary structure is defined as the unique sequence of amino acids in a polypeptide chain. For example, the insulin hormone has two polypeptide chains, A and B, formed by unique sequences of 21 and 30 amino acids respectively and joined by two disulfide bonds between the cystein amino acids. There is also a third disulfide bond in the A chain, between two cysteine amino acids, which enables the molecule to fold into the suitable shape.

The unique sequence of every protein is ultimately determined by the genetic coding of that protein. Any alteration in the nucleotide^[1] sequence, which constitutes the gene, can produce different amino acids that will be, later, added to the polypeptide chain and consequently affect the activity and structure of the resulting protein.

Thus, a single change in one of the 600 amino acids that make up the haemoglobin molecule origins, such a variation in its structure and activity, produces the appareance of sickle cell anaemia^[2].Specifically, in the β chain of this protein, glutamic acid is substituted by valine.
More precisely, since every amino acid is made up of three nucleotide bases, those 600 amino acids generate 1,800 bases and sickle haemoglobin, the cause of sickle cell anaemia, arising from a single mutation in those 1,800 bases.

As a result of this, haemoglobin molecules form long fibres that deform the disc or biconcave shape of red blood cells, acquiring a sickle or crescent shape that blocks arteries. This obstruction causes a series of disorders such as headaches, abdominal pain, diziness or breathlessnes so typical of this sickness.

Secondary structure

Secondary structures are generated from the local folding of polypeptides in certain regions, the most common are the α-helix and β-pleated sheet structures.
The α-helix is formed from the primary structure by rolling and twisting helically, with 3.6 amino acid residues per turn. They are constituted by hydrogen bonds between the carbonyl group (C=O) of one amino acid and the amino group (N-H) of another amino acid located four positions further in the polypeptide chain.

In the beta formation, amino acids are arranged in a zigzag way, named the ‘pleated sheet’ arrangement. Also it is originated by hydrogen bonds between N-H and C=O groups, but in this case they belong to adjacent chains.

Tertiary structure

The tertiary structure informs how the secondary structure of a polypeptide folds up over itself giving rise to a globular conformation.
This configuration determines the physicochemical properties of proteins and assists their solubility in water, enabling, this way, it to carry out hormonal enzymatic and transport functions. This structure retains its stability due to the bonds among the R groups of amino acids. These bonds are created from the following types of interactions: disulfide bridges^[3], hydrogen bonds, ionic and hydrophobic interactions and Van der Waals forces (to refresh all these concepts, visit the post titled: “Chemical bonds”).
In this arrangement, the nonpolar amino acids are, generally, facing the inside of the protein and the polar amino acids facing the outside, interacting with the surrounding aqueous medium.

Quaternary structure

In nature, some proteins are formed from several polypeptides, known as ‘subunits’, and their interaction creates the quaternary structure. These are weak interactions that assist the stabilisation of the overall structure of the protein.
The quaternary structure, usually, gives the protein its function and also creates many crucial biological structures, e.g.: viral capsids^[4], microfilaments^[5], microtubules^[6] and collagen fibres of the connective tissue.

Denaturation and protein folding

Every protein has its own sequence and shape that are maintained through chemical interaction.

If the protein is exposed to chemicals or subjected to variations in pH or temperature, then its shape and structure can be modified without affecting its primary structure. This process is called denaturation, which can be reversible as long as the polypeptide primary structure is invariable during the process, allowing the protein to maintain its activity.
On the other hand, when the process is irreversible, the protein loses its function. This is what albumin (protein of egg white) experiences by being cooked, since it is denatured when it is exposed to high temperatures.

Protein folding plays a vital role for protein functions. This folding is made with the help of a protein family, named chaperones (or chaperonins), present in all cells. They are not part of functional proteins^[6], but they bind to them to assist in their folding, their assembly and their cell transport to other parts of the cell where the protein performs its activity.

[1] Monomers of nucleic acids (DNA and RNA).
[2] Crescent or disc-shaped structures.
[3] Strong covalent bond between thiol groups (-SH) in the cysteine amino acid.
[4] Protein coat of virus that protects the viral genome.
[5] Thin fibres of proteins which with microtubules form the cell structure.
[6] Tubular structures of cells. Main component of cytoskeleton in eukaryotic cells.
[7] Those proteins that have a biological activity.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
  http://www.profesorenlinea.cl/Ciencias/ProteinasEstruct.htm
  http://study.com/academy/lesson/proteins-iv-higher-order-structure.html
  http://quimica.laguia2000.com/conceptos-basicos/cadena-lateral-en-aminoacidos
  http://es.slideshare.net/carolinacisdel/biomoleculas-enfermera
  http://biologiavfe.blogspot.com.es/2010/04/anemia-falciforme.html
  http://sicklecellcurefoundation.org/living-with-sickle-cell-disease/national-sickle-cell-month
  http://www.slideshare.net/VijayP7/secondary-structure-prediction-of-proteins
  http://www.slideshare.net/thelawofscience/protein-structure-11543259
  http://www.mobi4health.ug.edu.pl/wp-content/uploads/2014/10/2ndMSW-Milla-Neffling-Primer.pdf

1.3.5. Novel drug delivery systems

noreply@blogger.com (David Funes) — Sat, 23 Jan 2016 17:30:00 +0000

Currently, there are two basic problems related to drug dosage.
The first when the drug is too soluble in water and a controlled release system is required to counteract this. The second when the drug, on the contrary, has no or very low solubility, and therefore, it does not dissolve in water.

This second problem is vital to solve because, today, it is estimated that between 75 and 80% of new chemical substances that come out of academic laboratories and pharmaceutical companies are not water-soluble.
This challenge is to get the main constituents of the drug to dissolve in the gastrointestinal tract, and, in this way, to perform their systematic action^[1].

Thanks to nanoparticle engineering technologies, new crystalline drugs can be designed with a really small size (between 200 and 300 nm), which enables them to have faster dissolution rates than larger particles.

Once they are manufactured, these nanoparticles are incorporated into the different drug delivery systems where they are dissolved. In this way the drug is available to be absorbed by the lungs or the digestive system, depending on which route of administration has been chosen.

An alternative would be a drug dissolved in an organic solvent (not water) such as acetonitrile, methanol or ethanol. The drug is then dissolved with some kind of stabiliser in order to then be rapidly frozen. As a result of this procedure we obtain non-crystalline pharmaceutical substances, named amorphous forms or amorphous morphology, which can be dissolved at much higher concentrations than their corresponding (water) crystalline forms.
Thus ‘super-saturation’ of an aqueous solution is achieved, which facilitates the drug’s dissolution at high concentrations in the stomach and in the upper small intestine to be subsequently absorbed and distributed through the whole organism.

Properties of crystalline and amorphous drugs
Crystalline state	Amorphous state
Long range translational, rotational and conformational order	May exhibit short range order
Well defined melting point	Glass transition point^[2]
Good flow properties^[3]	Poor flow properties
More stable	Less stable
Less hygroscopic^[4]	More hygroscopic
Relatively less soluble	Relatively more soluble

An application example of this type of particle engineering system has been developed by members of the medical school of the University of Texas in San Antonio and the Veterans Administration hospital, looking at a new administration route for immune-suppressing drugs.

This sort of medication prevents the rejection of transplanted organs, but at present, by being administered orally it can cause a series of undesirable side effects, like the development of different types of cancer.

The objective of the technologies described above was to administer a lower dosage of these drugs with accuracy to the site where its immune-suppressing action was necessary. In order to achieve this, prototypes of a new inhaled immune-suppressing drug were developed during a period of four to five years. The inhalation route of administration was selected because the patients who had undergone a lung transplant were the research target, and consequently the lungs were the aim of this medication.
Several tests were performed in mammals (mice and rats) where the product safety was proven. Low levels of the drug in the blood but high concentrations in the lungs were identified, which shows the validity of the inhalation route.
Then, the drug was tested in a small group of healthy human volunteers, under strict safety rules, and the results were similar to those in animals. Yet, this medication is still under development.

In conclusion, the goal is the research of new routes of drug administration that are not available presently to benefit the patients by minimising side effects and reducing the systemic drug exposure^[5].

[1] Action exerted by the drug once is absorbed and distributed by the bloodstream.
[2] Temperature at which an amorphous solid becomes soft and flexible upon heating or rigid and brittle upon cooling.
[3] These properties indicate a higher or lower fluidity of particles depending on their size, shape, absorbed moisture and density.
[4] Substances that absorb moisture from the surrounding environment.
[5] A type of exposure where, in contrast with topical or local exposure drugs, the entire organism is affected by a medication performance.

Sources: UTAustinX: UT.4.01x Take Your Medicine - The Impact of Drug Development.
http://www.slideshare.net/rcdreddi/limerick-meet-and-greet-ac-edited
http://chemistry.about.com/od/chemistryglossary/a/glasstransition.htm
https://docs.google.com/document/d/1TOwPCUSItCGOnUivKC_5kuTGw7kh9D5DPIK2U-V7D2A/edit#heading=h.90pmyxnddx1b
http://trasplante-de-organos.blogspot.com.es/2011/09/inmunologia-del-trasplante-la.html

2.2.2. Imaging diagnostics

noreply@blogger.com (David Funes) — Sat, 16 Jan 2016 16:44:00 +0000

Over the last few years, imaging diagnostics has significantly increased its relevance such that they have become an indispensable tool to diagnose innumerable diseases such as neurological syndromes, cardiovascular disorders or cancer.

The convergence between imaging diagnostics and nanotechnology enables the creation of a new series of tracers and contrast agents with a higher resolution and sensitivity, which allows illness detection at earlier stages at cellular or even molecular level. This advance makes the quick application of treatment possible, thus increasing the chances of treatment and survival.

Imaging diagnostic nanosystems are mainly based on three types of nanoparticles: semiconductor, metal and magnetic.

Semiconductor nanoparticles

This first sort of nanoparticles, also called quantum dots, consist of semiconductors^[1], whose size has been reduced to a few nanometres (1-10 nm). This produces changes in their electronic arrangement, which means they lose their distinctive band structure.
Because of this new structure, their optical response (specifically their fluorescence) is going to be closely related to the size variation of these nanoparticles. Consequently, these quantum dots emit light in a broad range of wavelengths (colours), depending on their size and not the material they are made of, which turns them into excellent biological markers.

While a wide variety of semiconductor materials have been examined, the most commonly used are cadmium selenide (CdSe) and cadmium telluride (CdTe), because in addition to be manufactured on a large scale, their sizes can be controlled perfectly. This enables us to see emission bandwiths with a full spectrum of different colours over a long lifetime.

But the semiconductor nanoparticles must also get to their defined targets. To achieve that, the quantum dots must be coated with polymers, like polyethylene glycol (PEG), which makes them invisible to macrophages and prevents immune system cells degrading and digesting them before reaching their target.

The next step is to make these nanoparticles able to identify their targets through bioreceptors (antibodies) that are bound to their surface, and these in turn will be bound to the specific antigens^[2] of the target cells (e.g.: cancer cells). The bioreceptors of quantum dots produce a biomolecular recognition reaction when they meet these antigens, being accumulated in that area, and they are observed by using ultraviolet light due to the peculiar fluorescence that they emit.

Lastly and once they have fulfilled their role, the quantum dots must be purged from the organism to avoid unwanted side effects. It seems that the quantum dots are excreted through the kidneys and the liver without difficulties in animal testing, but to be tested in humans some problems related to the aggregation process must be resolved before receiving authorisation from the health agencies for commercialisation.

Metal nanoparticles

The fact that metal nanoparticles have a resonance frequency (their colour) dependent on their size and shape makes them a second option for contrast agents.
This property provides enables them to be manufactured to absorb or reflect light in the spectrum of interest. Thus, gold nanoparticles can be designed to absorb or reflect light in the near-infrared band (700-900 nm) as biological tissues are more transparent in that electromagnetic spectrum band. Using techniques like optical coherence tomography (OCT)^[3], 3D maps are obtained of the areas where the nanoparticles are gathered.

Magnetic nanoparticles

A third alternative is magnetic nanoparticles (iron oxides like magnetite: Fe₃O₄), with the ability to increase the contrast in magnetic resonance imaging (MRI) tests.
Their transport through the organism can be carried out with the use of an external magnetic field (like an electromagnet) taking advantage of their magnetic properties. They may replace the old fashioned markers made of heavy metals in the near future, because they have lower toxicity.

Both magnetic and metal nanoparticles use identical or very similar methods to the ones described for the quantum dots in order to avoid degradation by the immune system, thus enabling them to locate and bind to the target tissues and cells.

Imaging nanosystems belong to the in vivo diagnostic category, therefore they must be injected into the human body. This implies, as we have seen, potential drawbacks (biocompatibility, sophisticated design) that have to be resolved to ensure an effective and safe use in the organism, minimising undesirable side effects.

[1] Material that behaves as an insulator or a conductive material depending on several factors such as environment temperature, pressure, surrounding electrical and magnetic fields or the incident radiation.
[2] External or toxic substances for the body (generally proteins) that lead to antibody production and causing an immune response.
[3] Non-invasive imaging test that uses light waves to take cross section images.

Sources: Informe de vigilancia tecnológica: nanomedicina. Fundación para el conocimiento madri+d. CEIM. José Manuel González, Marta López,
Gema Ruiz.
Nanomedicina: aplicación de la nanotecnología en la salud. Laura M. Lechuga. Grupo de Nanobiosensores y Aplicaciones Bioanalíticas
Centro de Investigación en Nanociencia y Nanotecnología (CIN2). CSIC
  http://www1.radiology.ucsf.edu/research/labs/hyperpolarized-mri-tech-2/facilities_equipment
  http://www.osiconference.org/osi2015/presentations/Tu2.3%20Zahn.pdf
  http://www.nanowerk.com/news/newsid=7441.php (Image: Penn State)
  http://slideplayer.com.br/slide/74350/

2.2.1. Introduction to biosensors

noreply@blogger.com (David Funes) — Sat, 09 Jan 2016 16:08:00 +0000

As a consequence of our current technology, the diagnosis of certain diseases is made at overly advanced stage of the disease.
Today, the main instrument used to diagnose illnesses is the biosensor, which enables the detection of specific substances, their composition and molecular features, or to identify the presence of microorganisms in certain environments.

This instrument is comprised of two main components:

▣ A reactive or sensory area formed by a biological receptor (antibodies, DNA, enzimes…) with the ability to join those substances to be detected.
▣ A transducer system that processes and quantifies the signal (mechanical, electrical, optical…), which is caused by the interaction between the analysed substance and the recognition element.

The integration of both components makes biosensors sensitive and selective.

Biosensors have a wide range of applications in fields like environment, healthcare, biotechnology and the food industry.

Among the most common biosensors we find microarrays or biochips which consist of hundreds or thousands of biomolecules (generally oligonucleotides^[1] or single-stranded DNA fragments, often known as ‘bioreceptor probes’) located on a solid surface (glass, silicon, gold) in particular positions and concentrations making a 2D matrix, with each molecule separated by a distance of between 100 and 150 μm from each other.

The examined sample is marked (normally with reactive fluorescent dyes), in such a way that, when it comes into direct contact with the sensory molecules of the biosensor, they are hybridised^[2] with their homologous sequences, producing a fluorescent signal in that location.
Later, the data obtained are analysed and interpreted using a scanner and computer tools.

Biochips have countless applications: from personalised medicine (to know our higher or lower predisposition to suffer cancer or specific genetic disorders), the detection of harmful bacteria in food or water, discovery of virus and bacteria mutations, which become drug resistant or examining the microbial diversity in certain environments.

Another type of microarray are the protein microarrays (or protein chips) which, in contrast to DNA fragments, have thousands of functionally active proteins anchored to their surface. This class of instrument has great potential in basic research applied to molecular biology, the identification of disease markers and the search for therapeutic targets.

Lastly, we find within microarray technology cell chips. Antigens, proteins or even lipids are placed on these detector surfaces, which interact with cells, not only capturing them, but also triggering responses on them, such as phenotypic changes^[3] or the segregation of particular substances.
These kinds of microarrays are used for toxicological analysis, identification of illness markers and the analysis of pathogenic agents.

Science keeps developing and it is designing smaller and more powerful devices, on the basis of the enormous potential that nanotechnology offers. These diagnostic nanosystems, whose main advantage is, in comparison to current biosensors, the early detection of diseases, depending on their working area, are divided into: imaging diagnostic nanosystems and nanobiosensors, which will be object of study in the coming posts within this section.

[1] General term for a short, single stranded DNA or RNA used in research, genetic testing and forensics.
[2] Recognition and combination of two complementary molecules
[3] Changes in those particular and genetically inherited features of an organism.

Sources: Informe de vigilancia tecnológica: nanomedicina. Fundación para el conocimiento madri+d. CEIM
Fundación española para la ciencia y la tecnología (FECYT). Nanociencia y nanotecnología. Entre la ciencia ficción del presente
y la tecnología del futuro, 2009.
  http://www.definicionabc.com/ciencia/fenotipo.php
  http://www.blogdefarmacia.com/biosensores-tecnologia-para-la-salud/
  http://www.pharmatutor.org/articles/applications-of-biosensors-technology-future-trends-development-and-new-intervation-in- biotechnology?page=0,1
  http://www.slideshare.net/ManjuAnshika/biosensor-dr-manju-jha
  http://www.sensorsmag.com/specialty-markets/medical/strong-growth-predicted-biosensors-market-7640

1.3.4. Safety and efficacy of drugs

noreply@blogger.com (David Funes) — Wed, 30 Dec 2015 16:23:00 +0000

In my second to last post (Routes of drug administration), we studied the different drug delivery systems and how one of them is selected to take a drug compound and make it into a drug product.

In the drug development process, researchers often use living organisms to find out if their compounds can go through the cell membranes and carry out their therapeutic function. However, there is a really delicate balance between those therapeutic effects and toxicity in lots of drugs. To avoid this conflict, previously, many studies must have been conducted to select the lead compound with the smallest toxicity and the top therapeutic potency.
Many of these evaluations can be assessed on the bench top in cell cultures. However, these experiments are often too simple to predict the compound efficacy in human beings. Hence, more complex living systems, like in rats or mice, are used in order to gain a greater understanding about their efficacy and biological distribution. To do this, researchers have been able to find several ways to model human diseases (e.g. cancer, certain infections and inflammations) in rodents.
Although these models are not completely reliable indicators of the drug efficacy in humans, they are another tool which helps to choose the lead compound with the greatest chance of success. These models can also be used to determine the optimal dosage of the drug needed to reach its target.

Despite the fact that in this phase, the pre-clinical efficacy is not considered as important as the pre-clinical safety, these studies are vital for the pharmaceutical developers to evaluate the likehood of the drug efficacy against a particular human disease. Once the lead compound has been selected and formulated, it is evaluated through animal testing to ensure it is safe for human use.

Although lots of ethical objections have arisen about animal testing, it is the best procedure that scientists and government officials have developed to protect humans against the unanticipated dangers of experimental drugs. In order to ensure the quality and validity of the obtained data in these trials, it has been set a strict group of regulations, called ‘good laboratory practice’ (GLP), which developers have to fulfil. These regulations establish an organisational framework for the new pharmaceutical substances, as well as the evaluation of any toxicity that may happen during examinations.

In addition to an understanding of the potential toxic effects, it is also critical to know how long the drug remains in the bloodstream, how it is metabolised and what tissues it can be collect in.

Next, I will explain how to obtain (on average) the permission of regulatory agencies to subject experimental drugs to clinical studies:
In the US, for instance, if the lead compound overcomes the safety exams, an IND (Investigational New Drug) application is submitted as evidence that the drug is reliable and that it can be put forward for experimentation in humans. Together with this data, the pharmacological product must be manufacturable by a reproducible and controlled method.

This stage in the drug development process, which can mean a financial layout of several million dollars for the pharmaceutical developers, can present an insurmountable obstacle for smaller companies and academic researchers.
In principle, the expenditure of such a huge amount of money on a new drug that has not been commercialised yet may appear excessive. Nevertheless, it is absolutely necessary to ensure that the drug will not cause harm to the humans that receive it during the clinical trials.

In the US, as I mentioned earlier, new drug products are given the denomination IND by the regulatory agency FDA (Food and Drug Administration), which does not mean that the new drug has been approved to be marketed, but the drug does then have authorisation to be put under clinical trial.
In order to get an IND, previously an IND application had to be submitted with all the required FDA information guaranteeing that the product is safe and ready to commence clinical trials.

During the first stage of clinical trials, the volunteers cannot be exposed, under any condition, to unnecessary health risks. To ensure this, the IND application must contain information about the safety test results of the drug, its manufacturing process and details about the proposed clinical study. As long as the provided information is clear and properly submitted, the FDA opens an IND application. In any case, the FDA could stop the investigation at any point if the study integrity is compromised or the new product turns out to be a health risk.

Sources: UTAustinX: UT.4.01x Take Your Medicine - The Impact of Drug Development.
  https://www.msdsalud.es/tu-salud-al-dia/biblioteca-recursos/guias-para-pacientes/proceso-investigacion-desarrollo-aprobacion-
farmaco.html
  http://www.thebody.com/content/art16845.html
  http://geneticayclonacion.blogspot.com.es/2015_04_01_archive.html
http://speakingofresearch.com/facts/statistics/
  http://www.slideshare.net/swati2084/ind-investigational-new-drug-application-and-nda-26468953

1.3.3. A case study: developing inhaled products

noreply@blogger.com (David Funes) — Wed, 23 Dec 2015 18:58:00 +0000

Delivering aerosols via inhalation has been practiced for millennia. As a matter of fact, since prehistory, where men began to use certain subtances with therapeutical and hallucinogenic purposes, such as cohoba (made from the beans of a mimosa species) or cannabis.

But aerosols only first replaced oral medication (tablets, pills, capsules…) in the 1950s for the treatment of respiratory diseases because oral medication caused severe side effects. The first meter-dose inhaler was made simply from a Coke bottle, some perfume valves and propellant systems.

The main challenge that aerosols face to penetrate the lungs and the upper airways (mouth, pharynx, larynx and trachea) is that these organs are very well equipped to filter dust and other particles. Hence, the challenge consists of designing the aerosol particles in such a way that they do not end up in the mouth or throat as opposed to the lungs.
These particles must be produced with a size or aerodynamic diameter between 1 to 5 μm, in order to pass beyond the upper airways, which act as a filter. As you can imagine, manufacturing such tiny particles is a hugely complex task.
For instance, those microscopic particles which are made of dry powder, have a very large surface area, which makes them very sticky. Consequently, the major force that acts on them is Van der Waals interaction (“Chemical bonds”) and not gravity (e.g. when you write with a chalk on a chalkboard it stays on and does not fall off due to these interactions rather than gravity). So, the objective is to overcome these Van der Waals forces and develop methods of deaggregating aerosol particles.

Lots of the aerosols used to treat pulmonary pathologies (e.g. cystic fibrosis, Chronic Obstructive Pulmonary Disease or COPD and asthma) are not really well designed for patient use.

For example, metre-dose inhalers have been used successfully for many years, however a lot of patients, especially children and the elderly, cannot or do not use them correctly. These inhalers present a major difficulty for those with coordination issues in activating the actuation manoeuvre with the inhalation manoeuvre.

On the other hand, the dry powder inhalers that we find currently on the market are all all active, which means that the patient needs to provide all the necessary energy so that the aerosol gets into the lungs via the airways.
The efficacy of these type of aerosols depends on how hard you inhale through the device. In this way, if you do not breath fast or strong enough, the aerosol particles will not be deaggregated enough to reach the lungs and they will mainly accumulate in the throat.
As a consequence, these sort of inhalers are not suitable for those patients who do not have proper lung function.

A third type of inhaler is a nebulizer or water-based nebulizer spray systems, usually bulky units, not very portable, used at home or in the clinic, although some technical developments have been made lately to make them more portable and battery operated.

So, these are the different devices for the treatment of respiratory diseases by the inhalation route and their selection is based upon the patient’s needs and the kind of drug to be delivered.

There is an interesting story about the formulation of the meter-dose inhalers. At the beginning, when these devices were invented, back in the 1950s, they used chlorofluorocarbon (CFCs) propellants into which the drug was suspended or dissolved. But, due to reports in the 1970s about ozone layer depletion because of the use of these propellant gases, and their subsequent ban in the late eighties, the pharmaceutical industry replaced them with hydrofluroalkanes (HFAs) with similar chemical and physical properties to the CFCs.

In spite of comparable properties, the HFAs behaved in a very different way with the pharmacological molecules and the rest of excipients. For example, some surfactants^[1] used in drug formulation, like oleic acid, were soluble in the CFCs but not in the HFAs.

Because of this pharmaceutical companies adopted several strategies to reformulate the same drug into these propellants gases.
A meaningful example is the product called QVAR^[2], in which the drug is dissolved, using a co-solvent (diluent), specifically ethanol, together with hydrofluroalkane propellants. And it turned out that the aerosol produced is a much finer mist with a much finer droplet size. In this way, the amount of aerosol particles deposited in mouth and throat decreased tremendously, but, on the other hand, there was a dramatic increase in lung delivery.
This new approach provided a competitive advantage for the company that created it in comparison to the competition, which tried to adapt the performance of the HFA propellants in a similar way to the older-style CFCs.

[1] Substance that act as a detergent, emulsifier or humectant and allows to reduce the surface tension in a fluid.
[2] Anti-inflammatory inhaler indicated for the treatment of asthma attacks.

Sources: UTAustinX: UT.4.01x Take Your Medicine - The Impact of Drug Development.
  http://www.telegraph.co.uk/news/newstopics/howaboutthat/3225729/Stone-Age-man-took-drugs-say-scientists.html
  http://es.slideshare.net/CAWIMECA/el-sistema-respiratorio-42882484
  http://blogs.20minutos.es/yaestaellistoquetodolosabe/tag/clorofluorocarbonos/
  http://definicion.de/surfactante/
  http://www.asthma.ca/adults/treatment/meteredDoseInhaler.php
  http://es.slideshare.net/luciagorreto/taller-asma-2015-inhaladores
  http://www.drtrust.in/products/nebuliser-machine

3.3. Lipids

noreply@blogger.com (David Funes) — Sun, 29 Nov 2015 18:26:00 +0000

Lipids are a type of hydrocarbon mostly made up of nonpolar C-C and C-H bonds, which makes them hydrophobic (they repel water) or insoluble in water.
Lipids (more commonly known as fats) constitute a basic element in many hormones, as well as in cell membranes. They are an important source of long-term fuel for cells, they provide heat insulation for both animals and plants and they form a protective hydrophobic outer layer over fur or feathers of aquatic birds and mammals.

Within lipids we find the following categories:

Fats and oils

Fats, due to their chemical structure, are also named triacylglycerols or triglycerides. They are made up of two basic components: glycerol and fatty acids.
Glycerol is an organic component (specifically, a kind of alcohol) with three carbon atoms, five hydrogen atoms and three hydroxyl groups (OH). Fatty acids, in turn, are composed of a long chain of hydrocarbons to which a carboxyl group is attached.
The number of carbon atoms in fatty acids generally vary between four and thirty-six, although the most common contain 12 to 18 carbons. Fatty acids’ three carbon atoms (in the glycerol molecule) are joined by an ester bond and an oxygen atom. During the formation of this bond three water molecules are released.

Fatty acids are divided into two groups: saturated and unsaturated.
The first only have simple bonds between the adjacent carbons of the hydrocarbon chain. An example is the stearic acid formed by 18 carbon atoms and in which the number of hydrogen atoms attached to the carbon backbone is the maximum (no more hydrogen atoms can be attached). This acid is present in oils and in animal and plant fats. Among its uses we find the manufacturing of soaps, candles, plastics, cosmetics and as a softening agent for rubber.

Unsaturated fatty acids, on the contrary, have double bonds between their carbon atoms. Among the most common unsaturated fatty acids we find oleic acid, known for its beneficial impact on blood vessels, thus decreasing the risk of liver and cardiovascular diseases.

Unsaturated fatty acids are popularly known as oils, which are liquid at room temperature and can be monounsaturated when they have one double carbon bond in the molecule (olive oil) or polyunsaturated if they have more than one (canola oil).

Plants store fats or oils in their seeds, which are used as energy deposits during their development. In mammals, however, fat constitute globules, which take up most of the volume of a very specialised kind of cell called an ‘adipocyte’.

Unsaturated fats or oils have usually a plant origin and contain cis unsaturated fatty acids (visit the entry “The carbon atom” to remember the concepts cis and trans configuration). This cis double bond causes a bend or “twist” which prevent fatty acids from being compacted, keeping them in liquid state at room temperature. As examples of unsaturated fats: olive, corn, canola and cod oils. These kinds of fats help us to reduce our cholesterol levels (in contrast to saturated fats which contribute to the formation of plaques in our arteries).

Trans fats

Trans fats are present in some food products like margarine or peanut butter and can lead to increased levels of low density lipoproteins (LDL), more commonly known as “bad” cholesterol, which is deposited on arteries walls as plaque, producing cardiovascular disorders.
These trans fats are manufactured by the food industry by a method called ‘hydrogenation’, in which the double bonds of the cis structure of the hydrocarbon chain become double bonds in the trans configuration. During this process, oils are solidified by injecting gaseous hydrogen into them, so that they can acquire that desirable semisolid consistency in many processed foods.

Omega fatty acids

Both omega-3 fatty acids and omega-6 fatty acids belong to the group of the essential fatty acids that our organism requires but is not able to synthesise; thus we must ingest them through our diet.
The terms omega-3 and omega-6 indicate that the third and the sixth carbon, counting from the far end of the hydrocarbon chain, are the ones that are attached to their adjacent carbon by a double bond.

Among the food sources of omega-3 we find some oily fish like trout, tuna and salmon. These types of fatty acids reduce blood pressure and the triglyceride levels in blood, they help to prevent thrombosis, heart attacks and may help to decrease the risk of certain kinds of cancer.

Fats are not only excellent energy deposits in the long term and provide isolation for the body, but also enable us to digest fat-soluble vitamins. Therefore, despite the bad publicity that they have received, the “healthy” fats must be consumed in balanced diets regularly.

Waxes

Waxes are comprised long chains of fatty acids esterified^[1] to long-chain alcohol.
Because of their hydrophobic nature, they perform a protective function on the outer coating of the leaves of some plants and the feathers of aquatic birds.

Phospholipids

Phospholipids are formed by fatty acid chains attached to a glycerol or sphingosine^[2] backbone, where two fatty acids make up a diacylglycerol molecule and the third carbon of the glycerol backbone is occupied by a modified phosphate group.
The compound formed by the diacylglycerol molecule and the phosphate group constitute the phosphatidates, which are the precursors of phospholipids.
Phospholipids are part of the outer layer of animal cells and the main element of plasma membranes, which play a fundamental role in the cellular communication.

Phospholipids make up these membranes in such a way that the phospholipid tails (which are hydrophobic fatty acids that cannot interact with water) face internally and the phospholipid head (which is the hydrophilic phosphate group that interacts with water) faces externally, in contact with the aqueous environment. By having a hydrophobic and a hydrophilic part, phospholipids are classified as amphiphatic molecules.

The dynamic nature of plasma membranes is due to their being formed by phospholipids. In contact with water, phospholipid molecules are ordered spontaneously in a spherical structure called a micelle, with the heads (polar) facing the outside and the tails (nonpolar) facing the inside of these structures, just like plasma membranes.

Steroides

In spite of the fact that steroids are not very similar to the rest of the lipids, on account of the fact that they have a fused ring structure, they are included within this category as they are insoluble in water.
All steroids are made up of four carbon rings and many of them also have the –OH functional group, which allows them to be classified as alcohols (sterols).

In addition, some of them, like the cholesterol molecule, have a short hydrocarbon tail. Cholesterol is the most common steroid in the human being and animals, it is synthesised in the liver and is the precursor to vitamin D and also of bile salts, which help to metabolise the fats we ingest to be absorbed by cells afterwards.
Cholesterol is secreted by the endocrine glands and the gonads, as it is the precursor of steroid hormones like estradiol^[3] and testosterone. Therefore, despite the bad name that cholesterol has among lay people, this molecule plays a vital role for the proper functioning of our organism.

[1] Esterification reactions are those by an ester is obtained, usually from the reaction of a carboxylic acid and an alcohol.
Esters are organic compounds made by substituting an acid by an alkyl or other organic group.
[2] Amino alcohol made up of 18 carbons, forming an unsaturated hydrocarbon chain.
[3] Female sex hormone.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
  http://www.genomasur.com/BCH/BCH_libro/capitulo_02.htm
  http://www.salud180.com/sustancias/acido-estearico
  http://herbolaria.wikia.com/wiki/%C3%81cido_oleico
  https://www.flickr.com/photos/thame/3302072732
  http://www.eufic.org/article/es/artid/La-importancia-de-los-acidos-grasos-omega-3-y-omega-6/
  http://www.uhu.es/08007/documentos%20de%20texto/apuntes/2005/pdf/tema_03_lipidos.pdf
  http://www.fisicanet.com.ar/biologia/introduccion_biologia/ap11_lipidos.php
  http://brainly.com.br/tarefa/453268
  http://www.calpoly.edu/~jfernsle/Research/Biophysics/BiophysResearch.html