Modern Medical Laboratory Journal

A western blot is a laboratory method used to detect specific protein molecules from among a mixture of proteins. This mixture can include all of the proteins associated with a particular tissue or cell type. Western blots can also be used to evaluate the size of a protein of interest, and to measure the amount of protein expression. This procedure was named for its similarity to the previously invented method known as the Southern blot.

The first step in a western blot is to prepare the protein sample by mixing it with a detergent called sodium dodecyl sulfate, which makes the proteins unfold into linear chains and coats then with a negative charge. Next, the protein molecules are separated according to their sizes using a method called gel electrophoresis. Following separation, the proteins are transferred from the gel onto a blotting membrane. Although this step is what gives the technique the name "western blotting," the term is typically used to describe the entire procedure.

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The novel respiratory disease COVID-19 has reached the status of worldwide pandemic and large efforts are currently being undertaken in molecularly characterizing the virus causing it, SARS-CoV-2. The genomic variability of SARS-CoV-2 specimens scattered across the globe can underly geographically specific etiological effects. Italian scientists analyzed and annotated all SARS-CoV-2 mutations compared with the reference Wuhan genome NC_045512.2, observing an average of 7.23 mutations per sample. Their analysis shows the prevalence of single nucleotide transitions as the major mutational type across the world. SARS-CoV-2 is a single-stranded RNA beta-coronavirus with a compact 29,903 nucleotides-long genome. Genetic variance analyses must now play a crucial role in expanding knowledge on this new virus to adopt measures to contain its outbreak. The existing detected mutations allow to group the samples into five distinct clades, G, GH, GR, S, and V, characterized by a collection of specific mutations. The clades can be further characterized by the most recent mutations and will likely be split even further in the future.
Moreover, the Centers for Disease Control and Prevention (CDC) released an update on MAY 5 2021, about SARS-CoV-2 variant classifications and definitions which shows that genetic variants of SARS-CoV-2 have been emerging and circulating around the world throughout the COVID-19 pandemic. 
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Modern gas chromatography (GC) was invented by Martin and James in 1952 [1], and has become one of the most important and widely applied analytical techniques in modern chemistry. Major milestones in the development of GC, especially in column technology, detection and sample introduction are described in this historical review. Many trends in current progress can be seen to originate in the first two decades of the history of GC, but the invention of fused-silica capillary columns greatly increased the application of high-resolution GC across the field of organic analysis; the development of low-cost, bench-top mass spectrometers led to further advances. Progress continues to be rapid in comprehensive 2D GC, fast analysis, detection by atomic emission and time-of-flight mass spectrometry, and in applications to process analysis.
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Wounds are injuries that break the skin, leading to disruption of its normal anatomic structure and function. Wound healing is a dynamic and complex process. Hydrogels are three-dimensional (3D) networks consisting of physically or chemically cross-linked bonds of hydrophilic polymers. The insoluble hydrophilic structures demonstrate a remarkable potential to absorb wound exudates and allows oxygen diffusion to accelerate healing. Importantly, hydrogels possess a highly hydrated 3D polymeric network and can bind several-fold more water as compared to their dry weight and can thereby maintain a high moisture level of the wound bed. Due to these unique physical properties, hydrogel networks can be casted into various sizes and shapes. Therefore, hydrogel-based materials are the most suitable dressings to cover skin wounds. Furthermore, hydrogels offer a platform to load cells, antibacterial agents, growth factors, as well as distinct supplementary and biomacromolecules. With regard to ECM similarity, hydrogels used for wound healing applications should provide a cell-friendly 3D environment to promote tissue regeneration, with or without the presence of cells embedded in the scaffold. Importantly, all hydrogels need to satisfy the basic requirements of biocompatibility in clinical use as well as possess unique physical and mechanical properties suited for skin wound applications. Moreover, they also need to provide the appropriate microenvironment for vessel ingrowth and cellular proliferation.
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Electrophoresis is a chromatography technique by which a mixture of charged molecules is separated according to size when placed in an electric field. 
Electrophoresis is a general term that describes the migration and separation of charged particles (ions) under the influence of an electric field. An electrophoretic system consists of two electrodes of opposite charge (anode, cathode), connected by a conducting medium called an electrolyte. The separation effect on the ionic particles results from differences in their velocity (v), which is the product of the particle's mobility (m) and the field strength (E):
v=mE
The mobility (m) of an ionic particle is determined by particle size, shape, and charge, and the temperature during the separation, and is constant under defined electrophoretic conditions.
 The ability of electrophoresis to separate charged species ranges from small inorganic or organic ions to charged biopolymers (like DNA or proteins), or even chromosomes, microorganisms, or whole cells.
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Bioprinting technologies have been advancing at the convergence of automation, digitalization, and new tissue engineering (TE) approaches. In situ bioprinting may be favored during certain situations when compared with the conventional in vitro bioprinting when de novo tissues are to be printed directly on the intended anatomical location in the living body. 
The portable bioprinting approach involves a thoroughly portable device that allows the deposition of bio-inks (biomaterials and cells) in a direct-write fashion. The portable bioprinter has many advantages such as ease of use and high speed. However, the most prominent advantage of portable bioprinters is that the operator can hold the bioprinter to print bio-ink directly at the wound site, without needing a computer system and defect scanning. 
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Flow cytometry is a technology that provides rapid multi-parametric analysis of single cells in solution. Flow cytometers utilize lasers as light sources to produce both scattered and fluorescent light signals that are read by detectors such as photodiodes or photomultiplier tubes. These signals are converted into electronic signals that are analyzed by a computer and written to a standardized format (.fcs) data file. Cell populations can be analyzed and/or purified based on their fluorescent or light scattering characteristics. A variety of fluorescent reagents are utilized in flow cytometry. These include, fluorescently conjugated antibodies, DNA binding dyes, viability dyes, ion indicator dyes, and fluorescent expression proteins.
The instrumentation used for flow cytometry has evolved over the last several decades. Multiple laser systems are common as are instruments that are designed for specific purposes, such as systems with 96-well loaders designed for bead analysis, systems that combine microscopy and flow cytometry and systems that combine mass spectrometry and flow cytometry.
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Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) have been proposed that are effective in the regeneration of damaged lungs in severe COVID-19 patients. In this regard, In a randomized, double-blind, placebo-controlled phase 2 trial, Shi et al. illustrated that UC-MSCs treatment is a safe and potentially effective therapeutic approach for COVID-19 patients with lung damage. 
Mesenchymal stem cells (MSCs) are non-hematopoietic cells with immune-modulatory, regenerative, and differentiation properties. And their efficacy as a therapeutic method against pathological changes of the lung induced by the influenza virus. moreover, The safety and potential efficacy of MSC have also been evaluated in patients with acute respiratory distress syndrome (ARDS). The immunomodulatory and regenerative properties of MSCs offer a potential cellular therapeutic option for lung damage in patients with COVID-19.
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Nested polymerase chain reaction (Nested PCR) is a modification of PCR that was designed to improve sensitivity and specificity. This technique reduces nonspecific amplification of the DNA template. Nested PCR involves the use of two primer sets and two successive PCR reactions. The first set of primers are designed to anneal to sequences upstream from the second set of primers and are used in an initial PCR reaction. The first reaction is performed with primers that cover the target sequence and some additional sequence flanking both ends of the target sequence. After the first reaction, a second reaction is performed on the products of the first PCR with primers that bind to the target sequence and are within the amplified sequence of the first PCR. This reduces the amount of nonspecific binding because in the second reaction, most of the amplicons of the first reaction only contain the target sequence and its surrounding sequences.
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Decellularized extracellular matrices (dECMs) from mammalian tissues and organs as scaffolds have revolutionized tissue engineering by their ability to retain chemical compositions and three-dimensional microstructures that are similar to native ECMs. These bioscaffolds are subsequently repopulated with patient‐derived cells, thus constructing a personalized neo‐organ and ideally eliminating the need for immunosuppression. The technique of de‐ and recellularization has achieved substantial advances in the field of organ bioengineering.
Among different organs and tissues, whole heart tissue engineering has remained a challenge due to its architecture and biochemistry. The field of whole heart tissue engineering has been revolutionized since the 2008 publication of the first perfusion-decellularized whole heart. A decellularized heart composed of native extracellular matrix has been shown to offer a complex, unique, and natural scaffold that provides both physical and chemical cues required for cardiac function.
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Polymerase chain reaction (PCR) is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the researcher wants to amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies (amplicons).
PCR consist of three main stages: Denaturing – when the double-stranded template DNA is heated to separate it into two single strands. Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA. Extending – when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.
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Intravenous injection of mesenchymal stem cells leads to considerable improvements in the ability to walk and to use their hands in patients with spinal cord injuries.

Fluorescence in situ hybridization (FISH) is widely utilized in genomic and cell biological research as well as for diagnostic applications in preventive and reproductive medicine, and oncology. It is the gold standard technique for the detection of chromosomal abnormalities. FISH is a very straightforward technique that essentially consists in hybridizing a DNA probe to its complementary sequence on chromosomal preparations previously fixed on slides. Probes are labeled either directly, by incorporation of fluorescent nucleotides, or indirectly, by incorporation of reporter molecules that are subsequently detected by fluorescent antibodies or other affinity molecules. Probes and targets are finally visualized in situ by microscopy analysis. 
Despite the high specificity of FISH and the possibility of direct quantitative imaging, some of its key limitations prevent its regular use in diagnostics. 
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Numerous oncogenic and oncolytic viruses (HBV, HCV, HPV, EBV, HIV, Coxsackievirus, reovirus, vaccinia virus, adenovirus) are known to cause and regress various cancer types. Whether SARS-CoV-2 infection could cause or increase the risk of cancer has been of much debate!

The Ames test, Salmonella typhimurium reverse mutation assay, is a bacterial short-term test accomplished in vitro to evaluate the mutagenicity of various environmental carcinogens and toxins such as drugs, dyes, reagents, cosmetics, pesticides and other substances which are easily solubilized in a liquid suspension. It was named to honor Bruce Ames, who first identified and reported the utility of this assay in detecting mutations in 1974. Then, the protocol had been revised by Ames et al. in 1983.
The test employs several histidine-dependent Salmonella strains each carrying different mutations in various genes in the histidine operon. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When the Salmonella tester strains are grown on a minimal media agar plate containing a trace of histidine, only those bacteria that revert to histidine independence (his(+)) are able to form colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner. The Ames test is used as an initial screen to determine the mutagenic potential of new chemicals and drugs. 
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World Oral Health Day is celebrated globally every year on 20 March according to the burden of oral diseases.
The oral cavity has the second largest and diverse microbiota with over 700 species of bacteria. Oral diseases pose a major health burden for many countries and affect people throughout their lifetime, causing pain, discomfort, disfigurement, and even death. The majority of oral health conditions are: dental caries (tooth decay), periodontal diseases, oral cancers (cancer of the lip or mouth), oral manifestations of HIV, oro-dental trauma, cleft lip and palate, and noma (severe gangrenous disease starting in the mouth mostly affecting children). Most oral health conditions are largely preventable and can be treated in their early stages.
The burden of oral diseases can be reduced through public health interventions by addressing common risk factors including:

• promoting a well-balanced diet low in free sugars;
• stopping use of all forms of tobacco, including chewing of areca nuts;
• reducing alcohol consumption; 
• Adequate exposure to fluoride; 
• twice-daily tooth brushing; and
• encouraging the use of protective equipment when doing sports and traveling on bicycles and motorcycles (to reduce the risk of facial injuries).

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay is the first widely accepted colorimetric assay to measure cell proliferation and viability. The use of MTT tetrazolium was first developed by Mossman in 1983. The mechanism of this assay based on the reduction of MTT, a yellow water-soluble tetrazolium dye, primarily by the mitochondrial dehydrogenases (nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes), to insoluble formazan crystals with purple color. Then, a solubilization solution should be added to dissolve the insoluble purple formazan into a colored solution. The absorbance of this colored solution can be quantified by measuring between 500 and 600 nm wavelength by a spectrophotometer.

The “cytokine storm” results from a sudden rise in circulating levels of some pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1), tumor necrosis factor-α (TNF- α), and interferon-γ (IFN- γ). These high levels of cytokines lead to the influx of mast cells that contribute to inflammation into the site of infection. Cytokine storms can occur in different pathological contexts: malignancy, infections, sepsis, acute respiratory distress syndrome (ARDS), and even in auto-inflammatory diseases. Lung injury is one consequence of the cytokine storm that can progress into acute lung injury or its more severe form ARDS.
The results of numerous studies illustrated that cytokine storm is associated with COVID‐19 severity and is also a crucial cause of death from COVID‐19. Nevertheless, Existing evidence indicates relatively low incidence and mild severity of COVID-19 in pediatrics compared with adults. Many hypotheses have been proposed as reasons for the mild to moderate severity of COVID-19 in children and the absence of cytokine storm is one of these hypotheses.

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Enzyme-linked immunosorbent assay (ELISA) as a method that revolutionized medicine discovered by Eva Engvall and Peter Perlman in 1971. 
ELISA is a labeled immunoassay that is considered the gold standard of immunoassays. This immunological test is very sensitive and is commonly used to measure antibodies, antigens, proteins, glycoproteins, and hormones in biological samples. Some examples include diagnosis of HIV infection, pregnancy tests, and measurement of cytokines or soluble receptors in cell supernatant or serum. The detection of these products is accomplished by complexing antibodies and antigens to produce a measurable result. ELISA assays are generally carried out in 96 well plates, allowing multiple samples to be measured in a single experiment.
The four main types of ELISAs are indirect, direct, sandwich, and competitive.
The most straightforward version of these assays is the direct ELISA, a test capable of identifying antigens in a sample by optimizing the formation of antigen-antibody complexes. In a direct ELISA, the primary detection antibody binds directly to the protein of interest. Next, the plate is rewashed to remove any unbound antibody and followed by the addition of a substrate.

Read more about the other types of ELISA