Application Of Molecular Biological Methods Research Articles

Current knowledge of the taxonomy and classification of representatives from the order Legionellales (Legionellaceae, Coxiellaceae) Phylum Pseudomonadota

Since the development of microbiology as a science, the identification, systematics and classification of microorganisms have been carried out based on the study of phenotypic characteristics. In the second half of the 20th century, after the discovery of the structure of DNA and polymerase chain reaction, the era of molecular biological research began in microbiology. Since 2002, the use of a complex of molecular biological and phylogenetic methods has been recommended as a guiding approach in the classification and taxonomy of prokaryotes, which has led to reclassification in some taxa. The representation of the taxonomy of Legionella pneumophila (order Legionellales, class Gammaproteobacteria) was formulated after the outbreak of legionnaires’ disease (legionellosis) in the USA in 1976. The description of the causative agent of Q-fever – Coxiella burnetii occurred 40 years earlier, but its classification and taxonomic status in the order Rickettsiales of the class Alphaproteobacteria were presented incorrectly based on the study of available phenotypic characteristics in the 1930s and 1950s. This determined the «fate» of C. burnetii and Q-fever for half a century, included in the list of rickettsiae and rickettsioses traditionally studied by rickettsiologists. As a result of the application of molecular biological methods in the 1990s, in the order Legionellales was replenished by several representatives of the order Rickettsiales, three representatives of which (C. burnetii, Rickettsiella grilli and Wolbachia persica) were moved to the class Gammaproteobacteria. Analysis of the 16S rRNA gene sequence led to the reclassification of Coxiella into a separate genus of gamma-proteobacteria of the phylum Proteobacteria (now Pseudomonadota) along with the genera Legionella, Francisella and Rickettsiella with representatives of which have the greatest phylogenetic relationship. Sequencing of the 16S rRNA gene of C. burnetii isolates from different geographical regions revealed only three nucleotide substitutions, indicating that the nucleotide sequences of these strains are closely related (the degree of homology is 99%), which confirms the phylogenetic uniformity of the genus Coxiella within a single species. A comparative analysis of the proteome of representatives of the order Legionellales and a phylodendrogramm constructed during the study of the main proteins of representatives of the genera Legionella, Coxiella, Acquicella and Rickettsiella (Diplorickettsia) allowed the formation of various clades confirming their monophyla. The C. burnetii genome encodes 24 similar components out of 27 involved in the pathogenesis of L. pneumophila by type IVB secretory system (type IVB secretory systems). Verification of the taxonomic position of C. burnetii is an example of the use of molecular biological methods to solve problems of taxonomy and classification of prokaryotes. It is advisable to use an integrated approach based on the application of «classical» methods of bacteriology and new molecular biological and phylogenetic methods, which will allow to obtain an objective understanding of the microorganisms associated with the human population.

Hepatitis A virus subgenotypes in Latvia, 2008–2021

BackgroundIn Latvia outbreaks of the HAV were observed between 2008 and early 2010 and again in 2017–2018. However, the risks of introducing and spreading infection still exist, as the virus spreads easily when personal hygiene is not followed. MethodsTo determine the spread of HAV subgenotypes in the territory of Latvia the VP1/P2A genomic region of HAV was amplified and sequenced for 259 case serum samples. The study carried out a molecular biological investigation and molecular epidemiological investigation. Demographic data (sex, age), disease data (hepatitis symptoms, hospitalization, vaccination) and epidemiology data (part of the outbreak, possible source of infection, recent travel) were collected. Based on the obtained sequences, the phylogenetic tree was built and analyzed for the homology and belonging to different isolated HAV clusters from other countries. ResultsFrom the obtained data, it was concluded that HAV subgenotype IA had 13 clusters and 12 sporadic cases, HAV subgenotype IB had eight clusters and 11 sporadic cases, HAV subgenotype IIIA had one cluster and nine sporadic cases. It was found that the sources of infection among the investigated cases were different, they were mostly associated with contact with a patient with HAV, travel, as well as between persons who inject drugs and men who have sex with men, and the prevalence of HAV similar sequences was observed in different years. It was concluded that patients with HAV subgenotype IA had the longest hospitalization duration and averaged 9.3 days, while patients with subgenotype IB - 7.3 days, subgenotype IIIA - 7.7 days. Analyzing the data on vaccination, it was found that mostly all were not vaccinated or had an unknown vaccination status. ConclusionsAll of this has led to the conclusion that the application of molecular biological methods of the HAV and a careful analysis of epidemiological data can help to better understand the ways of spreading the infection, investigate local outbreaks, detect cases of imported infection and track the recirculation of the virus.

Impact of the Diet on the Formation of Oxidative Stress and Inflammation Induced by Bacterial Biofilm in the Oral Cavity.

The diet is related to the diversity of bacteria in the oral cavity, and the less diverse microbiota of the oral cavity may favor the growth of pathogenic bacteria of all bacterial complexes. Literature data indicate that disturbances in the balance of the bacterial flora of the oral cavity seem to contribute to both oral diseases, including periodontitis, and systemic diseases. If left untreated, periodontitis can damage the gums and alveolar bones. Improper modern eating habits have an impact on the oral microbiome and the gut microbiome, which increase the risk of several chronic diseases, including inflammatory bowel disease, obesity, type 2 diabetes, cardiovascular disease and cancer. The subject of our consideration is the influence of the traditional diet on the formation of oxidative stress and inflammation caused by bacterial biofilm in the oral cavity. Through dental, biomedical and laboratory studies, we wanted to investigate the effect of individual nutrients contained in specific diets on the induction of oxidative stress inducing inflammation of the soft tissues in the oral cavity in the presence of residual supra- and subgingival biofilm. In our research we used different types of diets marked as W, T, B, F and noninvasively collected biological material in the form of bacterial inoculum from volunteers. The analyzed material was grown on complete and selective media against specific strains of all bacterial complexes. Additionally, the zones of growth inhibition were analyzed based on the disc diffusion method. The research was supplemented with dental and periodontological indicators. The research was supplemented by the application of molecular biology methods related to bacterial DNA isolation, PCR reactions and sequencing. Such selected methods constitute an ideal screening test for the analysis of oral bacterial microbiota. The obtained results suggest that certain types of diet can be an effective prophylaxis in the treatment of civilization diseases such as inflammation of the oral cavity along with periodontal tissues and gingival pockets.

Editorial: Biographical Memoirs, Volume 69

Editorial: Biographical Memoirs, Volume 69

Molecular biological methods in environmental engineering.

Microbes are sensitive to environmental changes and can respond in a short time. Genomics, proteomics, transcriptomics, metabolomics, and multigroup association are used to characterize the composition, function, and metabolism of microorganisms, and to evaluate the environment according to the changes in microorganisms, which has important reference and guiding significance of environmental monitoring, management, and repair. In this paper, the application of molecular biological methods to study environmental microorganisms in the fields of wastewater treatment, pollution control, soil improvement, and environmental monitoring in 2019 is reviewed.

Eric A. Barnard. 2 July 1927—23 May 2018

Eric Barnard was a protein biochemist who played a leading role in the delineation of the molecular components of neuromuscular transmission and the emergence of molecular neuroscience as a scientific discipline. He began his career at King's College London, moving to the State University of Buffalo, New York, in 1965 before returning to Imperial College, London, in 1975. In 1985 he became the Director of the Medical Research Council (MRC) Molecular Neurobiology Unit in Cambridge. Upon retirement from the MRC, he moved to the Royal Free Hospital in London where he continued as Director of Molecular Neurobiology, but in 1998 returned to the University of Cambridge (Department of Pharmacology) as Emeritus Professor. In 2014, at the age of 86, he finally retired from active research. Although Eric was elected FRS for his early pioneering work on the protein chemistry of enzymes and the nicotinic acetylcholine receptor, his seminal contribution, initiated during his time at Imperial, was the application of molecular biological methods to the study of many neurotransmitter receptors. With Ricardo Miledi FRS (and later David Brown FRS and colleagues), he developed the Xenopus oocyte system for the expression of receptors from total tissue mRNA. His was the first group to clone a neurotransmitter receptor subunit cDNA, the nicotinic acetylcholine receptor α subunit of Torpedo marmorata . This was followed by purification and subsequent cloning of inhibitory γ-aminobutyric acid (GABA) A receptor subunit cDNAs. This achievement, driven by Eric and aided by his collaborator Peter Seeburg, led to the discovery of the ligand-gated ion channel superfamily, the discovery of neurotransmitter receptor heterogeneity, and the development of concepts of receptor families and superfamilies. His pioneering work was pivotal for the foundation of modern central nervous system drug discovery.

Detection of rabies virus antigen by the indirect rapid immunohistochemistry test in equines and comparisons with other diagnostic techniques.

Laboratory diagnosis of rabies in equines is essential for distinguishing the disease from other sources of encephalitis. Diagnosis by conventional techniques such as a direct fluorescent antibody test (dFAT) or viral isolation in mice or cell culture can be difficult, and the application of molecular biological methods may be necessary. We performed an indirect rapid immunohistochemistry test (iRIT) for the detection of the rabies virus (RABV) antigen in the central nervous system (CNS) of equines and compared the results with those of other diagnostic techniques. We reviewed result records from the Rabies Diagnosis Laboratory at Instituto Pasteur, São Paulo, Brazil, of 174 samples of equine CNS from July 2014 to June 2016, which were investigated by dFAT, rabies tissue culture infection test (RTCIT), mouse inoculation test (MIT) and reverse transcription-polymerase chain reaction (RT-PCR) followed by genetic sequencing. These samples, 29 presented divergent results among techniques and were selected for the performed in the iRIT. The detected positivity rate was 4/29 (14%) by dFAT, 5/28 (18%) by RTCIT, 10/29 (35%) by MIT and 26/27 (96%) by RT-PCR. We analysed 29 samples through imprints of the cortex, hippocampus, cerebellum and brainstem in slides fixed in 10% buffered formaldehyde. Eighteen samples were identified as positive (62%) by iRIT assay, representing a greater number of positive cases than that detected by dFAT, MIT and RTCIT but not by RT-PCR. Among the brain regions, the brainstem presented the highest positivity (78%), followed by the hippocampus (69%), cerebellum (67%) and cortex (67%). Our results provide evidence that iRIT can contribute to a rapid diagnosis of rabies in equines and that complementary tests should be used to improve diagnostic accuracy in this species.

Molecular biological methods in environmental engineering.

Environmental bioremediation technologies are undoubtedly more economical and environmental friendly, and present a good application prospect. Microorganisms can be used to monitor environmental changes for their quickly respond to environmental changes. Microbiological molecular biology methods have attracted more and more attention from human beings, and their applications in the environment is also expanding. Genomics, transcriptomics, proteomics, and metabolomics are four major molecular biology techniques. This review summarizes application of molecular biological methods in environmental engineering in 2017. The summary includes methods for DNA, RNA, and protein extraction, the application of four major technologies in the environmental field, and commonly used methods for in situ monitoring in the environment, including fluorescence in situ hybridization, real-time quantitative PCR, and amplicons sequencing technology.

Establishment of a genomic DNA extraction protocol for bamboo species

The bamboos Bambusa vulgaris and Guadua angustifolia are considered the most important species in South America. Generally, bamboo taxonomy is conducted by analysis of plants morphology, which sometimes may present morphological similarities, complicating the achievement of precise results. This fact can be changed by application of molecular biology methods; however, the Brazilian research of bamboo in molecular level is still limited. The Brazilian state of Mato Grosso do Sul has fragments of a natural bamboo forest with external characteristics similar to Guadua. In order to provide information about bamboo species, this paper evaluates the application of a modified protocol for DNA extraction from young leaves of two accesses of Guadua cultivated in Campo Grande/MS. It also evaluates the amplification efficiency of the matK and rbcL regions of the DNA isolated. It was possible to extract a mean of 487.5 ng μL-1 of DNA for both species with a purity level of 1.91. The molecular marker rbcL amplified the regions of 300 bp for both species, while the marker matK amplified the regions of 850 bp, also for both accesses. Results show that the modified protocol is useful for bamboo’s DNA extraction, with high concentration, quality, and able to be amplified by PCR.

How Microtubules Activate Kinesin & Dynein Atpase Activity

Understanding the role of cytoskeletal filaments in motility could be essential to the complete elucidation of the motility mechanism, because the movements of motors involve an intimate and concerted partnership between the motor and the cytoskeletal filaments. However, with microtubules, the technical difficulty involved in generating recombinant tubulin has impeded the application of molecular biological methods to the study of the structure-function relationship in microtubules in vitro. We developed experiment systems to purify recombinant tubulins (yeast and human tubulin) and analyzed how microtubule binding activates the ATPase activity of kinesin and dynein motors. The charged-to-alanine scanning mutagenesis analyses of tubulin showed that the H11’-H12 loop and H12 in alpha-tubulin, consisting of only four amino acids (415EEGE), accelerates the rate of ATP hydrolysis in both motors, despite the distinct molecular mechanisms in each kinesin and dynein motor. In both motors, the H12 of beta-tubulin contributes to the stability of the motor-microtubule interactions. Our results indicate that the two motors have independently evolved to use the same loop in alpha-tubulin for ATPase activation, probably because the loop plays, either architecturally or mechanically, an essential role in the motility mechanism. (The talk will also include a practical guide to the use of recombinant tubulin.)

Microbiological Advances in Biohydrometallurgy

The most exciting advances in biohydrometallurgy are occurring in the field of microbiology. The two main technologies employed in biohydrometallurgy, agitated tanks for the processing of refractory concentrates and heaps and dumps for the processing of low-grade ores, are technologically sound and widely practised at commercial scale, but their development began at a time when very little was known of the microorganisms that assisted metals extraction from sulfide ores. During and subsequent to those developments it has been shown that microbial communities in metals extraction are more diverse than originally thought, and extremely robust and adaptable to different and variable environments. Recent advances in genomics and proteomics, exploiting hugely increased computing power and speed, have made it possible to describe not only which microorganisms are present in bioleaching systems, but also what physiological functions are being exercised. The body of knowledge being acquired through the application of molecular biology methods will be used increasingly to monitor microbial behaviour, optimise conditions for more appropriate microbiological activity and/or infer the “microbiological health” of bioreactors (tanks and heaps).

Molecular tools for bathing water assessment in Europe: Balancing social science research with a rapidly developing environmental science evidence-base.

The use of molecular tools, principally qPCR, versus traditional culture-based methods for quantifying microbial parameters (e.g., Fecal Indicator Organisms) in bathing waters generates considerable ongoing debate at the science–policy interface. Advances in science have allowed the development and application of molecular biological methods for rapid (~2 h) quantification of microbial pollution in bathing and recreational waters. In contrast, culture-based methods can take between 18 and 96 h for sample processing. Thus, molecular tools offer an opportunity to provide a more meaningful statement of microbial risk to water-users by providing near-real-time information enabling potentially more informed decision-making with regard to water-based activities. However, complementary studies concerning the potential costs and benefits of adopting rapid methods as a regulatory tool are in short supply. We report on findings from an international Working Group that examined the breadth of social impacts, challenges, and research opportunities associated with the application of molecular tools to bathing water regulations.

Chapter Seven - Characean Internodal Cells as a Model System for the Study of Cell Organization

Giant internodal cells of characean green algae have been widely used for studying cellular physiology. This review emphasizes their significance for understanding cytoarchitecture and cytoplasmic reorganization. The cytoarchitecture of internodal cells undergoes pronounced, cytoskeleton-dependent changes during development and in response to environmental cues. Under bright light, internodes develop alternating bands of acid and alkaline pH at their surface that correlate with the differential size and abundance of cortical organelles and, in the genus Chara, with the size and distribution of convoluted plasma membrane domains known as charasomes. Wounding induces responses ranging from chloroplast detachment to deposition of wound walls. These properties and the possibility for mechanical manipulation make the internodal cell ideal for exploring plasma membrane domains, organelle interactions, vesicle trafficking, and local cell wall deposition. The significance of this model system will further increase with the application of molecular biological methods in combination with metabolomics and proteomics.

Progress in activity-dependent structural plasticity of neural circuits in cortex

Neural circuits of mammalian cerebral cortex have exhibited amazing abilities of structural and functional plasticity in development, learning and memory, neurological and psychiatric diseases. With the new imaging techniques and the application of molecular biology methods, observation neural circuits' structural dynamics within the cortex in vivo at the cellular and synaptic level was possible, so there were many great progresses in the field of the activity-dependent structural plasticity over the past decade. This paper reviewed some of the aspects of the experimental results, focused on the characteristics of dendritic structural plasticity in individual growth and development, rich environment, sensory deprivation, and pathological conditions, as well as learning and memory, especially the dynamics of dendritic spines on morphology and quantity; after that, we introduced axonal structural plasticity, the molecular and cellular mechanisms of structural plasticity, and proposed some future problems to be solved at last.

Natural focality of infections: Current problems and prospects of research

Consideration is given to basic concepts of natural focality that remain debatable (natural focus, epizootic and epidemic processes) and to the most relevant problems that largely determine the pathways of further research in this field, such as application of molecular biological methods to the study of natural foci, modes and forms of pathogen existence at the reservation phase, ways of emergence of “new” diseases with natural focality, and current strategy in disease prevention.

Application of Molecular Biology Methods for Detection of Influenza Viruses in Bulgaria

ABSTRACTInfluenza viruses A and B are world-wide major causative agents of human acute respiratory infections. Three subtypes of HA (H1 to H3) and two subtypes of NA (N1 and N2) are found among influenza A viruses that have caused epidemics and pandemics among humans. For the rapid isolation and identification of influenza viruses in clinical samples variety of methods are available but the use of nucleic acid amplification techniques like RT-PCR has made sensitive diagnosis of influenza virus infection feasible, with the possibility of type and subtype determination. During the season 2002/2003 the laboratory of Influenza and ARD detected 7 type A and 4 type B influenza viruses by RT-PCR in 15 simulated samples from the respiratory panel from SEQC. In the season 2003/2004 - 42 positive results for influenza viruses subtype A/H3N2/were obtained by RT-PCR using specific HA and NA primer pairs from 50 nasal and throat swabs from patients with influenza-like diseases. The RT-PCR provides a sensitive and specific method for detecting, typing and subtyping influenza viruses A and B in clinical samples and can be used in the routine diagnosis of the disease in Bulgaria together with a large complex of classical methods.

Progress in research on iron nutrition and interactions in plants

Iron is an essential microelement for plants and can be a limiting or toxic element according to the environmental growth conditions. Plants have therefore developed a large range of physiological mechanisms to cope with Fe deficiency or Fe overload. The application of molecular biological methods have shed light on the genes, gene products and regulatory mechanisms involved in Fe stress responses, however, the acquisition of physiological data now begins to lagg behind the progress gained by molecular approaches. This review highlights and summarizes the recent progress in plant iron research achieved from the molecular level to the field scale, communicated at the “XIIth International Symposium on Iron Nutrition and Interactions in Plams” held in 2004 in Tokyo.

New insights into transmission of tuberculosis

Considering the fact that tuberculosis is a global problem, that one third of the world population is infected with tuberculosis and that 8-10 million of newly recognized cases are detected per year, the need for acquiring new knowledge on pathogenesis and transmission of tuberculosis becomes obvious. Application of molecular biology methods in clinico-epidemiology practice has identified transmission chain of tuberculous bacilli in numerous intrahospital outbreaks of tuberculosis. CONDITIONS FAVORING TRANSMITION OF TUBERCULOUS: The possibility of transmission primarily depends on the following conditions: contagiousness of the patient who is a source of infection, conditions in which the exposure takes place, length of exposure, as well as the immune status of the exposed individual. RISK FACTORS FOR TRANSSMITION OF TUBERCULOSIS: Unrecognized, i.e., untreated tuberculosis represents a major risk factor for environmental spread of the bacillus. Therefore, early diagnosis and initiation of adequate therapy are priority tasks. High risk of tuberculous bacilli transmission is associated with all diagnostic and therapeutic procedures inducing cough, such as bronchoscopy, endotracheal intubation, induction of sputum, inhalation therapy as well as work in microbiological laboratories dealing with identification of M tuberculosis. Results of numerous studies indicate that there is significant risk for transmission and infection of individuals exposed to the source of infection in family settings, work environment or other close contacts. The problem of exposure of health workers at their workplaces represents a problem in institutions dealing with diagnosis and treatment of tuberculosis. A more comprehensive insight into significance of the problem and application of measures aimed at preventing transmission of tuberculosis should lead to practical application of preventive measures proposed by the International Union Against Tuberculosis and Lung Disease, in both hospital and non-hospital environments.

Problems and perspectives of molecular epidemiology of infectious diseases.

Current tendencies and problems of molecular biology and its application in infectious diseases are discussed. Basic stages of the development of molecular epidemiology as a leading tool for epidemiological studies are reviewed. More than 30 years ago, molecular epidemiology was first used to study nosocomial infections. Special attention is given to the achievements of Russian scientists, especially in the discovery of Astrakhan spotted fever Rickettsia, noncultured forms of Vibrio cholerae, and the microorganism "Montezuma." Also, the development of PCR-based methods of identification and typing of Mycobacterium tuberculosis by Russian specialists and its significant role in strategies for diagnosing and treatment of tuberculosis are discussed. Now, the most important field of application of molecular biology methods is bacterial evolution, especially with regard to pathogenic microorganisms and emerging infections. Searching for the novel pathogenic agents, establishment of the infectious nature of diseases with unclear origin, and determining the precise mechanisms of pathogenicity are most intriguing issues. So, molecular biology should play a major role both in clinical and research fields.

Application of molecular biological methods for studying probiotics and the gut flora.

Increasingly, the microbiological scientific community is relying on molecular biology to define the complexity of the gut flora and to distinguish one organism from the next. This is particularly pertinent in the field of probiotics, and probiotic therapy, where identifying probiotics from the commensal flora is often warranted. Current techniques, including genetic fingerprinting, gene sequencing, oligonucleotide probes and specific primer selection, discriminate closely related bacteria with varying degrees of success. Additional molecular methods being employed to determine the constituents of complex microbiota in this area of research are community analysis, denaturing gradient gel electrophoresis (DGGE)/temperature gradient gel electrophoresis (TGGE), fluorescent in situ hybridisation (FISH) and probe grids. Certain approaches enable specific aetiological agents to be monitored, whereas others allow the effects of dietary intervention on bacterial populations to be studied. Other approaches demonstrate diversity, but may not always enable quantification of the population. At the heart of current molecular methods is sequence information gathered from culturable organisms. However, the diversity and novelty identified when applying these methods to the gut microflora demonstrates how little is known about this ecosystem. Of greater concern is the inherent bias associated with some molecular methods. As we understand more of the complexity and dynamics of this diverse microbiota we will be in a position to develop more robust molecular-based technologies to examine it. In addition to identification of the microbiota and discrimination of probiotic strains from commensal organisms, the future of molecular biology in the field of probiotics and the gut flora will, no doubt, stretch to investigations of functionality and activity of the microflora, and/or specific fractions. The quest will be to demonstrate the roles of probiotic strains in vivo and not simply their presence or absence.