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3D Bioprinting - The Medical Futurist

2021-03-11T15:06:58.222Z

Three dimensional (3D) bioprinting is the utilization of 3D printing–like techniques to combine cells, growth factors, and biomaterials to fabricate biomedical parts that maximally imitate natural tissue characteristics. Generally, 3D bioprinting utilizes the layer-by-layer method to deposit materials known as bioinks to create tissue-like structures that are later used in medical and tissue engineering fields. Bioprinting covers a broad range of biomaterials.

source: Freepik

Innovative handheld 3D bioprinter treats serious burns

The new system which is in the early stages of development may become a way to treat patients whose burn injuries are too extensive to allow skin grafts.

Printing new skin cells on a burn injury may eventually become the new treatment to treat burns, shared a team of researchers from Canada. They have successfully tested the newly developed 'handheld 3D printer'.

The new system which is in the early stages of development may become a way to treat patients whose burn injuries are too extensive to allow skin grafts. The results are reported on Monday (local time) in the IOP publishing journal Biofabrication.

Senior author and professor Axel Gunther, from the University of Toronto, said, "Skin grafts, where the damaged tissue is removed and replaced with skin taken from another area of the patient's body, are a standard treatment for serious burns.

The senior author also shared that while there are alternatives - including scaffolds using bovine collagen or engineered skin substitutes grown in vitro - none are ideal. To overcome these challenges, the research team designed the handheld device to deposit precursor sheets directly onto wounds of any size, shape or topography.

Co-author Dr Marc Jeschke, medical director of the Ross Tilley Burn Centre at Sunnybrook Health Sciences Centre in Toronto, said, "In general, the wound surfaces we designed this device for are not flat, nor are they oriented horizontally. One of the most important advantages of the device is that it should allow for the uniform deposition of a bioink layer onto inclined surfaces.

Marc continued saying that in this study, we tested whether the device could do this effectively by using it to treat full-thickness burns in pigs. We found the device successfully deposited the 'skin sheets' onto the wounds uniformly, safely and reliably, and the sheets stayed in place with only very minimal movement.

"Most significantly, our results showed that the MSC-treated wounds healed extremely well, with a reduction in inflammation, scarring, and contraction compared with both the untreated wounds and those treated with a collagen scaffold," the co-author opined.

The researchers are extremely pleased by the success as well as the excellent healing outcomes of the test.

"However, in cases where a patient has extensive full-thickness burns — which destroy both the upper and lower layers of the skin -— here is not always sufficient healthy skin left to use," Axel added.

Bioinformatics & DNA sequencing

2021-03-05T11:43:40.463Z

Bioinformatics is an amalgamation of biology and information technology, which uses software to link biological data with techniques for information storage, distribution and analysis to support multiple areas of scientific research, including biomedicine.

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Before sequences can be analyzed they have to be obtained from the data storage bank example the Genbank. DNA sequencing is still a non-trivial problem as the raw data may be noisy or afflicted by weak signals. Algorithms have been developed for base calling for the various experimental approaches to DNA sequencing.

source: Freepik

Sequence assembly

Most DNA sequencing techniques produce short fragments of sequence that need to be assembled to obtain complete gene or genome sequences. The so-called shotgun sequencing technique (which was used, for example, by The Institute for Genomic Research (TIGR) to sequence the first bacterial genome, Haemophilus influenzae) generates the sequences of many thousands of small DNA fragments (ranging from 35 to 900 nucleotides long, depending on the sequencing technology).

The ends of these fragments overlap and, when aligned properly by a genome assembly program, can be used to reconstruct the complete genome. Shotgun sequencing yields sequence data quickly, but the task of assembling the fragments can be quite complicated for larger genomes.

For a genome as large as the human genome, it may take many days of CPU time on large-memory, multiprocessor computers to assemble the fragments, and the resulting assembly usually contains numerous gaps that must be filled in later. Shotgun sequencing is the method of choice for virtually all genomes sequenced today, and genome assembly algorithms are a critical area of bioinformatics research.

Genome annotation

In the context of genomics, annotation is the process of marking the genes and other biological features in a DNA sequence. This process needs to be automated because most genomes are too large to annotate by hand, not to mention the desire to annotate as many genomes as possible, as the rate of sequencing has ceased to pose a bottleneck. Annotation is made possible by the fact that genes have recognisable start and stop regions, although the exact sequence found in these regions can vary between genes.

The first description of a comprehensive genome annotation system was published in 1995 by the team at The Institute for Genomic Research that performed the first complete sequencing and analysis of the genome of a free-living organism, the bacterium Haemophilus influenzae.

Owen White designed and built a software system to identify the genes encoding all proteins, transfer RNAs, ribosomal RNAs (and other sites) and to make initial functional assignments. Most current genome annotation systems work similarly, but the programs available for analysis of genomic DNA, such as the GeneMark program trained and used to find protein-coding genes in Haemophilus influenzae, are constantly changing and improving.

Whole Exome Sequencing: Rising Need of Molecular Diagnosis

2021-03-04T10:24:08.282Z

Exome sequencing, also known as whole exome sequencing (WES), is a genomic technique for sequencing all of the protein-coding regions of genes in a genome (known as the exome). It consists of two steps: the first step is to select only the subset of DNA that encodes proteins.

These regions are known as exons – humans have about 180,000 exons, constituting about 1% of the human genome, or approximately 30 million base pairs. The second step is to sequence the exonic DNA using any high-throughput DNA sequencing technology.

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The goal of this approach is to identify genetic variants that alter protein sequences, and to do this at a much lower cost than whole-genome sequencing. Since these variants can be responsible for both Mendelian and common polygenic diseases, such as Alzheimer's disease, whole exome sequencing has been applied both in academic research and as a clinical diagnostic.

source: Freepik

Comparison with other technologies

Microarray-based genotyping

Microarrays use hybridization probes to test the prevalence of known DNA sequences, thus they cannot be used to identify unexpected genetic changes. In contrast, the high-throughput sequencing technologies used in exome sequencing directly provide the nucleotide sequences of DNA at the thousands of exonic loci tested. Hence, WES addresses some of the present limitations of hybridization genotyping arrays.

Although exome sequencing is more expensive than hybridization-based technologies on a per-sample basis, its cost has been decreasing due to the falling cost and increased throughput of whole genome sequencing.

Whole-genome sequencing

Exome sequencing is only able to identify those variants found in the coding region of genes which affect protein function. It is not able to identify the structural and non-coding variants associated with the disease, which can be found using other methods such as whole genome sequencing.

There remains 99% of the human genome that is not covered using exome sequencing. Presently, whole genome sequencing is rarely practical in the clinical context due to the high costs and time associated with sequencing full genomes.

Exome sequencing allows sequencing of portions of the genome over at least 20 times as many samples compared to whole genome sequencing, at the same cost. For translation of identified rare variants into the clinic, sample size and the ability to interpret the results to provide a clinical diagnosis indicates that with the current knowledge in genetics, exome sequencing may be the most valuable.

DNA Sequencing - Evolutionary Biology

2021-03-02T11:53:34.343Z

DNA sequencing may be used to determine the sequence of individual genes, larger genetic regions (i.e. clusters of genes or operons), full chromosomes, or entire genomes of any organism. DNA sequencing is also the most efficient way to indirectly sequence RNA or proteins (via their open reading frames). In fact, DNA sequencing has become a key technology in many areas of biology and other sciences such as medicine, forensics, and anthropology.

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Sequencing is used in molecular biology to study genomes and the proteins they encode. Information obtained using sequencing allows researchers to identify changes in genes, associations with diseases and phenotypes, and identify potential drug targets.

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Evolutionary biology

Since DNA is an informative macromolecule in terms of transmission from one generation to another, DNA sequencing is used in evolutionary biology to study how different organisms are related and how they evolved. In February 2021, scientists reported, for the first time, the sequencing of DNA from animal remains, a mammoth in this instance, over a million years old, the oldest DNA sequenced to date.

Metagenomics

The field of metagenomics involves identification of organisms present in a body of water, sewage, dirt, debris filtered from the air, or swab samples from organisms. Knowing which organisms are present in a particular environment is critical to research in ecology, epidemiology, microbiology, and other fields. Sequencing enables researchers to determine which types of microbes may be present in a microbiome, for example.

Virology

As most viruses are too small to be seen by a light microscope, sequencing is one of the main tools in virology to identify and study the virus. Viral genomes can be based in DNA or RNA. RNA viruses are more time-sensitive for genome sequencing, as they degrade faster in clinical samples.

Traditional Sanger sequencing and next-generation sequencing are used to sequence viruses in basic and clinical research, as well as for the diagnosis of emerging viral infections, molecular epidemiology of viral pathogens, and drug-resistance testing. There are more than 2.3 million unique viral sequences in GenBank. Recently, NGS has surpassed traditional Sanger as the most popular approach for generating viral genomes.

Medicine

Medical technicians may sequence genes (or, theoretically, full genomes) from patients to determine if there is risk of genetic diseases. This is a form of genetic testing, though some genetic tests may not involve DNA sequencing. Also, DNA sequencing may be useful for determining a specific bacteria, to allow for more precise antibiotics treatments, hereby reducing the risk of creating antimicrobial resistance in bacteria populations.

Human Embryonic Stem Cell Sparked a Revolution

2021-03-01T14:44:57.316Z

Embryonic stem cells (ESCs) are the cells of the inner cell mass of a blastocyst, formed prior to implantation in the uterus. In human embryonic development the blastocyst stage is reached 4–5 days after fertilization, at which time it consists of 50–150 cells.

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The human embryonic stem cells are obtained from the undifferentiated inner mass cell of the human embryo and human fetal tissue. The human embryonic stem cell can replicate indefinitely and produce non-regenerative tissue such as myocardial and neural cells. This potential of human embryonic stem cell allows them to provide an unlimited amount of tissue for transplantation therapies to treat a wide range of degenerative diseases. Hence, human embryonic stem cells are used in the treatment of various diseases such as Alzheimer's disease, cancer, blood and genetic disorders related to the immune system and others.

ESCs are pluripotent and give rise during development to all derivatives of the three germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extraembryonic membranes or to the placenta.

source: freepik

During embryonic development the cells of the inner cell mass continuously divide and become more specialized. For example, a portion of the ectoderm in the dorsal part of the embryo specializes as 'neurectoderm', which will become the future central nervous system. Later in development, neurulation causes the neurectoderm to form the neural tube. At the neural tube stage, the anterior portion undergoes encephalization to generate or 'pattern' the basic form of the brain. At this stage of development, the principal cell type of the CNS is considered a neural stem cell.

The neural stem cells self-renew and at some point transition into radial glial progenitor cells (RGPs). Early-formed RGPs self-renew by symmetrical division to form a reservoir group of progenitor cells. These cells transition to a neurogenic state and start to divide asymmetrically to produce a large diversity of many different neuron types, each with unique gene expression, morphological, and functional characteristics.

The process of generating neurons from radial glial cells is called neurogenesis. The radial glial cell, has a distinctive bipolar morphology with highly elongated processes spanning the thickness of the neural tube wall. It shares some glial characteristics, most notably the expression of glial fibrillary acidic protein (GFAP). The radial glial cell is the primary neural stem cell of the developing vertebrate CNS, and its cell body resides in the ventricular zone, adjacent to the developing ventricular system. Neural stem cells are committed to the neuronal lineages (neurons, astrocytes, and oligodendrocytes), and thus their potency is restricted.

Genetic Engineering Leads to Medicine Revolution

2021-02-25T12:10:14.421Z

Genetic engineering, also called genetic modification or genetic manipulation, is the direct manipulation of an organism's genes using biotechnology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. New DNA is obtained by either isolating and copying the genetic material of interest using recombinant DNA methods or by artificially synthesising the DNA. A construct is usually created and used to insert this DNA into the host organism.

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The first recombinant DNA molecule was made by Paul Berg in 1972 by combining DNA from the monkey virus SV40 with the lambda virus. As well as inserting genes, the process can be used to remove, or "knock out", genes. The new DNA can be inserted randomly, or targeted to a specific part of the genome.

Medicine

Genetic engineering has many applications to medicine that include the manufacturing of drugs, creation of model animals that mimic human conditions and gene therapy. One of the earliest uses of genetic engineering was to mass-produce human insulin in bacteria. This application has now been applied to, human growth hormones, follicle stimulating hormones (for treating infertility), human albumin, monoclonal antibodies, antihemophilic factors, vaccines and many other drugs.

Mouse hybridomas, cells fused together to create monoclonal antibodies, have been adapted through genetic engineering to create human monoclonal antibodies. In 2017, genetic engineering of chimeric antigen receptors on a patient's own T-cells was approved by the U.S. FDA as a treatment for the cancer acute lymphoblastic leukemia. Genetically engineered viruses are being developed that can still confer immunity, but lack the infectious sequences.

Genetic engineering is also used to create animal models of human diseases. Genetically modified mice are the most common genetically engineered animal model. They have been used to study and model cancer (the oncomouse), obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging and Parkinson disease. Potential cures can be tested against these mouse models. Also genetically modified pigs have been bred with the aim of increasing the success of pig to human organ transplantation.

Gene therapy is the genetic engineering of humans, generally by replacing defective genes with effective ones. Clinical research using somatic gene therapy has been conducted with several diseases, including X-linked SCID, chronic lymphocytic leukemia (CLL), and Parkinson's disease.

Restriction Enzyme: Tools to Change The Genome

2021-02-19T12:08:18.689Z

A restriction enzyme, restriction endonuclease, or restrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes.

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Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix...

These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction digestion; meanwhile, host DNA is protected by a modification enzyme (a methyltransferase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system.

source: freepik

Artificial restriction enzymes

Artificial restriction enzymes can be generated by fusing a natural or engineered DNA binding domain to a nuclease domain (often the cleavage domain of the type IIS restriction enzyme FokI). Such artificial restriction enzymes can target large DNA sites (up to 36 bp) and can be engineered to bind to desired DNA sequences.

Zinc finger nucleases are the most commonly used artificial restriction enzymes and are generally used in genetic engineering applications, but can also be used for more standard gene cloning applications. Other artificial restriction enzymes are based on the DNA binding domain of TAL effectors.

Artificial ribonucleases that act as restriction enzymes for RNA are also being developed.[needs update] A PNA-based system, called PNAzymes, has a Cu(II)-2,9-dimethylphenanthroline group that mimics ribonucleases for specific RNA sequence and cleaves at a non-base-paired region (RNA bulge) of the targeted RNA formed when the enzyme binds the RNA. This enzyme shows selectivity by cleaving only at one site that either does not have a mismatch or is kinetically preferred out of two possible cleavage sites.

Metabolomics: An Emerging Powerful Tool

2021-02-17T12:08:24.765Z

Metabolomics is the scientific study of chemical processes involving metabolites, the small molecule substrates, intermediates and products of cell metabolism. Specifically, metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind", the study of their small-molecule metabolite profiles.

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The metabolome represents the complete set of metabolites in a biological cell, tissue, organ or organism, which are the end products of cellular processes. Messenger RNA (mRNA), gene expression data and proteomic analyses reveal the set of gene products being produced in the cell, data that represents one aspect of cellular function.

Conversely, metabolic profiling can give an instantaneous snapshot of the physiology of that cell, and thus, metabolomics provides a direct "functional readout of the physiological state" of an organism. One of the challenges of systems biology and functional genomics is to integrate genomics, transcriptomic, proteomic, and metabolomic information to provide a better understanding of cellular biology.

source: Freepik

Key applications

Toxicity assessment/toxicology by metabolic profiling (especially of urine or blood plasma samples) detects the physiological changes caused by toxic insult of a chemical (or mixture of chemicals). In many cases, the observed changes can be related to specific syndromes, e.g. a specific lesion in liver or kidney. This is of particular relevance to pharmaceutical companies wanting to test the toxicity of potential drug candidates: if a compound can be eliminated before it reaches clinical trials on the grounds of adverse toxicity, it saves the enormous expense of the trials.

For functional genomics, metabolomics can be an excellent tool for determining the phenotype caused by a genetic manipulation, such as gene deletion or insertion. Sometimes this can be a sufficient goal in itself-for instance, to detect any phenotypic changes in a genetically modified plant intended for human or animal consumption. More exciting is the prospect of predicting the function of unknown genes by comparison with the metabolic perturbations caused by deletion/insertion of known genes. Such advances are most likely to come from model organisms such as Saccharomyces cerevisiae and Arabidopsis thaliana. The Cravatt laboratory at The Scripps Research Institute has recently applied this technology to mammalian systems, identifying the N-acyltaurines as previously uncharacterized endogenous substrates for the enzyme fatty acid amide hydrolase (FAAH) and the monoalkylglycerol ethers (MAGEs) as endogenous substrates for the uncharacterized hydrolase KIAA1363.

Metabologenomics is a novel approach to integrate metabolomics and genomics data by correlating microbial-exported metabolites with predicted biosynthetic genes. This bioinformatics-based pairing method enables natural product discovery at a larger-scale by refining non-targeted metabolomic analyses to identify small molecules with related biosynthesis and to focus on those that may not have previously well known structures.

Fluxomics is a further development of metabolomics. The disadvantage of metabolomics is that it only provides the user with steady-state level information, while fluxomics determines the reaction rates of metabolic reactions and can trace metabolites in a biological system over time.

Nutrigenomics is a generalised term which links genomics, transcriptomics, proteomics and metabolomics to human nutrition. In general a metabolome in a given body fluid is influenced by endogenous factors such as age, sex, body composition and genetics as well as underlying pathologies. The large bowel microflora are also a very significant potential confounder of metabolic profiles and could be classified as either an endogenous or exogenous factor. The main exogenous factors are diet and drugs. Diet can then be broken down to nutrients and non-nutrients. Metabolomics is one means to determine a biological endpoint, or metabolic fingerprint, which reflects the balance of all these forces on an individual's metabolism.

Western Blotting: Molecular biology on Hight

2021-02-12T12:12:12.419Z

Some clinical research and medical therapies arising from molecular biology are covered under gene therapy whereas the use of molecular biology or molecular cell biology in medicine is now referred to as molecular medicine.

Molecular biology also plays important role in understanding formations, actions, and regulations of various parts of cells which can be used to efficiently target new drugs, diagnose disease, and understand the physiology of the cell.

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Macromolecule blotting and probing

Northern blotting

The northern blot is used to study the expression patterns of a specific type of RNA molecule as relative comparison among a set of different samples of RNA. It is essentially a combination of denaturing RNA gel electrophoresis, and a blot. In this process RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled complement of a sequence of interest.

The results may be visualized through a variety of ways depending on the label used; however, most result in the revelation of bands representing the sizes of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed.

The procedure is commonly used to study when and how much gene expression is occurring by measuring how much of that RNA is present in different samples. It is one of the most basic tools for determining at what time, and under what conditions, certain genes are expressed in living tissues.

Western blotting

In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE. The proteins in the gel are then transferred to a polyvinylidene fluoride (PVDF), nitrocellulose, nylon, or other support membrane.

This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence, or autoradiography. Often, the antibodies are labeled with enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection.

Using western blotting techniques allows not only detection but also quantitative analysis. Analogous methods to western blotting can be used to directly stain specific proteins in live cells or tissue sections.

Eastern blotting

The eastern blotting technique is used to detect post-translational modification of proteins. Proteins blotted on to the PVDF or nitrocellulose membrane are probed for modifications using specific substrates.

Southern blotting

Named after its inventor, biologist Edwin Southern, the Southern blot is a method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples before or after restriction enzyme (restriction endonuclease) digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action. The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest.

Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as PCR, to detect specific DNA sequences from DNA samples. These blots are still used for some applications, however, such as measuring transgene copy number in transgenic mice or in the engineering of gene knockout embryonic stem cell lines.

Automation in Western Blot Technique

Western blot is the major analytical tool to assess the molecular function in basic cell biology research, drug discovery and pathology. There have been significant improvement in the technical aspect of western blot which has led to improvement in efficiency, reproducibility, ease of use. For example, iBind Western Systems launched by ThermoFisher Scientific Corporation which is and automated western blot device which does not require continuous monitoring. Further the turnaround time is reduced compared to traditional method where it involves the various steps and four hours for final results. In iBind all the solution is required to be loaded rest all process is automated by sequential lateral flow technology (SLF) which does not require any electricity or batteries. The significant advantage provided by automated western blot system will contribute in the growth of the western blot market.

Bioinformatics for Molecular Biology

2021-02-10T12:19:04.095Z

Molecular biology is the branch of biology that concerns the molecular basis of biological activity in and between cells, including molecular synthesis, modification, mechanisms and interactions. The central dogma of molecular biology describes the process in which DNA is transcribed into RNA, then translated into protein.

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Relationship to Other Biological Sciences

The following list describes a viewpoint on the interdisciplinary relationships between molecular biology and other related fields.

Molecular biology is the study of the molecular underpinnings of the processes of replication, transcription, translation, and cell function.
Biochemistry is the study of the chemical substances and vital processes occurring in living organisms. Biochemists focus heavily on the role, function, and structure of biomolecules such as proteins, lipids, carbohydrates and nucleic acids.
Genetics is the study of how genetic differences affect organisms. Genetics attempts to predict how mutations, individual genes and genetic interactions can affect the expression of a phenotype

While researchers practice techniques specific to molecular biology, it is common to combine these with methods from genetics and biochemistry. Much of molecular biology is quantitative, and recently a significant amount of work has been done using computer science techniques such as bioinformatics and computational biology. Molecular genetics, the study of gene structure and function, has been among the most prominent sub-fields of molecular biology since the early 2000s.

Other branches of biology are informed by molecular biology, by either directly studying the interactions of molecules in their own right such as in cell biology and developmental biology, or indirectly, where molecular techniques are used to infer historical attributes of populations or species, as in fields in evolutionary biology such as population genetics and phylogenetics. There is also a long tradition of studying biomolecules "from the ground up", or molecularly, in biophysics.