Molecular Biology Tools Research Articles

Fine-scale evaluation of two standard 16S rRNA gene amplicon primer pairs for analysis of total prokaryotes and archaeal nitrifiers in differently managed soils.

The advance of high-throughput molecular biology tools allows in-depth profiling of microbial communities in soils, which possess a high diversity of prokaryotic microorganisms. Amplicon-based sequencing of 16S rRNA genes is the most common approach to studying the richness and composition of soil prokaryotes. To reliably detect different taxonomic lineages of microorganisms in a single soil sample, an adequate pipeline including DNA isolation, primer selection, PCR amplification, library preparation, DNA sequencing, and bioinformatic post-processing is required. Besides DNA sequencing quality and depth, the selection of PCR primers and PCR amplification reactions arguably have the largest influence on the results. This study tested the performance and potential bias of two primer pairs, i.e., 515F (Parada)-806R (Apprill) and 515F (Parada)-926R (Quince) in the standard pipelines of 16S rRNA gene Illumina amplicon sequencing protocol developed by the Earth Microbiome Project (EMP), against shotgun metagenome-based 16S rRNA gene reads. The evaluation was conducted using five differently managed soils. We observed a higher richness of soil total prokaryotes by using reverse primer 806R compared to 926R, contradicting to in silico evaluation results. Both primer pairs revealed various degrees of taxon-specific bias compared to metagenome-derived 16S rRNA gene reads. Nonetheless, we found consistent patterns of microbial community variation associated with different land uses, irrespective of primers used. Total microbial communities, as well as ammonia oxidizing archaea (AOA), the predominant ammonia oxidizers in these soils, shifted along with increased soil pH due to agricultural management. In the unmanaged low pH plot abundance of AOA was dominated by the acid-tolerant NS-Gamma clade, whereas limed agricultural plots were dominated by neutral-alkaliphilic NS-Delta/NS-Alpha clades. This study stresses how primer selection influences community composition and highlights the importance of primer selection for comparative and integrative studies, and that conclusions must be drawn with caution if data from different sequencing pipelines are to be compared.

A Beginner's Guide to Cell Culture: Practical Advice for Preventing Needless Problems.

The cultivation of cells in a favorable artificial environment has become a versatile tool in cellular and molecular biology. Cultured primary cells and continuous cell lines are indispensable in investigations of basic, biomedical, and translation research. However, despite their important role, cell lines are frequently misidentified or contaminated by other cells, bacteria, fungi, yeast, viruses, or chemicals. In addition, handling and manipulating of cells is associated with specific biological and chemical hazards requiring special safeguards such as biosafety cabinets, enclosed containers, and other specialized protective equipment to minimize the risk of exposure to hazardous materials and to guarantee aseptic work conditions. This review provides a brief introduction about the most common problems encountered in cell culture laboratories and some guidelines on preventing or tackling respective problems.

Secreted Protein Acidic and Rich in Cysteine (SPARC)—Mediated Exercise Effects: Illustrative Molecular Pathways against Various Diseases

The strong benefits of exercise, in addition to the development of both the therapeutic applications of physical activity and molecular biology tools, means that it has become very important to explore the underlying molecular patterns linking exercise and its induced phenotypic changes. Within this context, secreted protein acidic and rich in cysteine (SPARC) has been characterized as an exercise-induced protein that would mediate and induce some important effects of exercise. Herein, we suggest some underlying pathways to explain such SPARC-induced exercise-like effects. Such mechanistic mapping would not only allow us to understand the molecular processes of exercise and SPARC effects but would also highlight the potential to develop novel molecular therapies. These therapies would be based on mimicking the exercise benefits via either introducing SPARC or pharmacologically targeting the SPARC-related pathways to produce exercise-like effects. This is of a particular importance for those who do not have the ability to perform the required physical activity due to disabilities or diseases. The main objective of this work is to highlight selected potential therapeutic applications deriving from SPARC properties that have been reported in various publications.

Increasing in situ bioremediation effectiveness through field-scale application of molecular biological tools.

Leveraging the capabilities of microorganisms to reduce (degrade or transform) concentrations of pollutants in soil and groundwater can be a cost-effective, natural remedial approach to manage contaminated sites. Traditional design and implementation of bioremediation strategies consist of lab-scale biodegradation studies or collection of field-scale geochemical data to infer associated biological processes. While both lab-scale biodegradation studies and field-scale geochemical data are useful for remedial decision-making, additional insights can be gained through the application of Molecular Biological Tools (MBTs) to directly measure contaminant-degrading microorganisms and associated bioremediation processes. Field-scale application of a standardized framework pairing MBTs with traditional contaminant and geochemical analyses was successfully performed at two contaminated sites. At a site with trichloroethene (TCE) impacted groundwater, framework application informed design of an enhanced bioremediation approach. Baseline abundances of 16S rRNA genes for a genus of obligate organohalide-respiring bacteria (i.e., Dehalococcoides) were measured at low abundances (101-102 cells/mL) within the TCE source and plume areas. In combination with geochemical analyses, these data suggested that intrinsic biodegradation (i.e., reductive dechlorination) may be occurring, but activities were limited by electron donor availability. The framework was utilized to support development of a full-scale enhanced bioremediation design (i.e., electron donor addition) and to monitor remedial performance. Additionally, the framework was applied at a second site with residual petroleum hydrocarbon (PHC) impacted soils and groundwater. MBTs, specifically qPCR and 16S gene amplicon rRNA sequencing, were used to characterize intrinsic bioremediation mechanisms. Functional genes associated with anaerobic biodegradation of diesel components (e.g., naphthyl-2-methyl-succinate synthase, naphthalene carboxylase, alkylsuccinate synthase, and benzoyl coenzyme A reductase) were measured to be 2-3 orders of magnitude greater than unimpacted, background samples. Intrinsic bioremediation mechanisms were determined to be sufficient to achieve groundwater remediation objectives. Nonetheless, the framework was further utilized to assess that an enhanced bioremediation could be a successful remedial alternative or complement to source area treatment. While bioremediation of chlorinated solvents, PHCs, and other contaminants has been demonstrated to successfully reduce environmental risk and reach site goals, the application of field-scale MBT data in combination with contaminant and geochemical data analyses to design, implement, and monitor a site-specific bioremediation approach can result in more consistent remedy effectiveness.

Efficient Synthesis of α‐P‐Modified Nucleoside Triphosphates and Dinucleoside Tetraphosphates via Linear PVPVPIII‐Nucleoside Intermediates

AbstractAs an important class of nucleoside triphosphate analogues, α‐P‐modified nucleoside triphosphates (NTPαXs) have long been recognized as versatile tools in molecular biology and medicinal chemistry. However, the available synthetic methods based on modified nucleoside monophosphate and cyclic PVPVPIII‐nucleoside intermediates are only low to moderate‐yielding. Inspired by recent progress on PV‐PIII coupling reactions, we attempted to access NTPαXs via the putative linear PVPVPIII‐nucleoside intermediates. The current research found that a specific type of nucleoside phosphoramidites, fluorenylmethyl (Fm) nucleosidyl‐phosphoromorpholidites, are highly reactive in coupling with nBu4NH3P2O7 even without acidic activators. In situ oxidation of linear PVPVPIII‐nucleosides with specific sulfurizing, boranylating, and selenylating reagents followed by deprotection of α‐P and nucleoside moieties afforded NTPαXs (X=S, Se, and BH3) in excellent isolated yields. By exploiting dicyanoimidazole (DCI)’s promoting effect on PV‐PIII coupling, the current linear PVPVPIII‐nucleoside‐based method was further developed into an efficient route to α‐P‐modified dinucleoside tetraphosphates.

Emerging Molecular Targets for Anti-Epileptogenic and Epilepsy Modifying Drugs.

The pharmacological treatment of epilepsy is purely symptomatic. Despite many decades of intensive research, causal treatment of this common neurologic disorder is still unavailable. Nevertheless, it is expected that advances in modern neuroscience and molecular biology tools, as well as improved animal models may accelerate designing antiepileptogenic and epilepsy-modifying drugs. Epileptogenesis triggers a vast array of genomic, epigenomic and transcriptomic changes, which ultimately lead to morphological and functional transformation of specific neuronal circuits resulting in the occurrence of spontaneous convulsive or nonconvulsive seizures. Recent decades unraveled molecular processes and biochemical signaling pathways involved in the proepileptic transformation of brain circuits including oxidative stress, apoptosis, neuroinflammatory and neurotrophic factors. The "omics" data derived from both human and animal epileptic tissues, as well as electrophysiological, imaging and neurochemical analysis identified a plethora of possible molecular targets for drugs, which could interfere with various stages of epileptogenetic cascade, including inflammatory processes and neuroplastic changes. In this narrative review, we briefly present contemporary views on the neurobiological background of epileptogenesis and discuss the advantages and disadvantages of some more promising molecular targets for antiepileptogenic pharmacotherapy.

Clustering Highly Divergent Homologous Proteins: An Alignment‐Free Method

The comparative analysis of amino acid sequences is an important tool in molecular biology that often requires multiple sequence alignments. In comparisons between less closely related genomes, however, it becomes more difficult to accurately align protein-coding sequences, or even to identify homologous regions in different genomes. In this article, we describe an alignment-free method for the classification of homologous protein-coding regions from different genomes. This methodology was originally developed for comparing genomes within virus families, but may be adapted for other organisms. We quantify sequence homology from the overlap (intersection distance) of the k-mer (word) frequency distributions for different protein sequences. Next, we extract groups of homologous sequences from the resulting distance matrix using a combination of dimensionality reduction and hierarchical clustering methods. Finally, we demonstrate how to generate visualizations of the composition of clusters with respect to protein annotations, and by coloring protein-coding regions of genomes by cluster assignments. These provide a useful means to quickly assess the reliability of the clustering results based on the distribution of homologous genes among genomes. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Data collection and processing Basic Protocol 2: Calculating k-mer distances Basic Protocol 3: Extracting clusters of homology Support Protocol: Genome plot based on clustering results.

The Breadth of Bacteriophages Contributing to the Development of the Phage-Based Vaccines for COVID-19: An Ideal Platform to Design the Multiplex Vaccine.

Phages are highly ubiquitous biological agents, which means they are ideal tools for molecular biology and recombinant DNA technology. The development of a phage display technology was a turning point in the design of phage-based vaccines. Phages are now recognized as universal adjuvant-free nanovaccine platforms. Phages are well-suited for vaccine design owing to their high stability in harsh conditions and simple and inexpensive large-scale production. The aim of this review is to summarize the overall breadth of the antiviral therapeutic perspective of phages contributing to the development of phage-based vaccines for COVID-19. We show that phage vaccines induce a strong and specific humoral response by targeted phage particles carrying the epitopes of SARS-CoV-2. Further, the engineering of the T4 bacteriophage by CRISPR (clustered regularly interspaced short palindromic repeats) presents phage vaccines as a valuable platform with potential capabilities of genetic plasticity, intrinsic immunogenicity, and stability.

Immunoinformatic-Based Multi-Epitope Vaccine Design for Co-Infection of Mycobacterium tuberculosis and SARS-CoV-2.

(1) Background: Many co-infections of Mycobacterium tuberculosis (MTB) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have emerged since the occurrence of the SARS-CoV-2 pandemic. This study aims to design an effective preventive multi-epitope vaccine against the co-infection of MTB and SARS-CoV-2. (2) Methods: The three selected proteins (spike protein, diacylglycerol acyltransferase, and low molecular weight T-cell antigen TB8.4) were predicted using bioinformatics, and 16 epitopes with the highest ranks (10 helper T lymphocyte epitopes, 2 CD8+ T lymphocytes epitopes, and 4 B-cell epitopes) were selected and assembled into the candidate vaccine referred to as S7D5L4. The toxicity, sensitization, stability, solubility, antigenicity, and immunogenicity of the S7D5L4 vaccine were evaluated using bioinformatics tools. Subsequently, toll-like receptor 4 docking simulation and discontinuous B-cell epitope prediction were performed. Immune simulation and codon optimization were carried out using immunoinformatics and molecular biology tools. (3) Results: The S7D5L4 vaccine showed good physical properties, such as solubility, stability, non-sensitization, and non-toxicity. This vaccine had excellent antigenicity and immunogenicity and could successfully simulate immune responses in silico. Furthermore, the normal mode analysis of the S7D5L4 vaccine and toll-like receptor 4 docking simulation demonstrated that the vaccine had docking potential and a stable reaction. (4) Conclusions: The S7D5L4 vaccine designed to fight against the co-infection of MTB and SARS-CoV-2 may be safe and effective. The protective efficacy of this promising vaccine should be further verified using in vitro and in vivo experiments.

Branched Linkers for Site-Specific Fluorescent Labeling of Antibodies.

Fluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of which increases along with the degree of labeling. In this work, we propose a methodology for the synthesis of site-specific antibody-dye conjugates with a high degree of labeling. To this end, we synthesized two oxyamine-based branched triazide linkers and coupled them with a periodate-oxidized anti-PRAME antibody 6H8; two oxyamine-based linear monoazide linkers of similar structure were used as controls. The azide-labeled antibodies were subsequently conjugated with fluorescent dyes via SPAAC, a copper-free click reaction. Compared to their counterparts made with linear linkers, the branched conjugates possessed a higher degree of labeling. The utility of the methodology was demonstrated in the detection of the PRAME protein on the surface of the cell by flow cytometry.

A Sweet Method of Modeling Restriction Endonuclease-Based Molecular Cloning

Molecular cloning is an invaluable research tool in modern molecular biology. However, it is often difficult for students to grasp conceptually without visual aids and even more difficult to understand how to successfully set up a cloning experiment. Here, we describe a flipped classroom activity that simulates cloning using donuts as models of plasmids. Students noted in semistructured interviews that the interactive nature of this activity made it an engaging introduction to molecular cloning.

Focused Engineering of Pyrrolysyl-tRNA Synthetase-Based Orthogonal Translation Systems for the Incorporation of Various Noncanonical Amino Acids.

The expansion of the genetic code has become a valuable tool for molecular biology, biochemistry, and biotechnology. The pyrrolysyl-tRNA synthetase (PylRS) variants with their cognate tRNAPyl derived from methanogenic archaea of the genus Methanosarcina are the most popular tools for ribosomally mediated site-specific and proteome-wide statistical incorporation of noncanonical amino acids (ncAAs) into proteins. The incorporation of ncAAs can be used for numerous biotechnological and even therapeutically relevant applications. Here we present a protocol of engineering PylRS for novel substrates with unique chemical functionalities. These functional groups can act as intrinsic probes, especially in complex biological environments such as mammalian cells, tissues, and even whole animals.

Bioinformatic prediction and experimental validation of RiPP recognition elements.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products for which discovery efforts have rapidly grown over the past decade. There are currently 38 known RiPP classes encoded by prokaryotes. Half of the prokaryotic RiPP classes include a protein domain called the RiPP Recognition Element (RRE) for successful installation of post-translational modifications on a RiPP precursor peptide. In most cases, the RRE domain binds to the N-terminal "leader" region of the precursor peptide, facilitating enzymatic modification of the C-terminal "core" region. The prevalence of the RRE domain renders it a theoretically useful bioinformatic handle for class-independent RiPP discovery; however, first-in-class RiPPs have yet to be isolated and experimentally characterized using an RRE-centric strategy. Moreover, with most known RRE domains engaging their cognate precursor peptide(s) with high specificity and nanomolar affinity, evaluation of the residue-specific interactions that govern RRE:substrate complexation is a necessary first step to leveraging the RRE domain for various bioengineering applications. This chapter details protocols for developing custom bioinformatic models to predict and annotate RRE domains in a class-specific manner. Next, we outline methods for experimental validation of precursor peptide binding using fluorescence polarization binding assays and in vitro enzyme activity assays. We anticipate the methods herein will guide and enhance future critical analyses of the RRE domain, eventually enabling its future use as a customizable tool for molecular biology.

Polyploid SNP Genotyping Using the MassARRAY System.

Molecular marker discovery and genotyping are major challenges in polyploid breeding programs incorporating molecular biology tools. In this context, this work describes a method for single nucleotide polymorphism (SNP) genotyping in polyploid crops using matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry, the MassARRAY System.

Small RNA interfering effects in chicken embryonic fibroblasts using two sites 8664/p201 and 2122/pll3.7 of chicken s1p1/edg 1 gene

Introduction: Currently, short interfering RNA (siRNA) is an experimental tool in molecular biology to study gene function in cell culture and in vivo in model of organisms (mouse and chicken embryos, etc.) Method: Lentivirus production technique by human embryonic kidney 293T cell lines (HEK293T) with two (2) sites 8664/P201 and 2122/Pll.3.7 of the sphingosine 1 phosphate receptor gene and endothelial differentiation 1 gene (S1P1/ Chicken EDG1) was used to show the effects of small interfering RNA in chicken embryonic fibroblasts during embryogenesis. After preparation of plasmids 8664/P201 and 2122/Pll3.7, lentivirus packaging, transfection with 2xHBS, virus titration and chicken egg preparation, microinjection of lentivirus was performed in the embryos chicken. Results: The results obtained after the microinjection of lentivirus into chicken embryos from the second day to the fourth day of incubation showed the effects of small interfering RNA in the embryonic development of chicken materialized by bleeding in the yolk sac of embryos therefore a decrease in gene function. Conclusion: Previous studies have already proven that expression of small interfering RNA decreases in embryos of certain vertebrates such as mammals.

DNA Nanoribbon for Efficient Anti-miRNA Peptide Nucleic Acid Delivery and Synergistic Enhancement of Cancer Cell Apoptosis.

Antisense peptide nucleic acid (asPNA), an effective antisense drug, has been employed as a gene therapy agent and a useful tool in molecular biology. Gaining control over the delivery of asPNA to target tissues has been a major hindrance to its wide application in clinical practice. A simple and efficient DNA nanoribbon (DNR)-based drug delivery process has been designed in this study that releases the asPNA agent to inhibit oncogenic microRNAs (miRNAs). Furthermore, we demonstrated how the AS1411 aptamer that binds nucleolin on the cell membranes works as a control mechanism capable of identifying target cancer cells and enhancing the enrichment capacity of DNR. With the biodegradability of DNR, we can efficiently initiate the release of asPNA into the cytoplasm, particularly targeting the intended miR-21 and synergistically increasing programmed cell death 4 (PDCD4) expression to enhance cell apoptosis. We assume that this well-defined delivery mechanism will aid in designing antisense site-specific treatments for various diseases, including cancer.

<i>Lentinus squarrosulus</i> Mont. Mushroom: Molecular Identification, In vitro Anti-Diabetic, Anti-Obesity, and Cytotoxicity Assessment

Mushrooms are fungi with nutritional and health benefits. Lentinus squarrosulus Mont., an edible fungus, has traditional usage and relevance in local therapy for managing metabolic diseases. In that view, this study aimed to evaluate the in vitro anti-obesity, anti-diabetic, and cytotoxic potential of the chloroform/methanol extract (CME) and aqueous extract (AE) of the mushroom. L. squarrosulus was identified using molecular biology tools. The CME and AE were obtained sequentially and, then, subjected to α-amylase, α-glucosidase, and lipase inhibitory enzyme assays as well as total phenolic content (TPC) and flavonoid content (TFC) estimations. The cytotoxic potential of extract fractions of L. squarrosulus was assessed using the brine shrimp lethality assay. The molecular identification of the mushroom displayed that the internal transcribed spacer sequence was an equivalent match to that of L. squarrosulus with a high percentage similarity, and thus assigned a unique accession number (KT120043.1). The CME of L. squarrosulus had higher TPC, TFC, and α-glucosidase inhibitory activity than AE. Furthermore, AE of the mushroom showed a higher lipase inhibitory potential with an IC50 value of 22.28 ± 0.65 μg/mL than the CME, while that of the reference, i.e. orlistat was 2.28 ± 0.34 μg/mL. However, these extracts exhibited very low or no α-amylase inhibitory and cytotoxic activity at the tested concentrations. This study reveals that CME of L. squarrosulus, rich in polyphenols and flavonoids, possesses considerable α-glucosidase and lipase inhibitory activities.

First Detection of KRAS Mutation in Colorectal Cancer Patients in Côte d’Ivoire

Advances in molecular biology tools have made it possible to make progress in terms of therapeutics by acting specifically on the molecular mechanisms involved in the virulence of pathogens or in the development of the disease. In the case of cancer, new therapeutics have been developed thanks to these advances. This is called targeted therapy. Targeted therapy molecules specifically act as a molecule, or a molecular pathway involved in the development of the tumor. The use of targeted therapy drugs therefore requires molecular characterization of tumors. In developing countries, the reference tool is high-throughput sequencing, but due to the high cost, this strategy remains inaccessible for the majority of African populations. It is remains necessary to implement alternative molecular diagnostic tools in order to reduce the disparities currently observed in access to cancer care. The High-Resolution Melting analysis (HRM) method is based on real-time PCR and is described as a simple, rapid and specific method for the detection of somatic mutations predictive of anti-tumor therapeutic response. We demonstrated in this study that this method could be implemented in a lower middle-income country like Côte d’Ivoire, and we used it to detect KRAS mutations in colorectal cancer patients for the first time in Cote d’Ivoire.

A non-destructive coconut fruit and seed traits extraction method based on Micro-CT and deeplabV3+ model.

With the completion of the coconut gene map and the gradual improvement of related molecular biology tools, molecular marker-assisted breeding of coconut has become the next focus of coconut breeding, and accurate coconut phenotypic traits measurement will provide technical support for screening and identifying the correspondence between genotype and phenotype. A Micro-CT system was developed to measure coconut fruits and seeds automatically and nondestructively to acquire the 3D model and phenotyping traits. A deeplabv3+ model with an Xception backbone was used to segment the sectional image of coconut fruits and seeds automatically. Compared with the structural-light system measurement, the mean absolute percentage error of the fruit volume and surface area measurements by the Micro-CT system was 1.87% and 2.24%, respectively, and the squares of the correlation coefficients were 0.977 and 0.964, respectively. In addition, compared with the manual measurements, the mean absolute percentage error of the automatic copra weight and total biomass measurements was 8.85% and 25.19%, respectively, and the adjusted squares of the correlation coefficients were 0.922 and 0.721, respectively. The Micro-CT system can nondestructively obtain up to 21 agronomic traits and 57 digital traits precisely.

Bacillus: a eco-friendly source for plant development and sanity

Bacillus are non-pathogenic gram-positive bacteria capable of producing resistant spores. They have several beneficial characteristics to crop production, such as increased plant growth, resistance to abiotic/biotic stresses, nutritional improvement, and potential for bioremediation of contaminated soils. The use of microorganisms in agriculture has increased due to the reduction in the use of agrochemicals, and sustainable production. Therefore, the present study was based on bibliographical research on the current state of the art of biotechnological prospecting and obtaining commercial products of Bacillus for the agricultural sector. The search was conducted through the Google Academic platform limiting the search to the years 2015 to 2020. Papers that explained in vitro or in vivo prospecting methodologies of Bacillus with promoter or protective activity of plants and those that described microbial inoculants with this genus were selected. Different species of Bacillus can modulate plant growth producing plant hormones. Molecular biology tools such as DNA sequencing and PCR are applied to the study of genes involved in plant growth modulation by Bacillus such as AcPho, ipdC and asbA. For the elaboration of agricultural bioproducts, vegetative structures or endospores are used, under an optimal concentration of 1 x 108 CFU/mL. In the face of species diversity, many have not yet been studied and are promising to become bioproducts. This fact is enhanced by the immeasurable applicability of Bacillus.