Molecular Biology Protocols Research Articles

Practical laboratory class to assess gene silencing using CRISPR interference (CRISPRi) technology in the archaeon Haloferax volcanii.

Perturbation of gene expression using RNA interference (RNAi) or CRISPR interference (CRISPRi) is a useful strategy to explore the function of essential genes. In the archaeon Haloferax volcanii, the CRISPR-Cas system has been adapted as a CRISPRi tool to silence the expression of specific genes. We developed a laboratory class (LC) to conceptualize gene silencing through inactivation of the H. volcanii LonB protease gene, a negative regulator of carotenoid pigments biosynthesis, using CRISPRi. This LC has been successfully applied in the Biology and Biochemistry of Microorganisms course for undergraduate students of Biology in 2022 and 2023. The following objectives were proposed: (a) generate H. volcanii mutant strains with reduced expression of the lonB gene using CRISPRi; (b) examine the effect of lonB gene silencing on cell pigmentation and growth rate; (c) assess lonB gene repression by Western blotting (WB). This LC allows students to obtain and screen CRISPRi silenced-mutants by means of simple procedures using a non-pathogenic organism as well as handle basic microbiology, biochemistry and molecular biology protocols. Additionally, the LC fosters social actions through collaborative work (experimental work), the interpretation and discussion of data and the ability to communicate outcomes orally and in a written format (scientific report).

Resolution of RHCE Haplotype Ambiguities in Transfusion Settings.

Red blood cell (RBC) transfusion, limited by patient alloimmunization, demands accurate blood group typing. The Rh system requires specific attention due to the limitations of serological phenotyping methods. Although these have been compensated for by molecular biology solutions, some RhCE ambiguities remain unresolved. The RHCE mRNA length is compatible with full-length analysis and haplotype discrimination, but the RHCE mRNA analyses reported so far are based on reticulocyte isolation and molecular biology protocols that are fastidious to implement in a routine context. We aim to present the most efficient reticulocyte isolation method, combined with an RT-PCR sequencing protocol that embraces the phasing of all haplotype configurations and identification of any allele. Two protocols were tested for reticulocyte isolation based either on their size/density properties or on their specific antigenicity. We show that the reticulocyte sorting method by antigen specificity from EDTA blood samples collected up to 48 h before processing is the most efficient and that the combination of an RHCE-specific RT-PCR followed by RHCE allele-specific sequencing enables analysis of cDNA RHCE haplotypes. All samples analyzed show full concordance between RHCE phenotype and haplotype sequencing. Two samples from the immunohematology laboratory with ambiguous results were successfully analyzed and resolved, one of them displaying a novel RHCE allele (RHCE*03 c.340C>T).

An Automated Versatile Diagnostic Workflow for Infectious Disease Detection in Low-Resource Settings.

Laboratory automation effectively increases the throughput in sample analysis, reduces human errors in sample processing, as well as simplifies and accelerates the overall logistics. Automating diagnostic testing workflows in peripheral laboratories and also in near-patient settings -like hospitals, clinics and epidemic control checkpoints- is advantageous for the simultaneous processing of multiple samples to provide rapid results to patients, minimize the possibility of contamination or error during sample handling or transport, and increase efficiency. However, most automation platforms are expensive and are not easily adaptable to new protocols. Here, we address the need for a versatile, easy-to-use, rapid and reliable diagnostic testing workflow by combining open-source modular automation (Opentrons) and automation-compatible molecular biology protocols, easily adaptable to a workflow for infectious diseases diagnosis by detection on paper-based diagnostics. We demonstrated the feasibility of automation of the method with a low-cost Neisseria meningitidis diagnostic test that utilizes magnetic beads for pathogen DNA isolation, isothermal amplification, and detection on a paper-based microarray. In summary, we integrated open-source modular automation with adaptable molecular biology protocols, which was also faster and cheaper to perform in an automated than in a manual way. This enables a versatile diagnostic workflow for infectious diseases and we demonstrated this through a low-cost N. meningitidis test on paper-based microarrays.

Improving an rRNA depletion protocol with statistical design of experiments.

In prokaryotic RNA-seq library preparation, rRNA depletion is required to remove highly abundant rRNA transcripts from total RNA. rRNA is so abundant that small improvements in depletion efficiency lead to large increases in mRNA sequencing coverage. The current gold-standard method for rRNA depletion makes rRNA depletion the most expensive step in prokaryotic RNA-seq library preparation. A variety of commercial and home-made methods exist to lower the cost or increase the efficiency of rRNA removal. Many of these techniques are suboptimal when applied to new species of bacteria or when the protocol or reagents need to be changed. Re-optimizing a protocol by trial-and-error is an expensive and laborious process. Systematic frameworks like the statistical design of experiments (DOE) can efficiently improve processes by exploring the quantitative relationship between multiple factors. DOE allows experimenters to find factor interactions that may not be apparent when factors are studied in isolation. We used DOE to optimize an rRNA depletion protocol by updating reagents and identifying factors that maximize rRNA removal and minimize cost. The optimized protocol more efficiently removes rRNA, uses fewer reagents, and is less expensive than the original protocol. Our optimization required only 36experiments and identified two significant interactions among three protocol factors. Overall, our approach demonstrates the utility of a rational, DOE framework for improving complex molecular biology protocols.

Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids

Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function, and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine, and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of several hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent invitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.

An efficient and cost-effective method for directed mutagenesis at multiple dispersed sites-a case study with Omicron Spike DNA.

Site-directed mutagenesis is an invaluable technique that enables the elucidation of the contribution of specific residues to protein structure and function. The simultaneous introduction of mutations at a large number of sites (>10), singly and in multiple combinations, is often necessary to fully understand the functional contributions. We report a simple, efficient, time and cost-effective method to achieve this using commonly available molecular biology reagents and protocols, as an alternative to gene synthesis. We demonstrate this method using the Omicron Spike DNA construct as an example, and create a construct bearing 37 mutations (as compared to wild-type Spike DNA), as well as 4 other constructs bearing subsets of the full spectrum of mutations. We believe that this method can be an excellent alternative to gene synthesis, especially when three or more variants are required.

Cloning and Expression Analysis of bZIP Transcription Factor from Finger Millet (Eleusine coracana L.) Under Abiotic Stress Conditions

Basic leucine zipper (bZIP) transcription factors comprise one of the largest gene families in plants. They play a key role in almost every aspect of plant growth and development and also in biotic and abiotic stress tolerance. In this study, we were attempted to study characterization of bZIP, a transcription factor from a climate smart cereal finger millet (Eleusine coracana L.). Seeds of Eleusine coracana (finger millet) was purchase from local market and were grown under field conditions drought and salt stress conditions. In this study, EcbZIP gene was isolated from finger millet, cloned into DH5α cells, screened by using colony PCR and expression analysis in response to two abiotic stresses was carried out by using qRT PCR. EcbZIP coding DNA sequence and protein sequence were retrieved from NCBI Nucleotide Database and Genpept of Accession number KP033192.1 and AJP67539.1 and validated by using SMART (simple modular architecture tool) Domain Tool. Cloning and expression studies were carried out using standardized molecular biology protocol. Results depicted that EcbZIP transcription factor showed significant upregulation under both salt and drought stress conditions, indicating that it plays an important role in tolerance towards these stresses. In conclusion, expression analysis of bZIP gene from finger millet seed cultivar ML-365 showed 5-fold upregulation to salt stress to drought stress and 8-fold upregulation to salt stress. Hence, it can serve as a candidate gene for improving abiotic stress tolerance and can be helpful in enhancing the crop productivity under stress conditions.

Auto-qPCR; a python-based web app for automated and reproducible analysis of qPCR data

Quantifying changes in DNA and RNA levels is essential in numerous molecular biology protocols. Quantitative real time PCR (qPCR) techniques have evolved to become commonplace, however, data analysis includes many time-consuming and cumbersome steps, which can lead to mistakes and misinterpretation of data. To address these bottlenecks, we have developed an open-source Python software to automate processing of result spreadsheets from qPCR machines, employing calculations usually performed manually. Auto-qPCR is a tool that saves time when computing qPCR data, helping to ensure reproducibility of qPCR experiment analyses. Our web-based app (https://auto-q-pcr.com/) is easy to use and does not require programming knowledge or software installation. Using Auto-qPCR, we provide examples of data treatment, display and statistical analyses for four different data processing modes within one program: (1) DNA quantification to identify genomic deletion or duplication events; (2) assessment of gene expression levels using an absolute model, and relative quantification (3) with or (4) without a reference sample. Our open access Auto-qPCR software saves the time of manual data analysis and provides a more systematic workflow, minimizing the risk of errors. Our program constitutes a new tool that can be incorporated into bioinformatic and molecular biology pipelines in clinical and research labs.

Optimized Protocol for Cyanobacterial 16S rRNA Analysis in Danube Delta Lakes

Molecular biology protocols have been more and more accessible to researchers for ecological investigations. However, these protocols always require optimization steps for the analysis of specific types of samples. This study aimed to optimize a molecular protocol to analyze cyanobacterial 16S rRNA in Danube Delta shallows lakes. In this regard, several commercial DNA extraction kits were tested compared to the potassium ethyl xanthogenate extraction method on different matrices. The obtained DNA was further used for 16S rRNA PCR optimization. Finally, an optimized protocol is proposed for the molecular analysis of the cyanobacteria group in freshwater samples. The best DNA extraction method was the potassium xanthogenate extraction from dried cyanobacterial biomass. A dynamic in total genomic eDNA was observed, reflecting the seasonal difference in phytoplankton biomass from the studied lakes. The PCR protocol optimized by us can be successfully applied for the identification of a broad range of cyanobacterial genetic markers.

The Clinical Characteristics and Survival Profiles of Wilms Tumor in the United Arab Emirates: A Single-center Retrospective Analysis

Background: Wilms Tumor (WT) is the most common pediatric kidney tumor, yet the disease has variable clinical characteristics and prognostic outcomes across different populations. Objectives: This study aims to review the clinical characteristics and survival outcomes of children with WT who have received treatment at Dubai Hospital, UAE. Methods: A retrospective study was conducted involving the medical records of confirmed WT children (aged < 14 years) who had received treatment between 2013 and 2018. Diagnosis should have been established based on the histopathological examination of operable tumors and needle biopsy for inoperable tumors. The Société Internationale d’Oncologie Pédiatrique (SIOP) WT 2001 protocol was used for patient management, and the UMBRELLA SIOP–RTSG 2016 Wilms tumor pathology and molecular biology protocol was used for histopathologic classification and clinical staging. The presenting sign and symptoms, the findings of diagnostic imaging techniques, histological grading, the received treatments, and follow-up outcomes were collected and analyzed. Kaplan-Meier survival analysis was used to carry out the survival analysis. Results: Ten children were diagnosed with WT (median age of 3.40 years, 60% males). All patients presented with abdominal masses without prominent pain. Synchronous bilateral lesions were found at diagnosis in one patient and metastatic lesions in three patients. Four children were discharged against medical advice; five received treatment according to the SIOP WT 2001 regimen, while the remaining patient was managed in the United States according to the National Wilms Tumor Study Group (NWTSG) protocol. The overall 6-year and relapse-free survival rates were 90% and 80%, respectively. Conclusion: The clinical characteristics and managemental outcomes of children presenting with WT are promising, possibly owing to adopting the SIOP protocol. Considering the small sample size, more large-scale, nation-wide studies are warranted.

Caracterización fenotípica y genotípica de cultivares de cacao (Theobroma cacao L.) de Dibulla, La Guajira, Colombia

En la Sierra Nevada de Santa Marta los cultivos de cacao están conformados mayoritariamente por cultivares híbridos comerciales y, aunque se encuentran cacaos nativos, estos son poco cultivados. Dada la necesidad de verificar si estos cultivares de cacao encontrados en la Sierra pertenecen al grupo genético tipo Criollo, se realizó una caracterización fenotípica y genotípica de cacaos del municipio Dibulla, La Guajira. Para esto, se muestrearon 11 cultivares en Mingueo. Los rasgos fenotípicos se evaluaron empleando descriptores UPOV para cacao. Los parámetros cualitativos y cuantitativos se cotejaron por análisis de conglomerado y análisis de componentes principales (ACP), y las variables cuantitativas se compararon a través de la prueba no paramétrica test de Mann-Whitney. Para evaluar las relaciones genéticas, se estandarizaron protocolos de biología molecular y se secuenció la región ITS. A partir de las secuencias, se realizaron agrupamientos por métodos de distancia y filogenéticos. Finalmente, se encontraron diferencias significativas entre las semillas (p = 0,01), y resalta la coloración blanca del cotiledón de los criollos en contraste con la coloración púrpura oscura de los híbridos. Asimismo, los análisis de conglomerados, ACP y los análisis de secuencias demostraron diferencias entre el grupo de los cacaos nativos y los híbridos comerciales cultivados; además, los cacaos nativos se emparentan con el grupo de cacao tipo Criollo.

Development of an Apolipoprotein AI Chimera for Targeted Nanodisc Drug Delivery to Breast Cancer Cells

A current challenge in cancer treatment is the ability to selectively target diseased cells with chemotherapeutics. Several promising treatments suffer this limitation and have toxic off target effects on healthy cells. Our overall objective is to generate a platform for targeted drug delivery employing self‐assembling nanodiscs comprised of phospholipids and apolipoprotein AI (apoAI). ApoAI is an exchangeable apolipoprotein located on high density lipoproteins (HDL), which promotes cholesterol efflux from macrophages and facilitates selective uptake of cholesterol by binding scavenger receptor, class B type 1 (SR‐B1), also known as the HDL receptor. We hypothesize that a chimeric protein of apoAI with MT1‐AF7p (apoAI‐AF7p), a peptide ligand that binds membrane metalloproteinase 14 (MMP14), will enhance the targeting capability of the nanodiscs to MDA‐MB‐231 breast cancer cells. MMP14 is a cell surface protein that is overexpressed in many types of cancers responsible for metastasis and linked to poor prognosis in patients. We designed a construct bearing the coding sequence for human apoAI with a hexa‐His‐tag at the N‐terminal end to facilitate protein purification by affinity chromatography, and MT1‐AF7p (HWKHLHNTKTFL) at the C‐terminal end with an intervening linker segment ([G4S]3). The construct was designed and custom‐generated with optimized codon sequence in a pCR2.1 vector. We successfully sub‐cloned the chimeric insert into a pET20b(+) expression vector employing standard molecular biology protocols and verified the resulting plasmid through Sanger sequencing. Recombinant apoAI and apoAI‐AF7p proteins were over‐expressed in E. coli and purified using a nickel affinity chromatography. The purified proteins were visualized by 15% acrylamide SDS‐PAGE, which revealed major bands at ~ 29 kDa band for apoAI and 32 kDa band for apoAI‐AF7p consistent with their expected size difference. We obtained ~ 10 mg purified protein/L culture medium, which is sufficient to carry out structural and functional characterization. Chimeric apoAI‐AF7p nanodiscs have potential not only for targeting, but also for drug delivery since MDA‐MB‐231 cells are reported to have high expression levels of both SR‐B1 and MMP14 which mediates homing and selective uptake of the hydrophobic core of HDL respectively. Preliminary data from fluorescence microscopy indicates the presence of both these proteins as well as the LDLr. Additionally, preliminary circular dichroism data shows similar helical content in the chimera as apoA1. Our next steps are to characterize the expression of MMP14, SR‐B1, and LDLr on MDA‐MB‐231 cells via FACS, qPCR and immunoblotting as well prepare nanodiscs with the chimera. Once particle formation and protein surface expression is established, cells will be incubated with particles and we’ll assess cell binding characteristics. The chimeric nanodiscs bears potential not only for targeting breast cancer cells, but also for drug delivery since these cells are reported to have high expression levels of SRB1, which mediate uptake of the contents within the hydrophobic core.Support or Funding InformationThis project is supported by the National Institute of Health (NIH) under the award numbers GM105561 (VN), R25GM071638 (DK) and T34GM008074 (RM)ApoA1‐AF7p chimera design: hexa‐His tag at the N‐terminus of ApoA1 and the affinity peptide MT1‐AF7p at the C terminus.Figure 1

Addressing Long-Standing Questions with Advanced Approaches: The 4th B Chromosome Conference

B chromosomes (Bs) are enigmatic accessory genomic elements extensively characterized in diverse eukaryotes. Since their discovery in the beginning of the 20th century, B chromosomes have been the subject of investigation in laboratories all around the world. As a consequence, scientific meetings have dealt with B chromosomes, including the most specific and important conference in the field, “The B Chromosome Conference.” The 4th B Chromosome Conference (4BCC) took place in Botucatu, Brazil, in 2019 and was an excellent opportunity to discuss the latest developments in the B chromosome research field. B chromosome science has advanced from classical and molecular cytogenetics to genomics and bioinformatics approaches. The recent advances in next-generation sequencing technologies and high-throughput molecular biology protocols have led Bs to be the subject of massive data analysis, thus enabling the investigation of structural and functional issues not considered before. Although extensive progress has been made, questions are still remaining to be answered. The advances in functional studies based on RNA, epigenetics, and gene ontologies open the perspective to a better understanding of the complex biology of B chromosomes.

Drawing Flowcharts of Lab Protocols Helps Students Prepare for Biology Labs

With the publishing of the Vision and Change report, we know it is best practice to include authentic research experiences in our undergraduate science lab classes. One big challenge in teaching so-called "wet lab" classes is figuring out a way to make sure students come to lab prepared to successfully complete their experiments. Molecular biology protocols are particularly challenging as they are typically long, detailed, and have multiple steps to complete. The most successful teaching practice I have tried is having students prepare for lab by hand-drawing flowcharts of the lab protocols. Drawing is a proven way to increase scientific understanding and requires students to engage with the lab materials before class. These flowcharts are due when students walk in to lab and more importantly, students use their flowcharts during lab. This teaching tool is easy to teach to students, simple to assess, and does not rely on any pre-existing knowledge of molecular biology or artistic skill. I have had great success using flowcharts as a teaching tool in both upper division and lower division classes as well as with both life science major and non-major students. Flowcharts have many potential applications beyond undergraduate "wet lab" classes including discussion courses and graduate research projects.

Next Generation Sequencing (NGS) Application in Multiparameter Gene Expression Analysis.

Next generation sequencing (NGS) is routinely used in gene expression analyses. In particular, RNA-seq has been the method of choice for highly sensitive genome-wide quantification of RNA expression. The method can be used in a wide variety of model systems, including studies to gain insight into underlying mechanisms of toxicologic processes and disease development induced by environmental toxicants. RNA-seq has also been coupled to many other molecular biology protocols to monitor specific aspects of the gene expression process. Here, we describe two such coupling-(a) global run-on sequencing (GRO-seq) that coupled it to the nuclear run-on (NRO), and (b) polysome profiling that coupled it to sucrose-gradient-based polysome isolation. Simultaneous RNA-seq, GRO-seq, and polysome profiling analyses enabled genome-wide analysis of the mode of stability control of individual RNA molecules.

Heterologous Biosynthesis of Type II Polyketide Products Using E. coli

The heterologous biosynthesis of complex natural products has enabled access to polyketide, nonribosomal peptide, isoprenoid, and other compounds with wide-spanning societal value. Though several surrogate host systems exist, Escherichia coli is often a preferred choice due to its rapid growth kinetics and extensive molecular biology protocols. However, a persistent challenge to the utilization of E. coli has been the successful in vivo reconstitution of type II polyketide synthase (PKS) systems. In particular, gene expression of the ketosynthase (KS) components of the minimal PKS has consistently yielded insoluble protein products. In the following report, two type II PKS systems were functionally reconstituted in E. coli. The approach to do so relied upon the utilization of the native transcriptional coupling between the dimeric KS subunits, leading to soluble recombinant protein products and successful polyketide biosynthesis. Resulting strains produced 10 mg/L TW95c and 25 mg/L dehydrorabelomycin. Hence, the strategy offers a new option in the biosynthetic engineering efforts for the heterologous production of type II polyketide products using E. coli.

A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field.

Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION sequencer. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure a reliable and robust performance under suboptimal field conditions without increasing costs. Here, we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding, and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 1000 reads per sample. ONTrack-derived consensus barcodes have a high accuracy, ranging from 99.8 to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions.

Transposon insertion profiling by sequencing (TIPseq) for mapping LINE-1 insertions in the human genome

BackgroundTransposable elements make up a significant portion of the human genome. Accurately locating these mobile DNAs is vital to understand their role as a source of structural variation and somatic mutation. To this end, laboratories have developed strategies to selectively amplify or otherwise enrich transposable element insertion sites in genomic DNA.ResultsHere we describe a technique, Transposon Insertion Profiling by sequencing (TIPseq), to map Long INterspersed Element 1 (LINE-1, L1) retrotransposon insertions in the human genome. This method uses vectorette PCR to amplify species-specific L1 (L1PA1) insertion sites followed by paired-end Illumina sequencing. In addition to providing a step-by-step molecular biology protocol, we offer users a guide to our pipeline for data analysis, TIPseqHunter. Our recent studies in pancreatic and ovarian cancer demonstrate the ability of TIPseq to identify invariant (fixed), polymorphic (inherited variants), as well as somatically-acquired L1 insertions that distinguish cancer genomes from a patient’s constitutional make-up.ConclusionsTIPseq provides an approach for amplifying evolutionarily young, active transposable element insertion sites from genomic DNA. Our rationale and variations on this protocol may be useful to those mapping L1 and other mobile elements in complex genomes.

Biopreservation potential of antimicrobial protein producing Pediococcus spp. towards selected food samples in comparison with chemical preservatives

The present study elucidates biopreservation potential of an antimicrobial protein; bacteriocin, producing Pediococcus spp. isolated from dairy sample and enhancement of their shelf life in comparison with two chemical preservatives. The antimicrobial protein producing Pediococcus spp. was isolated from selected diary samples and characterised by standard microbiology and molecular biology protocols. The cell free supernatant of Pediococcus spp. was applied on the selected food samples and monitored on daily basis. Antimicrobial potential of the partially purified protein from this bacterium was tested against clinical isolates by well diffusion assay. The preservation efficiency of bacteriocin producing isolate at various concentrations was tested against selected food samples and compared with two chemical preservatives such as sodium sulphite and sodium benzoate. The bacteriocin was partially purified and the microbiological qualities of the biopreservative treated food samples were assessed. The present study suggested that 100 μg/l of bacteriocin extract demonstrated antimicrobial potential against E. coli and Shigella spp. The treatment with the Pediococcus spp. showed enhanced preservation at 15 mL/kg of selected samples for a period of 15 days in comparison with sodium sulphite and sodium benzoate. The microbiological quality of food samples treated with biopreservative showed lesser total bacterial count (CFU/g) in comparison with the food samples applied with chemicals (p ≤ 0.05). Thus, the present study suggests that bacteriocin producing Pediococcus probably provides enhanced shelf life to the selected food samples and can be used as biopreservatives.

Visualizing biologically active small molecules in cells using click chemistry

Natural products and synthetic small molecules can be used to perturb, dissect and manipulate biological processes, thereby providing the basis for drug development. Over the past decades, the evolution of molecular biology protocols and microscopy techniques has made it possible to visually detect proteins in living systems with valuable spatiotemporal resolution, in which dynamic topological information has proved to be insightful. By contrast, although small molecules have become essential for biological studies, general methods to track them in cells remain underexplored. In this Review, we discuss how bioorthogonal chemistry, and click chemistry in particular, can be exploited to label and visualize almost any biologically active small molecule in cells and tissues. We review recent developments, highlighting cases in which visualizing small molecules has provided crucial mechanistic insights. This methodology is facile to implement, is versatile and is illuminating. Click chemistry enables efficient chemical labelling of small molecules in cells, providing a powerful method to visualize almost any biologically active compound. This versatile methodology can provide valuable information about the mechanisms of action of small molecules in various biological settings.