Molecular Biology Laboratory Research Articles

Interdisciplinary approaches to predicting evolutionary biology.

Interdisciplinary approaches to predicting evolutionary biology.

Primerdiffer: a python command-line module for large-scale primer design in haplotype genotyping.

Primer design is a routine practice for modern molecular biology labs. Bioinformatics tools like primer3 and primer-blast have standardized the primer design for a specific region. However, large-scale primer design, especially for genome-wide screening, is still a labor-intensive job for most wet-lab researchers using these pipelines. Here, we present the primerdiffer pipeline, which can be used to batch design primers that differentiate haplotypes on a large scale with precise false priming checking. This command-line interface (CLI) pipeline includes greedy primer search, local and global in silico PCR-based false priming checking, and automated best primer selection. The local CLI application provides flexibility to design primers with the user's own genome sequences and specific parameters. Some species-specific primers designed to genotype the hybrid introgression strains from Caenorhabditis briggsae and Caenorhabditis nigoni have been validated using single-worm PCR. This pipeline provides the first CLI-based large-scale primer design tool to differentiate haplotypes in any targeted region. The open-source python modules are available at github (https://github.com/runsheng/primerdiffer, https://github.com/runsheng/primervcf) and Python package index (https://pypi.org/project/primerdiffer/, https://pypi.org/project/primervcf/).

Interview with the 2023 Hooke medal winner Andrew Carter

ABSTRACT Andrew Carter studied biochemistry at the University of Oxford, where he discovered his love for structural biology, which prompted him to join Venki Ramakrishnan's lab at the MRC Laboratory of Molecular Biology (LMB) for his PhD. Here, he used X-ray crystallography to study the ribosome and its interactions with antibiotics. He then spent a year as a postdoc at Clare College, Cambridge, before moving to Ron Vale's lab at the University of California, San Francisco (UCSF), USA, where he first began his work on dyneins. Andrew has since devoted his research career to studying dynein, and in 2010 set up his own lab back at the MRC LMB, where his research has contributed significantly to the dynein and motor protein fields. In recognition of this work, Andrew was recently awarded the British Society of Cell Biology (BSCB) Hooke Medal award for 2023 and we caught up with him to ask about this award, his research and his career.

Directed evolution of glyphosate oxidase and a chemiluminescence system for glyphosate detection: A comprehensive practical laboratory experiment on biotechnology.

This article describes a comprehensive practical laboratory method for developing an enzyme to more easily measure glyphosate levels in solution. Through this article, undergraduate students of biology majors can conduct research experiments in critical fields by utilizing various techniques, such as chemiluminescence (CL) biosensors with engineered enzymes and are guided in molecular biology laboratories. A glyphosate oxidase mutant library was constructed by DNA shuffling, and a glyphosate oxidase variant with increased glyphosate degradation activity was selected by using a high-throughput screening assay. Following protein overexpression in Escherichia coli (DE3) and purification by affinity chromatography, the glyphosate oxidase variant protein combined with luminol-H2 O2 reaction was constructed as a new CL biosensor for detecting glyphosate in soils.

Drug repositioning prediction for psoriasis using the adverse event reporting database.

Inverse signals produced from disproportional analyses using spontaneous drug adverse event reports can be used for drug repositioning purposes. The purpose of this study is to predict drug candidates using a computational method that integrates reported drug adverse event data, disease-specific gene expression profiles, and drug-induced gene expression profiles. Drug and adverse events from 2015 through 2020 were downloaded from the United States Food and Drug Administration Adverse Event Reporting System (FAERS). The reporting odds ratio (ROR), information component (IC) and empirical Bayes geometric mean (EBGM) were used to calculate the inverse signals. Psoriasis was selected as the target disease. Disease specific gene expression profiles were obtained by the meta-analysis of the Gene Expression Omnibus (GEO). The reverse gene expression scores were calculated using the Library of Integrated Network-based Cellular Signatures (LINCS) and their correlations with the inverse signals were obtained. Reversal genes and the candidate compounds were identified. Additionally, these correlations were validated using the relationship between the reverse gene expression scores and the half-maximal inhibitory concentration (IC50) values from the Chemical European Molecular Biology Laboratory (ChEMBL). Inverse signals produced from a disproportional analysis can be used for drug repositioning and to predict drug candidates against psoriasis.

Study of the Genetic Diversity of Tiger Nut (<i>Cyperus esculentus</i>) Ecotypes of Niger Using SSR Markers

Tiger Nuts (<i>Cyperus esculentus </i>L.), a member of the Cyperaceae family, is a plant cultivated in Niger for its tubers. The present study focuses on the molecular characterization of 10 Tiger Nuts (<i>Cyperus esculentus</i> L.) ecotypes from Niger. These Tiger Nuts ecotypes were collected in the Tiger Nuts producing regions of Niger (Dosso and Maradi). DNA extraction and genetic diversity analysis were carried out at the molecular biology laboratories of ICRISAT, Hyderabad, India. Codominant nuclear and polymorphic microsatellite (SSR) markers were used. The genetic diversity parameters calculated are: polymorphism rate (P) at the 95% threshold, allelic diversity (A), observed (Ho) and expected (He) heterozygosity rate and the panmixy deviation in a subpopulation (Fis) under the Hardy-Weinberg hypotheses. The results showed that genetic diversity ranged from 0.03 (StvCyR_181a and StvCyR_327ska) to 0.60 (StvCyR_93ska) with a mean of 0.28 and. For the Tiger Nuts producing regions, the average deficit in heterozygotes is highly significant, with a mean Fis of 0.415 (Fis, 1000 permutation test, P<0.001). A significant amount of variability (27%) from differentiation between the ecotypes studied was observed in the analysis of molecular variance. These results clearly show a genetic differentiation between the populations of the large, small and wild Tiger Nuts, grouping them into three distinct groups. The study also showed that the genetic structure of the Tiger Nuts is not linked to a particular geographical origin and that the molecular tests give us evidence of conserved variability in the Niger Tiger Nuts ecotypes.

Simple and low-cost antibiotic susceptibility testing for Mycobacterium tuberculosis using screen-printed electrodes.

One quarter of the global population is thought to be latently infected by Mycobacterium tuberculosis (TB) with it estimated that 1 in 10 of those people will go on to develop active disease. Due to the fact that M. tuberculosis (TB) is a disease most often associated with low- and middle-income countries, it is critical that low-cost and easy-to-use technological solutions are developed, which can have a direct impact on diagnosis and prescribing practice for TB. One area where intervention could be particularly useful is antibiotic susceptibility testing (AST). This work presents a low-cost, simple-to-use AST sensor that can detect drug susceptibility on the basis of changing RNA abundance for the typically slow-growing M. tuberculosis (TB) pathogen in 96h using screen-printed electrodes and standard molecular biology laboratory reactionware. In order to find out the sensitivity of applied sensor platform, a different concentration (108 -103 CFU/mL) of M. tuberculosis was performed, and limit of detection and limit of quantitation were calculated as 103.82 and 1011.59 CFU/mL, respectively. The results display that it was possible to detect TB sequences and distinguish antibiotic-treated cells from untreated cells with a label-free molecular detection. These findings pave the way for the development of a comprehensive, low-cost, and simple-to-use AST system for prescribing in TB and multidrug-resistant tuberculosis.

Eddy Fischer-Renaissance scientist and bon viveur.

IUBMB LifeEarly View SPECIAL ISSUE Eddy Fischer—Renaissance scientist and bon viveur Tony Hunter, Corresponding Author Tony Hunter hunter@salk.edu orcid.org/0000-0002-7691-6993 Molecular and Cell Biology Laboratory, Salk Institute, La Jolla, California, USA Correspondence Tony Hunter, Molecular and Cell Biology Laboratory, Salk Institute, La Jolla, CA, USA. Email: hunter@salk.eduSearch for more papers by this author Tony Hunter, Corresponding Author Tony Hunter hunter@salk.edu orcid.org/0000-0002-7691-6993 Molecular and Cell Biology Laboratory, Salk Institute, La Jolla, California, USA Correspondence Tony Hunter, Molecular and Cell Biology Laboratory, Salk Institute, La Jolla, CA, USA. Email: hunter@salk.eduSearch for more papers by this author First published: 21 February 2023 https://doi.org/10.1002/iub.2708Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Early ViewOnline Version of Record before inclusion in an issue RelatedInformation

Stability and Molecular Characterization of Heat Tolerant Genotypes of Chickpea (Cicer arietinum L.)

Twenty eight diverse genotypes sown in three different dates were screened using thirty three SSR primers. Twelve morphological characters recorded. The current study was conducted at all India Coordinated Research Project on Chickpea at R.A.K., College of Agriculture, Sehore (M.P.) during Rabi 2020-21, and 2021-22. The molecular work was carried out at Plant Molecular Biology Laboratory, Department of Plant Molecular Biology and Biotechnology, College of Agriculture, Rajmata Vijayaraje Scindia Krishi Vishwa Vidyalaya, Gwalior (M.P.). The component G×E interaction were found significant for flower initiation, days to 50% flowering, days to pod initiation, plant height, days to maturity, number of pods per plant, number of empty pods per plant, number of seeds per plant, biological yield per plant, harvest index, 100 seed weight and seed yield per plant. The highest gene diversity was found in TA-135 (0.7474) followed by GAA-44 (0.7219), GAA-40 (0.7015), STMS-2 (0.6939), TA-71 (0.6709), NCPGR-1 (0.6403) and TA-18 (0.3648). Based on a dendrogram all the 28 genotypes were grouped into three major clusters, in which cluster I contained 2 genotypes, cluster II contained 5 genotypes and cluster III encompassed remaining 21 genotypes. Genotypes RVG 204, JG-14, and RVSSG-61 were found stable for favourable and unfavourable sowing conditions, while ICC-4958, JG-11, JG-12, RVG-203, RVG-204, RVSSG-52, JG-74, RVSSG-71 showed consistent performance during unfavourable sowing conditions for seed yield per plant. The important traits and marker based diversity and stability has been discussed in this research paper.

First person – Sherman Foo

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Sherman Foo is first author on ‘ Diacylglycerol at the inner nuclear membrane fuels nuclear envelope expansion in closed mitosis’, published in JCS. Sherman conducted the research described in this article while a PhD student in the labs of Snezhana Oliferenko (The Francis Crick Institute, London, UK, and Randall Centre for Cell and Molecular Biophysics, King's College London, UK) and Markus R. Wenk (Singapore Lipidomics Incubator, National University of Singapore). He is now a postdoc in the lab of Buzz Baum at Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK, where he is currently studying cellular organization of hyperthermophilic archaea, with a particular focus on the surface layer and cell cycle of Sulfolobus acidocaldarius.

BioBlocksLab: A portable DIY Bio Lab using BioBlocks language.

Standard molecular biology laboratories are usually made with complex, sophisticated, and expensive equipment. Unfortunately, most of these labs are not affordable for everyone. In this paper, we show how we built a portable bio lab BioBlocksLab, made of four modules: a centrifuge, a thermocycler, electrophoresis, and an incubator. We also propose a new version of a blockly programming language to describe experimental lab protocols, called BioBlocks 2.0, which is based on the Microsoft MakeCode platform from the open-source project Microsoft Programming Experience Toolkit (PXT). We run BioBlocks programs of real lab protocols to control different hardware modules with biological reagents and get positive results. We offer an easy, affordable, and open-source way for everyone to do experiments with Do-It-Yourself (DIY) portable bio-labs.

Matrix Metalloproteinase-3 but Not Matrix Metalloproteinase-9, Implicated in the Manifestation of Chronic Periodontitis.

Periodontal disease is an inflammatory condition affecting the tooth's supporting tissues, resulting in gradual loss of periodontal ligament (PDL), alveolar bone, and gum resorption. Neutrophil and monocyte/macrophage, destructive proteases like matrix metalloproteinase (MMP)-3 and MMP-9 play pivotal roles in such lesions in periodontitis. Therefore, this study aims to compare the level of MMP-3 and MMP-9 gene expression in patients with or without periodontitis in an Iranian population. This cross-sectional study was carried out on 22 chronic periodontitis patients and 17 healthy control subjects referred to the department of periodontology, Mashhad Dental School. In both groups, the gingival tissue was removed during surgery and transferred to the Molecular Biology Laboratory for MMP-3 and MMP-9 gene expression evaluation. The qRT-PCR, TaqMan method was used for gene expression assessments. The average age of periodontitis patients was 33± 5 years, and in controls, 34.7± 6 with no significant differences. The mean MMP-3 expression in periodontitis patients was 146.67±38.7, and in controls, 63.4±9.1. The difference was statistically significant (P=0.04). The mean expression of MMP-9 in periodontitis patients and controls were 103.8± 21.66 and 87.57± 16.05, respectively. Although the target gene expression in patients was higher, the difference was insignificant. Furthermore, there was not any significant correlation between age or gender with the expression of MMP3 or MMP9. The study demonstrated that the MMP3 seems to have a destructive impact on the gingival tissue in chronic periodontitis, but not MMP9.

Using RNAi to Examine the Connection between Phenotype and Genotype in Caenorhabditis elegans to Enhance the Undergraduate Research Experience

Gene expression plays a pivotal role in the development, differentiation, and maintenance of organisms by allowing genes to encode for an observable trait (i.e., phenotype). To understand the function of a particular gene, several approaches can be taken, ranging from removing the gene entirely to targeting the product of the gene (i.e., the protein). RNA interference (RNAi) has been shown to be a powerful approach used to silence gene activity and examine the connection between DNA and protein along with controlling gene expression. The course-based undergraduate research experience (CURE) described in this article is a hands-on molecular biology lab–based lesson that allows students to examine the impact of RNAi on Caenorhabditis elegans reproduction and development through the examination of the central dogma of biology. Through these activities, students gain practice in the scientific method of inquiry by designing experiments to observe how genotype connects to phenotype and, subsequently, organism behavior.

Deteksi Plasmodium knowlesi Menggunakan Nested Polymerase Chain Reaction (PCR) di Kecamatan Muara Komam Kalimantan Timur

Plasmodium knowlesi is a parasite of the genus plasmodium that naturally infects long-tailed macaques (Macaca fascicularis), but currently reported has ability to infect humans. The identification/detection of P. knowlesi can be done using RDT, microscopic, or molecular examinations using nested PCR. Nested PCR is the most sensitive and specific method of examination to date. This study aimed to detect P. knowlesi in humans by RDT, microscopic, and nested PCR examinations. The study was descriptive with a cross-sectional approach, carried out from March to July 2019. The samples in this study were 123 patients who were suspected of being infected with malaria and who underwent laboratory tests at the Muara Komam Health Center. Microscopic examination and RDT examination were carried out at the Muara Komam Health Center, while nested PCR was carried out at the Eijkman Molecular Biology Laboratory Jakarta. The results of RDT and microscopic examinations showed as many as 16 of 123 (13%) malaria-positive samples of P. falciparum and P. vivax, and 10 of 123 (8.1%) malaria-positive samples of P. falciparum and P. vivax. Nested PCR tests targeting the rRNA SSU gene were able to identify P. knowlesi by 6 out of 123 (4.87%). In conclusion, the study showed that Plasmodium knowlesi was detected in humans in Muara Komam, East Kalimantan through nested PCR examination.

A research program-linked, course-based undergraduate research experience that allows undergraduates to participate in current research on mycobacterial gene regulation.

Undergraduate instructional biology laboratories are typically taught within two paradigms. Some labs focus on protocols and techniques delivered in "cookbook" format with defined experimental outcomes. There is increasing momentum to alternatively employ student-driven, open-ended, and discovery-based strategies, often via course-based undergraduate research experiences (CUREs) using crowd-sourcing initiatives. A fraction of students also participate in funded research in faculty research labs, where they have opportunities to work on projects designed to expand the frontiers of human knowledge. These experiences are widely recognized as valuable but are not scalable, as most institutions have many more undergraduates than research lab positions. We sought to address this gap through our department's curriculum by creating an opportunity for students to participate in the real-world research process within a laboratory course. We conceived, developed, and delivered an authentic, guided research experience to students in an upper-level molecular biology laboratory course. We refer to this model as a "research program-linked CURE." The research questions come directly from a faculty member's research lab and evolve along with that research program. Students study post-transcriptional regulation in mycobacteria. We use current molecular biology methodologies to test hypotheses like "UTRs affect RNA and protein expression levels," "there is functional redundancy among RNA helicases," and "carbon starvation alters mRNA 5' end chemistries." We conducted standard assessments and developed a customized "Skills and Concepts Inventory" survey to gauge how well the course met our student learning outcomes. We report the results of our assessments and describe challenges addressed during development and execution of the course, including organizing activities to fit within an instructional lab, balancing breadth with depth, and maintaining authenticity while giving students the experience of obtaining interpretable and novel results. Our data suggest student learning was enhanced through this truly authentic research approach. Further, students were able to perceive they were participants and contributors within an active research paradigm. Students reported increases in their self-identification as scientists, and a positive impact on their career trajectories. An additional benefit was reciprocation back to the funded research laboratory, by funneling course alumni, results, materials, and protocols.

Genesis-DB: a database for autonomous laboratory systems.

Artificial intelligence (AI)-driven laboratory automation-combining robotic labware and autonomous software agents-is a powerful trend in modern biology. We developed Genesis-DB, a database system designed to support AI-driven autonomous laboratories by providing software agents access to large quantities of structured domain information. In addition, we present a new ontology for modeling data and metadata from autonomously performed yeast microchemostat cultivations in the framework of the Genesis robot scientist system. We show an example of how Genesis-DB enables the research life cycle by modeling yeast gene regulation, guiding future hypotheses generation and design of experiments. Genesis-DB supports AI-driven discovery through automated reasoning and its design is portable, generic, and easily extensible to other AI-driven molecular biology laboratory data and beyond. Genesis-DB code and installation instructions are available at the GitHub repository https://github.com/TW-Genesis/genesis-database-system.git. The database use case demo code and data are also available through GitHub (https://github.com/TW-Genesis/genesis-database-demo.git). The ontology can be downloaded here: https://github.com/TW-Genesis/genesis-ontology/releases/download/v0.0.23/genesis.owl. The ontology term descriptions (including mappings to existing ontologies) and maintenance standard operating procedures can be found at: https://github.com/TW-Genesis/genesis-ontology.

Anatomical respiratory organ of Solen sp

Respiratory organs are important to investigate in Solen sp. due to the fact that literacy is hard to find beside unclear data information. Excellent knowledge of the respiratory organs in biota has become one of the impact factors for knowing its potential in adaptability and ecology. This study aimed to describe the anatomical respiratory organ by analyzing Solen sp. in the Madura Strait, which applied morphological and histological analysis at the Laboratory of Molecular Biology at the State University of Surabaya. The environmental parameters (temperature, pH, salinity, and substrate) and sample of Solen sp. was measured and collected around the Modung district, Bangkalan regency during July – August, 2023. The results showed conditions are within the range of environmental parameters and are reasonable and appropriate for the environment. The shape and structure of the respiratory organs Solen sp. described the shape of the folds with a fresh red color, as are generally of the normal and health gills. The relationship between environmental parameters and anatomical organ of Solen sp. should be concerning for the future research to protect Solen sp. from the overfishing.

Efficient Large DNA Fragment Knock-in by Long dsDNA with 3'-Overhangs Mediated CRISPR Knock-in (LOCK) in Mammalian Cells.

An efficient and precise genome-editing approach is in high demand in any molecular biology or cell biology laboratory worldwide. However, despite a recent rapid progress in the toolbox tailored for precise genome-editing, including the base editors and prime editors, there is still a need for a cost-effective knock-in (KI) approach amenable for long donor DNA cargos with high efficiency. By harnessing the high-efficient double-strand break (DSB) repair pathway of microhomology-mediated end joining, we previously showed that a specially designed 3'-overhang double-strand DNA (odsDNA) donor harboring 50-nt homology arm (HA) allows high-efficient exogenous DNA KI when combined with CRISPR-Cas9 technology. The lengths of the 3'-overhangs of odsDNA donors could be manipulated by the five consecutive phosphorothioate (PT) modifications. In this protocol, we detail the stepwise procedures to conduct the LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in) method for gene-sized (~1-3 kb) KI in mammalian cells.

Selection of Affibody Affinity Proteins from Phagemid Libraries.

Herein, we describe a general protocol for the selection of target-binding affinity protein molecules from a phagemid-encoded library. The protocol is based on our experience with phage display selections of non-immunoglobulin affibody affinity proteins but can in principle be applied to perform biopanning experiments from any phage-displayed affinity protein library available in a similar phagemid vector. The procedure begins with an amplification of the library from frozen bacterial glycerol stocks via cultivation and helper phage superinfection, followed by a step-by-step instruction of target protein preparation, selection cycles, and post-selection analyses. The described procedures in this standard protocol are relatively conservative and rely on ordinary reagents and equipment available in most molecular biology laboratories.

A Rapid Protocol for Preparing 8-Aminopyrene-1,3,6-Trisulfonate-Labeled Glycans for Capillary Electrophoresis-Based Enzyme Assays.

Purified glycan standards are required for glycan arrays, characterizing substrate specificities of glycan-active enzymes, and to serve as retention-time or mobility standards for various separation techniques. This chapter describes a method for the rapid separation, and subsequent desalting, of glycans labeled with the highly fluorescent fluorophore 8-aminopyrene-1,3,6-trisulfonate (APTS). By using fluorophore-assisted carbohydrate electrophoresis (FACE) on polyacrylamide gels, a technique amenable to equipment readily available in most molecular biology laboratories, many APTS-labeled glycans can be simultaneously resolved. Excising specific gel bands containing the desired APTS-labeled glycans, followed by glycan elution from the gel by simple diffusion and subsequent solid-phase extraction (SPE)-based desalting, affords a single glycan species free of excess labeling reagents and buffer components. The described protocol also offers a simple, rapid method for the simultaneous removal of excess APTS and unlabeled glycan material from reaction mixtures. This chapter describes a FACE/SPE procedure ideal for preparing glycans for capillary electrophoresis (CE)-based enzyme assays, as well as for the purification of rare, commercially unavailable glycans from tissue culture samples.