Molecular Genetic Tools Research Articles

Didactic strategy to increase knowledge of the clinical method in genetics

Introduction: the current challenges of Clinical Genetics include the integration into medical practice of the fundamental tools that constitute the diagnostic process, with the basic knowledge of the specialty. However, in training professionals, there is a tendency to prioritize the application of technology. Objective: to increase the knowledge that supports the application of the clinical method in residents of Clinical Genetics.Methods: a quasi-experimental study of longitudinal strategy, educational intervention type (before and after design) was carried out with 25 resident physicians of Clinical Genetics, who gave their consent to participate. Two equivalent instruments were applied, validated by expert criteria, which consisted of five forced choice questions, true and false type, where knowledge necessary for the diagnosis related to Medelian inheritance, congenital defects, molecular genetics, dysmorphic signs and diagnostic tools were explored. Mac Nemar's Chi-square was applied for dichotomous nominal qualitative variables in related or paired samples with a significance level of 0,05, in order to demonstrate the impact of the intervention on each of the evaluated topics. The educational intervention consisted of an interactive talk. Results: it was found that with the educational intervention the knowledge related to dysmorphic signs and diagnostic tools increased statistically significantly. Conclusion: the educational intervention raised the level of knowledge on the subject of dysmorphic signs, as well as on diagnostic tools

Polygenic Liability to Depression Is Associated With Multiple Medical Conditions in the Electronic Health Record: Phenome-wide Association Study of 46,782 Individuals

BackgroundMajor depressive disorder (MDD) is a leading cause of disease-associated disability, with much of the increased burden due to psychiatric and medical comorbidity. This comorbidity partly reflects common genetic influences across conditions. Integrating molecular-genetic tools with health records enables tests of association with the broad range of physiological and clinical phenotypes. However, standard phenome-wide association studies analyze associations with individual genetic variants. For polygenic traits such as MDD, aggregate measures of genetic risk may yield greater insight into associations across the clinical phenome. MethodsWe tested for associations between a genome-wide polygenic risk score for MDD and medical and psychiatric traits in a phenome-wide association study of 46,782 unrelated, European-ancestry participants from the Michigan Genomics Initiative. ResultsThe MDD polygenic risk score was associated with 211 traits from 15 medical and psychiatric disease categories at the phenome-wide significance threshold. After excluding patients with depression, continued associations were observed with respiratory, digestive, neurological, and genitourinary conditions; neoplasms; and mental disorders. Associations with tobacco use disorder, respiratory conditions, and genitourinary conditions persisted after accounting for genetic overlap between depression and other psychiatric traits. Temporal analyses of time-at-first-diagnosis indicated that depression disproportionately preceded chronic pain and substance-related disorders, while asthma disproportionately preceded depression. ConclusionsThe present results can inform the biological links between depression and both mental and systemic diseases. Although MDD polygenic risk scores cannot currently forecast health outcomes with precision at the individual level, as molecular-genetic discoveries for depression increase, these tools may augment risk prediction for medical and psychiatric conditions.

Distribution and characteristics of <i>Ashworthius sidemi</i> Schulz, 1933 (Nematoda, Trichostrongylidae) in wild ungulates in Belarus

The material was collected during the period 2018‒2021 by dissection of internal organs of European bisons, red deers, European roe deers and elks. The found helminths were identified morphologically and using molecular genetic tools. Analysis of 16 ND4 mDNA sequences of A. sidemi revealed low level of genetic diversity which may indicate that Ashworthius sidemi in Belarus spread from one source.

Elucidating miRNA Function in Cancer Biology via the Molecular Genetics' Toolbox.

Micro-RNA (miRNAs) are short non-coding RNAs of about 18–20 nucleotides in length and are implicated in many cellular processes including proliferation, development, differentiation, apoptosis and cell signaling. Furthermore, it is well known that miRNA expression is frequently dysregulated in many cancers. Therefore, this review will highlight the various mechanisms by which microRNAs are dysregulated in cancer. Further highlights include the abundance of molecular genetics tools that are currently available to study miRNA function as well as their advantages and disadvantages with a special focus on various CRISPR/Cas systems This review provides general workflows and some practical considerations when studying miRNA function thus enabling researchers to make informed decisions in regards to the appropriate molecular genetics tool to be utilized for their experiments.

A Saccharomyces eubayanus haploid resource for research studies

Since its identification, Saccharomyces eubayanus has been recognized as the missing parent of the lager hybrid, S. pastorianus. This wild yeast has never been isolated from fermentation environments, thus representing an interesting candidate for evolutionary, ecological and genetic studies. However, it is imperative to develop additional molecular genetics tools to ease manipulation and thus facilitate future studies. With this in mind, we generated a collection of stable haploid strains representative of three main lineages described in S. eubayanus (PB-1, PB-2 and PB-3), by deleting the HO gene using CRISPR-Cas9 and tetrad micromanipulation. Phenotypic characterization under different conditions demonstrated that the haploid derivates were extremely similar to their parental strains. Genomic analysis in three strains highlighted a likely low frequency of off-targets, and sequencing of a single tetrad evidenced no structural variants in any of the haploid spores. Finally, we demonstrate the utilization of the haploid set by challenging the strains under mass-mating conditions. In this way, we found that S. eubayanus under liquid conditions has a preference to remain in a haploid state, unlike S. cerevisiae that mates rapidly. This haploid resource is a novel set of strains for future yeast molecular genetics studies.

Tongue Ulcer as a Manifestation of Secondary HIV-Associated Multidrug-Resistant Tuberculosis

Tuberculosis of the tongue is one of the rare localizations of extrapulmonary tuberculosis. The article presents a clinical case of secondary tuberculous glossitis with concurrent multidrug-resistant pulmonary tuberculosis in a 35-year-old HIV-infected patient. Despite relatively rapid verification of the diagnosis, the clinical situation was difficult due to differential diagnosis with nonspecific lesions, unknown HIV status, and detection of drug resistance of Mycobacterium tuberculosis. This case illustrates the step-by-step diagnosis and treatment of tuberculous ulcers of the tongue and lungs. The comprehensive examination with the use of molecular genetic, bacteriological, histological, and X-ray diagnostic tools contributed to the timely prescription of the adequate anti-tuberculosis therapy regimen.

BinMat: A molecular genetics tool for processing binary data obtained from fragment analysis in R.

Processing and visualising trends in the binary data (presence or absence of electropherogram peaks), obtained from fragment analysis methods in molecular biology, can be a time-consuming and often cumbersome process. Scoring and analysing binary data (from methods, such as AFLPs, ISSRs and RFLPs) entail complex workflows that require a high level of computational and bioinformatic skills. The application presented here (BinMat) is a free, open-source and user-friendly R Shiny programme (https://clarkevansteenderen.shinyapps.io/BINMAT/) that automates the analysis pipeline on one platform. It is also available as an R package on the Comprehensive R Archive Network (CRAN) (https://cran.r-project.org/web/packages/BinMat/index.html). BinMat consolidates replicate sample pairs of binary data into consensus reads, produces summary statistics and allows the user to visualise their data as ordination plots and clustering trees without having to use multiple programmes and input files or rely on previous programming experience.

Myths and Realities about Genetically Modified Food: A Risk-Benefit Analysis

The development and consumption of genetically modified (GM) crops are surrounded by controversy. According to proponents, only molecular biology approaches and genetic engineering tools are realistic food shortage solutions for the world’s ever-growing population. The main purpose of this study is to review the impact of GM products on human, animal, and environmental health. People still reject GM crops not only because of safety concerns, but also for moral reasons. Toxicity, allergies, and possible horizontal gene transfer (HGT) to the environment or to other species have been associated with the marketing of GM products. Moreover, the scarce data available about the long-term implications of using GM crops is another opponent concern. Nevertheless, science has evidenced no harm from GM crops use to date but has, instead, reported several benefits that result from their commercialization, such as economic, environmental, and health benefits for the general public. Legislation and policies about GM product labeling standards are being discussed. To overcome emerging food security challenges, considering quality scientific information is essential rather than leaving the issue and merely moving toward moral discussion. Hence, a risk–benefit analysis is necessary.

An Algorithm for the Selection of Probes for Specific Detection of Human Disease Pathogens Using the DNA Microarray Technology.

The aim of the study was to develop an algorithm for the selection of discriminating probes to identify a wide range of causative agents of human infectious diseases.Materials and MethodsThe algorithm for selecting the probes was implemented in the form of the disprose (DIScrimination PRObe SElection) computer program written in the R language. Additionally, third-party software was used: the BLAST+ and ViennaRNA Package programs. The developed algorithm was tested by selecting specific probes for detecting Chlamydophila (Chlamydia) pneumoniae — an atypical bacterial pathogen causing community-acquired pneumonia (CAP). Nucleotide sequences for analysis were downloaded from the NCBI databank.ResultsAn algorithm for the selection of specific probes capable of detecting human infectious pathogens has been developed. The algorithm is implemented in the form of the disprose modular program, which allows for performing all stages of the probe selection process: loading the nucleotide sequences and their metadata from available databanks, creating local databases, forming a pool of probes, calculating their physicochemical parameters, aligning the probes and sequences contained in local databases, processing and evaluating the alignment results. The algorithm was successfully tested and its performance was confirmed by selecting a set of probes for the specific detection of Chlamydophila pneumoniae. The specificity of the selected probes calculated in silico indicated a low risk of their nonspecific binding and a high potential of using them as molecular genetic diagnostic tools (DNA microarrays, PCR).ConclusionAn algorithm for the selection of specific probes detecting a wide range of human pathogens in clinical biomaterial has been developed and implemented in the form of the disprose modular program. The probes selected using this program can serve as the functional basis of DNA-oriented microarrays able to identify causative agents of polyetiological diseases, such as CAP. Due to the flexibility and openness of the program, the scope of its application can be expanded.

Glial cell line derived neurotrophic factor in brain repair after focal ischemic stroke.

Glial cell line derived neurotrophic factor in brain repair after focal ischemic stroke.

The liverwort Marchantia polymorpha, a model for all ages.

The liverwort Marchantia polymorpha has been known to man for millennia due to its inclusion Greek herbals. Perhaps due to its familiarity and association with growth in, often, man-made disturbed habitats, it was readily used to address fundamental biological questions of the day, including elucidation of land plant life cycles in the late 18th century, the formulation of cell theory early in the 19th century and the discovery of the alternation of generations in land plants in the mid-19th century. Subsequently, Marchantia was used as model in botany classes. With the arrival of the molecular era, its organellar genomes, the chloroplast and mitochondrial, were some of the first to be sequenced from any plant. In the past two decades, molecular genetic tools have been applied such that genes may be manipulated seemingly at will. Here, are past, present, and some views to the future of Marchantia as a model.

Detecting Cell Cycle Stage and Progression in Fission Yeast, Schizosaccharomyces pombe.

We have previously described methods to synchronize cultures of fission yeast, Schizosaccharomyces pombe. In this chapter, we provide methods to detect cell cycle stage in cells and populations of S. pombe. These protocols used fixed samples. First, we describe sample preparation for flow cytometry of bulk DNA content. This technique allows users to monitor progression of DNA replication and detect any perturbation during the synthesis (S) phase of the cell cycle. Second, we describe methods to stain nuclei and septa of fixed yeast cells, and monitor proportions of cell cycle stages within cultures. Together, these methods provide the ability to compare cell cycle progression or delay between cultures, making use of the powerful molecular genetics tool that is S. pombe.

The Axolotl's journey to the modern molecular era.

The salamander Ambystoma mexicanum, commonly called "the axolotl" has a long, illustrious history as a model organism, perhaps with one of the longest track records as a laboratory-bred vertebrate, yet it also holds a prominent place among the emerging model organisms. Or rather it is by now an "emerged" model organism, boasting a full cohort molecular genetic tools that allows an expanding community of researchers in the field to explore the remarkable traits of this animal including regeneration, at cellular and molecular precision-which had been a dream for researchers over the years. This chapter describes the journey to this status, that could be helpful for those developing their respective animal or plant models.

Antibody Staining for Nematodes with Heat-induced Antigen Retrieval (HIAR).

Immunocytochemistry remains a valuable and necessary tool for biologists working with nematodes, even those nematode model organisms with advanced molecular genetic tools and transgenics. Because of the highly idiosyncratic nature of successful immunostaining procedures, innovations can still be found for this long-established technique. Heat-induced antigen retrieval (HIAR) is well known from other systems, but seems not to have been applied to antibody staining in nematodes. For some antigens, adding HIAR to an established antibody staining protocol for nematodes can reveal strong and reliable staining that without HIAR is poor or completely absent.

QTL MAPPING OF BROOMRAPE (OROBANCHE CUMANA WALLR.) RESISTANCE IN SUNFLOWER (HELIANTHUS ANNUUS L.) USING GBS-SNPS

Broomrape is one of the most important biotic stresses causing serious yield reductions in sunflower. Control of this parasitic plant is difficult and physical and chemical strategies are usually insufficient. Therefore, introduction of genetic resistance to broomrape in sunflower is a key breeding goal. Breeding efforts on broomrape resistance have been conducted for decades, however, new broomrape races, such as race F, have emerged and rapidly evolved to be more aggressive and devastating. Although a few quantitative trait loci (QTLs) were identified for race F resistance, none of these loci are suitable for marker assisted selection because of their small phenotypic effects. In the present study, three major QTLs for broomrape race F resistance were identified on LG7, LG11 and LG12 using a high density SNP map constructed with the genotyping by sequencing approach in an intraspecific F2 population. The population consisted of 300 individuals derived from a cross between susceptible Helianthus annuus cv. RHA 436 as the recipient parent and resistant H. annuus cv. H08 M1 as the donor parent. Breeder-friendly SNP-based cleaved amplified polymorphic sequence markers were developed for the QTLs. The QTLs and CAPS markers identified in this study will be valuable molecular genetic tools for sunflower breeding.

Laminar Organization of the Entorhinal Cortex in Macaque Monkeys Based on Cell-Type-Specific Markers and Connectivity.

The entorhinal cortex (EC) is a major gateway between the hippocampus and telencephalic structures, and plays a critical role in memory and navigation. Through the use of various molecular markers and genetic tools, neuron types constituting EC are well studied in rodents, and their layer-dependent distributions, connections, and functions have also been characterized. In primates, however, such cell-type-specific understandings are lagging. To bridge the gap between rodents and primates, here we provide the first cell-type-based global map of EC in macaque monkeys. The laminar organization of the monkey EC was systematically examined and compared with that of the rodent EC by using immunohistochemistry for molecular markers which have been well characterized in the rodent EC: reelin, calbindin, and Purkinje cell protein 4 (PCP4). We further employed retrograde neuron labeling from the nucleus accumbens and amygdala to identify the EC output layer. This cell-type-based approach enabled us to apply the latest laminar definition of rodent EC to monkeys. Based on the similarity of the laminar organization, the monkey EC can be divided into two subdivisions: rostral and caudal EC. These subdivisions likely correspond to the lateral and medial EC in rodents, respectively. In addition, we found an overall absence of a clear laminar arrangement of layer V neurons in the rostral EC, unlike rodents. The cell-type-based architectural map provided in this study will accelerate the application of genetic tools in monkeys for better understanding of the role of EC in memory and navigation.

Genome Sequence Resource for Erysiphe necator NAFU1, a Grapevine Powdery Mildew Isolate Identified in Shaanxi Province of China.

Erysiphe necator is an economically important biotrophic fungal pathogen responsible for powdery mildew disease on grapevine. Currently, genome sequences are available for only a few E. necator isolates from the United States. Based on the combination of Nanopore and Illumina sequencing technologies, we present here the complete genome assembly for an isolate of E. necator, NAFU1, identified in China. We acquired a total of 15.93 Gb of raw reads. These reads were processed into a 61.12-Mb genome assembly containing 73 contigs with an N50 of 2.06 Mb and a maximum length of 6.05 Mb. Combining the results of three gene-prediction modules (i.e., an evidence-based gene modeler [EVidenceModeler], an ab initio gene modeler, and a homology-based gene modeler), we predicted 7,235 protein-coding genes in the assembled genome of E. necator NAFU1. This information will facilitate studies of genome evolution and pathogenicity mechanisms of E. necator and other powdery mildew species through comparative genome sequence analysis and other molecular genetic tools.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Integrated molecular analysis identifies a conserved pericyte gene signature in zebrafish

ABSTRACT Pericytes reside in capillary beds where they share a basement membrane with endothelial cells and regulate their function. However, little is known about embryonic pericyte development, in part, due to lack of specific molecular markers and genetic tools. Here, we applied single cell RNA-sequencing (scRNA-seq) of platelet derived growth factor beta (pdgfrb)-positive cells to molecularly characterize pericytes in zebrafish larvae. scRNA-seq revealed zebrafish cells expressing mouse pericyte gene orthologs, and comparison with bulk RNA-seq from wild-type and pdgfrb mutant larvae further refined a pericyte gene set. Subsequent integration with mouse pericyte scRNA-seq profiles revealed a core set of conserved pericyte genes. Using transgenic reporter lines, we validated pericyte expression of two genes identified in our analysis: NDUFA4 mitochondrial complex associated like 2a (ndufa4l2a), and potassium voltage-gated channel, Isk-related family, member 4 (kcne4). Both reporter lines exhibited pericyte expression in multiple anatomical locations, and kcne4 was also detected in a subset of vascular smooth muscle cells. Thus, our integrated molecular analysis revealed a molecular profile for zebrafish pericytes and allowed us to develop new tools to observe these cells in vivo.

A OneStep Solution to Fix and Stain Cells for Correlative Live and Fixed Microscopy.

Correlating the location of subcellular structures with dynamic cellular behaviors is difficult when working with organisms that lack the molecular genetic tools needed for expressing fluorescent protein fusions. Here, we describe a protocol for fixing, permeabilizing, and staining cells in a single step while imaging on a microscope. In contrast to traditional, multi‐step fixing and staining protocols that take hours, the protocol outlined here achieves satisfactory staining within minutes. This approach takes advantage of well‐characterized small molecules that stain specific subcellular structures, including nuclei, mitochondria, and actin networks. Direct visualization of the entire process allows for rapid optimization of cell fixation and staining, as well as straightforward identification of fixation artifacts. Moreover, live imaging prior to fixation reveals the dynamic history of cellular features, making it particularly useful for model systems without the capacity for expressing fluorescent protein fusions. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Fixing, permeabilizing, and staining mammalian cells in one step on the microscope

Interactomics and personalized pharmacotherapy – present and looking to the future

The review highlights the advances in natural science that form the basis of the concept of personalized and precision medicine (PPM). The provisions of PPM (prediction, prevention, personalization) have been disclosed and modern molecular genetic tools have been shown, which are used in leading scientific and practical biomedical centers to improve the quality of treatment of patients with multifactorial diseases (MFD). The main principles of molecular genetic biomarking of MFDs, as well as gene technologies (CRISPR, non-coding RNA, etc.) used in medical practice and at the stage of clinical trials were highlighted. Particular attention has been paid to molecular genetic methods of targeted therapy for cancer, including antitumor vaccines. Scientific developments in the field of prediction and preventive treatment of MFD have been considered - precision healing technologies of tomorrow. The main provisions of interactomics as an interdisciplinary field of natural science have been highlighted, as well as the applied aspects of this section of fundamental science for the development of diagnostic and treatment-and-prophylactic technologies of a new generation.