Enzymatic Routes for Chiral Amine Synthesis: Protein Engineering and Process Optimization.

Chiral amines are essential motifs in pharmaceuticals, agrochemicals, and specialty chemicals. While traditional chemical routes to chiral amines often lack stereoselectivity and require harsh conditions, biocatalytic methods using engineered enzymes can offer high efficiency and selectivity under sustainable conditions. This review discusses recent advances in protein engineering of transaminases, oxidases, and other enzymes to improve catalytic performance. Strategies such as directed evolution, immobilization, and computational redesign have expanded substrate scope and enhanced efficiency. Furthermore, process optimization guided by techno-economic assessments has been crucial for establishing viable biomanufacturing routes. Combining state-of-the-art enzyme engineering with multifaceted process development will enable scalable, economical enzymatic synthesis of diverse chiral amine targets.

Bioremediation and bioscavenging for elimination of organophosphorus threats: An approach using enzymatic advancements.

Organophosphorus compounds (OP) are highly toxic pesticides and nerve agents widely used in agriculture and chemical warfare. The extensive use of these chemicals has severe environmental implications, such as contamination of soil, water bodies, and food chains, thus endangering ecosystems and biodiversity. Plants absorb pesticide residues, which then enter the food chain and accumulate in the body fat of both humans and animals. Numerous human cases of OP poisoning have been linked to both acute and long-term exposure to these toxic OP compounds. These compounds inhibit the action of the acetylcholinesterase enzyme (AChE) by phosphorylation, which prevents the breakdown of acetylcholine (ACh) neurotransmitter into choline and acetate. Thus, it becomes vital to cleanse the environment from these chemicals utilizing various physical, chemical, and biological methods. Biological methods encompassing bioremediation using immobilized microbes and enzymes have emerged as environment-friendly and cost-effective approaches for pesticide removal. Cell/enzyme immobilized systems offer higher stability, reusability, and ease of product recovery, making them ideal tools for OP bioremediation. Interestingly, enzymatic bioscavengers (stoichiometric, pseudo-catalytic, and catalytic) play a vital role in detoxifying pesticides from the human body. Catalytic bioscavenging enzymes such as Organophosphate Hydrolase, Organophosphorus acid anhydrolase, and Paraoxonase 1 show high degradation efficiency within the animal body as well as in the environment. Moreover, these enzymes can also be employed to decontaminate pesticides from food, ensuring food safety and thus minimizing human exposure. This review aims to provide insights to potential collaborators in research organizations, government bodies, and industries to bring advancements in the field of bioremediation and bioscavenging technologies for the mitigation of OP-induced health hazards.

Insights into the molecular mechanisms of H. pylori-associated B-cell lymphoma.

Cancer research has extensively explored various factors contributing to cancer development, including chemicals, drugs, smoking, and obesity. However, the role of bacterial infections in cancer induction remains underexplored. In particular, the mechanisms underlying H. pylori-induced B-cell lymphoma, a potential consequence of bacterial infection, have received little attention. In recent years, there has been speculation about contagious agents causing persistent inflammation and encouraging B-lymphocyte transition along with lymphomagenesis. MALT lymphoma associated with chronic H. pylori infection, apart from two other central associated lymphomas - Burkitt's Lymphoma and DLBCL, is well studied. Owing to the increasing colonization of H. pylori in the host gut and its possible action in the development of B-cell lymphoma, this review aims to summarize the existing reports on different B-cell lymphomas' probable association with H. pylori infections; also emphasizing the function of the organism in lymphomagenesis; including its interaction with the host, pathogen and host-specific factors, and tumor microenvironment.

Recombinant Organophosphorus acid anhydrolase (OPAA) enzyme-carbon quantum dot (CQDs)-immobilized thin film biosensors for the specific detection of Ethyl Paraoxon and Methyl Parathion in water resources.

Organophosphates pesticide (OP) toxicity through water resources is a large concern globally among all the emerging pollutants. Detection of OPs is a challenge which needs to be addressed considering the hazardous effects on the health of human beings. In the current research thin film biosensors of recombinant, Organophosphorus acid anhydrolase (OPAA) enzyme along with carbon quantum dots (CQDs) immobilized in thin films were developed. OPAA-CQDs thin film biosensors were used for the specific detection of two OPs Ethyl Paraoxon (EP) and Methyl Parathion (MP) in river water and household water supply. Recombinant OPAA enzyme was expressed in E. Coli, purified and immobilized on the CQD containing chitosan thin films. The CQDs used for this purpose were developed by a one-pot hydrothermal method from phthalic acid and Tri ethylene diamine. The properties of CQDs, OPAA and thin films were characterized using techniques like XPS, TEM, XRD, enzyme activity and CLSM measurements. Biosensing studies of EP and MP were performed by taking fluorescence measurements using a fiber optic spectrometer. The analytical parameters of biosensing were compared against an estimation carried out using the HPLC method. The biosensing performance indicates that the OPAA-CQDs thin film-based biosensors were able to detect both EP and MP in a range of 0-100 µM having a detection limit of 0.18 ppm/0.69 ppm for EP/MP, respectively with a response time of 5 min. The accuracy of estimation of EP/MP when spiked in water resources lie in the range of ∼100-102% which clearly indicates the OPAA-CQD based thin film biosensors can function as a point-of-use method for the detection of OP pesticides in complex water resources.

Activation-induced cytidine deaminase an antibody diversification enzyme interacts with chromatin modifier UBN1 in B-cells.

Activation-induced cytidine deaminase (AID) is the key mediator of antibody diversification in activated B-cells by the process of somatic hypermutation (SHM) and class switch recombination (CSR). Targeting AID to the Ig genes requires transcription (initiation and elongation), enhancers, and its interaction with numerous factors. Furthermore, the HIRA chaperon complex, a regulator of chromatin architecture, is indispensable for SHM. The HIRA chaperon complex consists of UBN1, ASF1a, HIRA, and CABIN1 that deposit H3.3 onto the DNA, the SHM hallmark. We explored whether UBN1 interacts with AID using computational and in-vitro experiments. Interestingly, our in-silico studies, such as molecular docking and molecular dynamics simulation results, predict that AID interacts with UBN1. Subsequently, co-immunoprecipitation and pull-down experiments established interactions between UBN1 and AID inside B-cells. Additionally, a double immunofluorescence assay confirmed that AID and UBN1 were co-localized in the human and chicken B-cell lines. Moreover, proximity ligation assay studies validated that AID interacts with UBN1. Ours is the first report on the interaction of genome mutator enzyme AID with UBN1. Nevertheless, the fate of interaction between UBN1 and AID is yet to be explored in the context of SHM or CSR.

Outer membrane proteins and vesicles as promising vaccine candidates against Vibrio spp. infections.

Indiscriminate use of antibiotics to treat bacterial infections has brought unmanageable antibiotic-resistant strains into existence. Vibrio spp. represents one such gram-negative enteric pathogenic group with more than 100 species, infecting humans and fish. The Vibrio spp. is demarcated into two groups, one that causes cholera and the other producing non-cholera or vibriosis infections. People who encounter contaminated water are at risk, but young children and pregnant women are the most vulnerable. Though controllable, Vibrio infection still necessitates the development of preventative measures, such as vaccinations, that can lessen the severity of the infection and reduce reliance on antibiotic use. With emerging multi-drug resistant strains, efforts are needed to develop newer vaccines, such as subunit-based or outer membrane vesicle-based. Thus, this review strives to bring together available information about Vibrio spp. outer membrane proteins and vesicles, encompassing their structure, function, and immunoprotective role.

Insights in detection and analysis of organophosphates using organophosphorus acid anhydrolases (OPAA) enzyme-based biosensors.

The human population is dependent on agriculture for its food requirements and survival. Several insecticides and pesticides have found their use for improvements in agricultural yields. Organophosphates (OP) are one of the many compounds used as insecticides and pesticides. OPs have also been used to develop G and V-series chemicals which act as highly toxic nerve agents that can severely influence the normal function of the nervous system in all living beings. Thus, OP compounds utilized as insecticides/pesticides and nerve agents are hazardous to the environment, lethal for humans and other non-target animals. To avoid their toxicity, approaches to detect and neutralize them have become essential. A variety of analytical procedures such as electrochemical processes and chromatography methods, namely liquid and gas chromatography, have been employed to detect OPs. Though these techniques are sensitive and highly accurate they suffer from drawbacks, for instance: their bulky nature and expensive instrumentation, the difficulty of operation, long detection times, and they can yield unpredictable results with variable sample complexities. With the advent of several types of biosensors, the assay of OP compounds has become simpler, faster, cost-effective with improved sensitivity, and provides the capability for onsite detection. OP biosensor assays typically utilize several enzymes with the capability to hydrolyze/degrade OP compounds, such as organophosphate hydrolase (OPH) and organophosphate acid hydrolase (OPAA). This review focuses on discussing various aspects of OPAA as biological recognition unit in terms of its: structure, properties, activity enhancement methods, and utilization for developing OPAA-based biosensing technologies for insecticides, pesticides, and nerve agents.

Biochemical and molecular mechanisms of antibiotic resistance in Salmonella spp.

Salmonella is a diverse Gram-negative bacterium that represents the major disease burden worldwide. According to WHO, Salmonella is one of the fourth global causes of diarrhoeal disease. Antibiotic resistance is a worldwide health concern, and Salmonella spp. is one of the microorganisms that can evade the toxicity of antimicrobials via antibiotic resistance. This review aims to deliver in-depth knowledge of the molecular mechanisms and the underlying biochemical alterations perceived in antibiotic resistance in Salmonella. This information will help understand and mitigate the impact of antibiotic-resistant bacteria on humans and contribute to the state-of-the-art research developing newer and more potent antibiotics.

Biosensors for detection of prostate cancer: a review.

Diagnosis of prostate cancer (PC) has posed a challenge worldwide due to the sophisticated and costly diagnostics tools, which include DRE, TRUS, GSU, PET/CT scan, MRI, and biopsy. These diagnostic techniques are very helpful in the detection of PCs; however, all the techniques have their serious limitations. Biosensors are easier to fabricate and do not require any cutting-edge technology as required for other imaging techniques. In this regard, point-of-care (POC) biosensors are important due to their portability, convenience, low cost, and fast procedure. This review explains the various existing diagnostic tools for the detection of PCs and the limitation of these methods. It also focuses on the recent studies on biosensors technologies as an alternative to the conventional diagnostic techniques for the detection of PCs.

Heterologous expression of hAID in E. coli leads to the production of a splice isoform of AID: hAIDδC, a mystery to be explored.

Activation-induced cytidine deaminase (AID) is a key player that initiates antibody diversification in activated B-cell. AID mediates somatic hypermutation (SHM) and class switch recombination (CSR) via the deamination of cytosine to uracil at the Ig locus, resulting in the production of high-affinity antibodies. AID is predominantly restricted to Ig genes, whereas off-targeting of AID leads to lymphocyte-related malignancies. Interestingly, apart from FL-AID other splice isoforms of AID are highly expressed in the lymphocyte malignancies. In our study, we found that the heterologous expression of hAID-FL in E. coli cells produced two induced bands of hAID as demonstrated by SDS-PAGE and western blotting. Remarkably, peptide mapping data predicted that one band is hAID-FL and the other is its splice isoform, hAIDδE4a. To get an insight into why E. coli cells expressed hAID-FL and hAID variant, we mutated the 5' and 3' splice site of a putative intron of hAID, but it failed to produce only hAID-FL. Incidentally, hAID expressed with fusion partners also displayed two bands, and peptide mapping data strongly suggest that besides hAID-FL, the lower band showed a significant number of amino acids missing towards the C-terminal domain (named as hAIDδC). Our results are the first report to show that expression of recombinant hAID alone or irrespective of solubilization tags in E. coli cells produced hAID-FL and hAIDδC. It will be fascinating to explore the potential mechanism underlying the expression of hAIDδC from recombinant hAID plasmid in E. coli cells.

HomA and HomB, outer membrane proteins of Helicobacter pylori down-regulate activation-induced cytidine deaminase (AID) and Ig switch germline transcription and thereby affect class switch recombination (CSR) of Ig genes in human B-cells.

H. pylori is one of the major causes of chronic gastritis, peptic ulcer disease (PUD), gastric mucosa-associated lymphoid tissue lymphoma (MALT) and gastric carcinoma. H. pylori toxin VacA is responsible for host cell apoptosis, whereas CagA is known to aberrantly induce expression of activation-induced cytidine deaminase (AID) in gastric epithelial cells that causes mutations in oncogenes and tumour suppressor genes, leading to the transformation of normal cells into cancerous cells. Although, a significant amount of research has been conducted to understand the role of bacterial factors modulating deregulated host cell pathways, the interaction between H. pylori and immune cells of the marginal zone and its consequences are still not well understood. HomB and HomA, outer membrane proteins (OMPs) from H. pylori, which assist in the adhesion of bacteria to host cells, are found to be associated with H. pylori virulent strains and promote inflammation. Interestingly, we observed that the interaction of HomB/HomA OMPs with B-cells transiently downregulates AID expression and Ig switch germline transcription. Downregulation of AID leads to impairment of class switch recombination (CSR), resulting in significantly reduced switching to IgG and IgA antibodies. Besides, we examined the immune-suppressive response of B-cells and observed that the cells stimulated with HomA/B show upregulation in the levels of IL10, IL35, as well as PDL1, a T-cell inhibition marker. Our study suggests the potential role of OMPs in immune response modulation strategies used by the pathogen to evade the immune response. These results provide a better understanding of H. pylori pathogenesis and assist in identifying novel targets for therapy.

Biophysical characterization of the homodimers of HomA and HomB, outer membrane proteins of Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that causes chronic inflammations in the stomach area and is involved in ulcers, which can develop into gastric malignancies. H. pylori attaches and colonizes to the human epithelium using some of their outer membrane proteins (OMPs). HomB and HomA are the most studied OMPs from H. pylori as they play a crucial role in adherence, hyper biofilm formation, antibiotic resistance and are also associated with severe gastric malignancies. The role of HomA and HomB in pathogenesis concerning their structure and function has not been evaluated yet. In the present study, we explored the structural aspect of HomA and HomB proteins using various computational, biophysical and small-angle X-ray scattering (SAXS) techniques. Interestingly, the in-silico analysis revealed that HomA/B consists of 8 discontinuous N and C terminal ß-strands forming a small ß-barrel, along with a large surface-exposed globular domain. Further, biophysical experiments suggested that HomA and HomB are dimeric and most likely the cysteine residues present on surface-exposed loops participate in protein-protein interactions. Our study provides essential structural information of unexplored proteins of the Hom family that can help in a better understanding of H. pylori pathogenesis.

Recombinant organophosphorus hydrolase (OPH) expression in E. coli for the effective detection of organophosphate pesticides.

Accumulation and exposure of organophosphate pesticides are of great concern today owing to their abundant usage and potential health hazards. Harmful effects of organophosphate pesticide exposure and limitations of the available treatment methods necessitate the development of reliable, selective, cost-effective, and sensitive methods of detection. We developed a novel biosensor based on the enzymatic action of recombinant organophosphorus hydrolase (OPH) expressed in E. coli. We report the development of colorimetric biosensors made of His-Nus-OPH as well as His-Nus-OPH loaded alginate microspheres. The colorimetric detection method developed using solution-phase and alginate-encapsulated His-Nus-OPH exhibited detection limits of 0.045 and 0.039 mM, respectively, for ethyl paraoxon, and 0.101 and 0.049 mM, respectively, for methyl parathion. Additionally, fluorescence measurement using pH-sensitive fluorescein isothiocyanate (FITC) was used to sense the quantity of organophosphorus pesticides. The fluorometric detection method using solution-phase His-Nus-OPH, with ethyl paraoxon and methyl parathion as the substrate, reveals the lower limit of detection as 0.014 mM and 0.044 mM, respectively. Our results demonstrate the viability of His-Nus-OPH for OP detection with good sensitivity, LOD, and linear range. We report the first use of N-terminal His-NusA-tagged OPH, which enhances solubility significantly and presents a significant advance for the scientific community.

Unfolding the Role of Splicing Factors and RNA Debranching in AID Mediated Antibody Diversification.

Activated B-cells diversify their antibody repertoire via somatic hypermutation (SHM) and class switch recombination (CSR). SHM is restricted to the variable region, whereas, CSR is confined to the constant region of immunoglobulin (Ig) genes. Activation-induced cytidine deaminase (AID) is a crucial player in the diversification of antibodies in the activated B-cell. AID catalyzes the deamination of cytidine (C) into uracil (U) at Ig genes. Subsequently, low fidelity repair of U:G mismatches may lead to mutations. Transcription is essential for the AID action, as it provides a transient single-strand DNA substrate. Since splicing is a co-transcriptional event, various splicing factors or regulators influence the transcription. Numerous splicing factors are known to regulate the AID targeting, function, Ig transcription, and AID splicing, which eventually influence antibody diversification processes. Splicing regulator SRSF1-3, a splicing isoform of serine arginine-rich splicing factor (SRSF1), and CTNNBL1, a spliceosome interacting factor, interact with AID and play a critical role in SHM. Likewise, a splicing regulator polypyrimidine tract binding protein-2 (PTBP2) and the debranching enzyme (DBR1) debranches primary switch transcripts which later forms G-quadruplex structures, and the S region guide RNAs direct AID to S region DNA. Moreover, AID shows several alternate splicing isoforms, like AID devoid of exon-4 (AIDΔE4) that is expressed in various pathological conditions. Interestingly, RBM5, a splicing regulator, is responsible for the skipping of AID exon 4. In this review, we discuss the role and significance of splicing factors in the AID mediated antibody diversification.

A novel biosensor for the detection of organophosphorus (OP)-based pesticides using organophosphorus acid anhydrolase (OPAA)-FL variant.

Indiscriminate use of organophosphorus (OP)-based insecticides is a great concern to human health because of bioaccumulation-induced health hazards. Potentially fatal consequences and limited treatment methods of OP poisoning necessitate the need for the development of reliable, selective, cost-effective, and sensitive methods of OP detection. To tackle this issue, the development of effective devices and methods is required to sensitively detect as well as degrade OPs. Enzymatic sensor systems have gained popularity due to high catalytic activity, enhanced detection limits, and high sensitivity with the environmentally benign operation. Organophosphorus acid anhydrolase (OPAA) from Alteromonas sp. JD6.5 is capable of hydrolyzing the P-F, P-O, P-S, and P-CN bonds, in OPs, including nerve agents of the G/V-series. Several mutants of OPAA are reported which have greater activity against various OPs. In this study, recombinant expression of the OPAA-FL variant in Escherichia coli was performed, purified, and subsequently tested for activity against ethyl paraoxon. OPAA-FL variant showed its optimum activity at pH 8.5 and 50 °C. Colorimetric and fluorometric assays were used for estimation of ethyl paraoxon based on p-nitrophenol and fluorescein isothiocyanate (FITC) fluorescence intensity, respectively. Colorimetric and fluorometric assay estimation indicates that ethyl paraoxon can be estimated in the linear range of 0.01 to 1 mM and 0.1 to 0.5 mM, with LOD values 0.04 mM and 0.056 mM, respectively. Furthermore, the OPAA-FL variant was immobilized into alginate microspheres for colorimetric detection of ethyl paraoxon and displayed a linear range of 0.025 to 1 mM with a LOD value of 0.06 mM. KEY POINTS: • Biosensing of paraoxon with purified and encapsulated OPAA-FL variant. • Colorimetric and fluorometric biosensing assay developed using OPAA-FL variant for paraoxon. • First report on alginate encapsulation of OPAA-FL variant for biosensing of paraoxon. Graphical abstract.

Exploring Computational and Biophysical Tools to Study the Presence of G-Quadruplex Structures: A Promising Therapeutic Solution for Drug-Resistant Vibrio cholerae.

Vibrio cholerae, a gram-negative bacterium that causes cholera, has already caused seven major pandemics across the world and infects roughly 1.3-4 million people every year. Cholera treatment primarily involves oral rehydration therapy supplemented with antibiotics. But recently, multidrug-resistant strains of V. cholerae have emerged. High genomic plasticity further enhances the pathogenesis of this human pathogen. Guanines in DNA or RNA assemble to form G-quadruplex (GQ) structures which have begun to be seen as potential drug targeting sites for different pathogenic bacteria and viruses. In this perspective, we carried out a genome-wide hunt in V. cholerae using a bio-informatics approach and observed ∼85 G-quadruplex forming motifs (VC-PGQs) in chromosome I and ∼45 putative G-quadruplexs (PGQs) in chromosome II. Ten putative G-quadruplex forming motifs (VC-PGQs) were selected on the basis of conservation throughout the genus and functional analysis displayed their location in the essential genes encoding bacterial proteins, for example, methyl-accepting chemotaxis protein, orotate phosphoribosyl transferase protein, amidase proteins, etc. The predicted VC-PGQs were validated using different bio-physical techniques, including Nuclear Magnetic Resonance spectroscopy, Circular Dichroism spectroscopy, and electrophoretic mobility shift assay, which demonstrated the formation of highly stable GQ structures in the bacteria. The interaction of these VC-PGQs with the known specific GQ ligand, TMPyP4, was analyzed using ITC and molecular dynamics studies that displayed the stabilization of the VC-PGQs by the GQ ligands and thus represents a potential therapeutic strategy against this enteric pathogen by inhibiting the PGQ harboring gene expression, thereby inhibiting the bacterial growth and virulence. In summary, this study reveals the presence of conserved GQ forming motifs in the V. cholerae genome that has the potential to be used to treat the multi-drug resistance problem of the notorious enteric pathogen.

G-quadruplex stabilization in the ions and maltose transporters gene inhibit Salmonella enterica growth and virulence.

The G-quadruplex structure is a highly conserved drug target for preventing infection of several human pathogens. We tried to explore G-quadruplex forming motifs as promising drug targets in the genome of Salmonella enterica that causes enteric fever in humans. Herein, we report three highly conserved G-quadruplex motifs (SE-PGQ-1, 2, and 3) in the genome of Salmonella enterica. Bioinformatics analysis inferred the presence of SE-PGQ-1 in the regulatory region of mgtA, SE-PGQ-2 in ORF of entA, and SE-PGQ-3 in the promoter region of malE and malK genes. The G-quadruplex forming sequences were confirmed by biophysical and biomolecular techniques. Cellular studies affirm the inhibitory effect of G-quadruplex specific ligands on Salmonella enterica growth. Further, PCR inhibition, reporter based assay, and RT-qPCR assays emphasize the biological relevance of G-quadruplexes in these genes. Thus, this study confirmed the presence of G-quadruplex motifs in Salmonella enterica and characterized them as a promising drug target.

SRSF1-3, a splicing and somatic hypermutation regulator, controls transcription of IgV genes via chromatin regulators SATB2, UBN1 and histone variant H3.3.

SRSF1, a member of the SR protein family, is an important splicing factor and regulator of splicing. Multiple splicing isoforms have been reported for this gene. SRSF1-3, a splicing isoform of SRSF1, is necessary for AID-dependent SHM of IgV genes. However, its precise role in SHM remains enigmatic. Transcriptomic analysis of SRSF1-3 reconstituted cells shows upregulation of transcription factor SATB2 and chromatin regulator UBN1. The increased SATB2 and UBN1 are strikingly enriched in the MAR and promoter regions of the IgL gene, respectively. Furthermore, UBN1 enrichment at the promoter region was coupled with a hundred-fold enhanced occupancy of the histone variant H3.3 at the IgL promoter, that is a hallmark of efficient SHM. The enhanced occupancy of SATB2 at the MAR, UBN1 and histone variant H3.3 at the IgL promoter leads to an increase in IgL transcription, revealing a role of SRSF1-3 in SHM. Thus, SRSF1-3 is likely involved in the regulation of SHM, via upregulation of a crucial transcription factor SATB2, as well as, by overexpression of a chromatin modulator of Ig genes, UBN1, which further assists in the recruitment of the histone variant H3.3. Furthermore, the splicing isoform SRSF1-3 regulates alternate splicing pattern of splicing isoforms for various crucial genes. The present study provides the first evidence that a splicing isoform of an SR protein can regulate the post-transcriptional processing of RNA in vivo.

Splicing regulator SRSF1-3 that controls somatic hypermutation of IgV genes interacts with topoisomerase 1 and AID.

Somatic hypermutation (SHM) of Ig genes is initiated by activation-induced cytidine deaminase (AID) and requires target gene transcription. A splice isoform of SRSF1, SRSF1-3, is necessary for AID-dependent SHM of IgV genes. Nevertheless, its exact molecular mechanism of action in SHM remains unknown. Our in silico studies show that, unlike SRSF1, SRSF1-3 lacks a strong nuclear localization domain. We show that the absence of RS domain in SRSF1-3 affects its nuclear localization, as compared to SRSF1. Consequently, SRSF1-3 is predominantly present in the cytoplasm. Remarkably, co-immunoprecipitation studies showed that SRSF1-3 interacts with Topoisomerase 1 (TOP1), a crucial regulator of SHM that assists in generating ssDNA for AID activity. Moreover, the immunofluorescence studies confirmed that SRSF1-3 and TOP1 are co-localized in the nucleus. Furthermore, Proximity Ligation Assay corroborated the direct interaction between SRSF1-3 and TOP1. An interaction between SRSF1-3 and TOP1 suggests that SRSF1-3 likely influences the TOP1 activity and consequently can aid in SHM. Accordingly, SRSF1-3 probably acts as a link between TOP1 and SHM, by spatially regulating TOP1 activity at the Ig locus. We also confirmed the interaction between SRSF1-3 and AID in chicken B-cells. Thus, SRSF1-3 shows dual-regulation of SHM, via interacting with AID as well as TOP1.

Advances in detection of hazardous organophosphorus compounds using organophosphorus hydrolase based biosensors.

Agricultural advancements focusing on increasing crop production have led to excessive usage of insecticides and pesticides, resulting in leaching and accumulation of these highly toxic chemicals in soil, water, and the food-chain. Organophosphorus (OP) compounds are the most commonly used insecticides and pesticides, which cause a wide range of long-lasting and life-threatening conditions. Due to the acute toxicity and long-term side effects of OP compounds, their timely, on-the-spot and rapid detection has gained importance, for efficient healthcare management. In this respect, several OP degrading enzymes have gained the spotlight in developing the enzyme-based biosensors, owing to their high activity and broad specificity. Among these enzymes, organophosphorus hydrolase (OPH) has emerged as a promising candidate for the detection of OP compounds, due to its ability to act on a broad range of substrates having a variety of bonds, like P─F, P─O, P─S, and P─CN. Various techniques employing OPH in free/immobilized/conjugated forms into sensing devices were reported to accurately detect OP compounds. The transduction mechanisms of bio-sensing are electrochemical, optical as well as novel methods like magnetoelastic/surface plasmon resonance. Furthermore, to improve the detection limits and sensitivity, nanoparticles and quantum dots are often employed in conjunction with OPH. Here, we highlight the recent advances in sensing OP compounds using OPH based biosensors, compare specifications of sensing methods, and evaluate the influence of different materials used in developing sensors. This review will also enable researchers to design and configure highly sensitive and accurate sensing systems, leading to the development of point-of-care devices for real-time analysis.