Regional Centre for Biotechnology

Botrytis cinerea is a necrotrophic fungal pathogen that poses a significant threat to many crops. Understanding the proteome dynamics of phytopathogens during infection can help combat plant diseases. However, most proteomics studies in phytopathogens face interference from abundant host proteins. Here, we optimized a solid media that better mimics in-planta conditions and used it to perform the temporal protein dynamics in Botrytis cinerea. An agar media with 20% tomato fruit extract and 2% deproteinised leaf extract was utilized for label-free quantitative proteomics at 12, 36, 72 and 120 hpi. Out of 3244 quantified proteins, 2045 showed differential regulation. Glycosyl hydrolases, pectin esterases, stress protein DDR48, RhoGEF and essential transcription factors were found to be upregulated during the early phase, highlighting their role in fungal virulence. Meanwhile, pathways such as macromolecule synthesis, purine, and carbohydrate metabolism were upregulated in the late-growth phase. Overall, the study provides a comprehensive understanding of proteome dynamics during Botrytis infection.

Severe dengue often presents as shock syndrome with enhanced vascular permeability and plasma leakage into tissue spaces. In vitro studies have documented the role of Src family kinases (SFKs) and RhoA-kinases (ROCK) in dengue virus serotype 2 (DENV2)-induced endothelial permeability. Here, we show that the FDA-approved SFK inhibitors Bosutinib, Vandetanib and Ponatinib, as well as the ROCK inhibitors, Netarsudil and Ripasudil significantly inhibit DENV2-induced endothelial permeability. In cultured telomerase immortalized human microvascular endothelial cells (HMEC-1), treatment with these inhibitors reduced the phosphorylation of VE-Cadherin, Src and myosin light chain 2 (MLC2) proteins that were upregulated during DENV2 infection. It also prevented the loss of VE-Cadherin from the inter-endothelial cell junctions induced by viral infection. In in-vivo studies using DENV2-infected AG129 IFN receptor-α/β/γ deficient mice, ponatinib, when administered 24 h post-infection onwards, demonstrated significant benefits in improving body weight, clinical outcomes, and survival rates. While all virus-infected, untreated mice died by day-10 post-infection, 80% of the ponatinib-treated mice survived, and approximately 60% were still alive at the end of the 15-day observation period. The treatment also significantly reduced disease severity factors such as vascular leakage, thrombocytopenia; mRNA transcript levels of proinflammatory cytokines such as IL-1β and TNF-α; and restored liver function. Comparable effects were observed even when ponatinib treatment was initiated after symptom onset. The results highlight ponatinib as an effective therapeutic option in severe dengue; and also a similar potential for other FDA- approved SFK and ROCK inhibitors.

Early interactions between tubercle bacilli and lung cells are critical in tuberculosis (TB) pathogenesis. Conventional two-dimensional cell cultures fail to replicate the multicellular complexity of lungs. We introduce a three-dimensional pulmosphere model for Mycobacterium tuberculosis infection in bovine systems, demonstrating through comprehensive transcriptome and proteome analyses that these multicellular spheroids closely mimic lung cell diversity, interactions, and extracellular matrix (ECM) composition. Cell viability, hypoxia, and reactive oxygen species assessments over three weeks confirm the model’s suitability. To establish infection, we employed M. bovis BCG—an attenuated vaccine strain, and M. tuberculosis H37Rv—a laboratory adapted human clinical strain that is attenuated for cattle infection compared to M. bovis. Both infection upregulated key host pathways; however, M. tuberculosis induced distinct responses, including enhanced ECM receptors expression, neutrophil chemotaxis, interferon signaling, and RIG-1 signaling. A six genes/protein signature- IRF1, CCL5, CXCL8, CXCL10, SERPINE1, and CFB -emerges as an early host response marker to M. tuberculosis infection. Infection with virulent M. bovis and M. orygis revealed a shared upregulated gene signature across Mycobacterium tuberculosis complex species, but with pathogen-specific variations. This study presents a robust ex vivo bovine pulmosphere TB model with implications in biomarkers discovery, high-throughput drug screening, and TB control strategies.

Diabetes foot ulcers (DFU) are the most common foot injuries leading to lower extremity amputation. Our study aimed to provide the first representative analysis highlighting the vital role of Tertiary Lymphoid Organs (TLO) inflammatory landscape in diabetic foot ulcers. The study explores mechanisms of TLO formation and the disease‐specific roles of TLOs in regulating peripheral inflammatory and immune responses. Additionally, comprehensive analysis of clinical data from DFU cases, focused on TLO pathophysiology and systemic immune‐inflammation landscape, is documented, aiming to identify the risk factors contributing to the development of DFUs. Our experimental results showed very significant differences were observed among the IL‐17 and IFN‐γ cytokine levels between the DFU vs. Control and DFU vs. NIDFU (Non‐Infectious Diabetic Foot Ulcers) groups, while minimal differences were observed in IL‐6 and TNF‐α cytokine levels. Immunohistochemistry staining or Immunophenotyping of DFU patient‐derived wound samples for TLO inflammatory stratification showed remarkable differences between DFU, NIDFU, and control groups both in CD3⁺ T Cells and CD20⁺ B cells. Overall, our study findings highlight the perspective role of TLO in DFU mechanisms and its prudent role in regulating peripheral inflammatory‐immune responses. TLO study‐related significant findings might be one of the important mechanisms, and its effective unveil might be a valuable treatment modality for DFU‐complications.

Delivery of an infant before the completion of 37 weeks of gestation is considered preterm birth (PTB). Admission of preterm infants to the neonatal intensive care unit (NICU) causes considerable stress that impedes their growth. A recent innovation in the neonatal care is the Mother Newborn Care Unit (MNCU), which is a facility where sick and small newborns are cared with their mothers 24 × 7 with all facilities of sick newborn care and provision for postnatal care to mothers. This study includes preterm babies (birth weight 1 to 1.8 kg) admitted to either MNCU or NICU. We collected saliva from these babies at admission and discharge and measured cortisol levels in these samples as an indicator of stress. Based on protein quantitation, we chose 12 pairs of salivary samples for MS-based proteomics and quantified 308 protein groups. Differential protein analysis of MNCU vs NICU revealed 44 and 41 differentially expressed proteins (DEPs) and in pathway enrichment of unique DEPs, 22 and 19 proteins were present in the admission and discharge group, respectively. We report protein changes involved in immune and metabolic pathways with important functions. Thus, our study shows evidence in favor of MNCU care that enhance the growth and lower the preterm stress. Graphical abstract

The eukaryotic 43S pre-initiation complex (PIC), containing methionyl initiator transfer RNA (Met-tRNAiMet) in a ternary complex (TC) with eIF2-GTP, scans the messenger RNA (mRNA) leader for an AUG start codon in favorable “Kozak” context. Recognition of AUG triggers the rearrangement of the PIC from an open scanning conformation to a closed arrested state with more tightly bound Met-tRNAiMet. Cryo-EM reconstructions of yeast PICs suggest remodeling of the interaction between 40S protein uS11/Rps14 with ribosomal RNA (rRNA) and mRNA between open and closed states; however, its importance in start codon recognition was unknown. uS11/Rps14-L137 substitutions disrupting rRNA contacts favored in the open complex increase initiation at suboptimal sites, and L137E stabilizes TC binding to PICs reconstituted in vitro with a UUG start codon, all indicating inappropriate rearrangement to the closed state at suboptimal initiation sites. Conversely, uS11/Rps14-R135 and -R136 substitutions perturbing interactions with rRNA exclusively in the closed state confer the opposite phenotypes of initiation hyperaccuracy, and for R135E, accelerated TC dissociation from reconstituted PICs. Thus, distinct interactions of uS11/Rps14 with rRNA stabilize first the open and then the closed conformation of the PIC to influence the accuracy of initiation in vivo.

RNA switches regulated by specific inducer molecules have become a powerful synthetic biology tool for precise gene regulation in mammalian systems. The engineered RNA switches can be integrated with natural RNA‐mediated gene regulatory functions as a modular and customizable approach to probe and control cellular behavior. RNA switches have been used to advance synthetic biology applications, including gene therapy, bio‐production, and cellular reprogramming. This review explores recent progress in the design and functional implementation of synthetic riboswitches in mammalian cells based on diverse RNA regulation mechanisms by highlighting recent studies and emerging technologies. We also discuss challenges such as off‐target effects, system stability, and ligand delivery in complex biological environments. In conclusion, this review emphasizes the potential of synthetic riboswitches as a platform for customizable gene regulation in diverse biomedical applications.

Biosensors have revolutionized the field of analytical chemistry by providing sensitive, selective, and real-time detection of various analytes. Among these analytes, electroactive molecules hold particular significance due to their diverse roles in biological, environmental, and industrial processes. This chapter provides a comprehensive overview of voltammetric biosensors tailored for the detection of electroactive molecules, focusing on recent advancements, key technologies, and emerging trends. It begins by elucidating the fundamental principles underlying voltammetric biosensor design, types, and operation, emphasizing the importance of transduction mechanisms, recognition elements, and signal amplification strategies. It then delves into the classification of electroactive molecules, encompassing neurotransmitters, redox-active species, and electroactive pharmaceuticals, among others, and their significance in different application domains. In addition, it critically examines various voltammetric biosensor platforms used for detecting electroactive molecules. This includes the development of multiplexed sensing platforms, the exploration of novel recognition elements, and the translation of laboratory-based prototypes into practical applications for healthcare, environmental monitoring, and industrial process control.

Background Liquid biopsy-based biomarkers offer several advantages since they are minimally invasive, can be useful in longitudinal monitoring of the disease and have higher patient compliance. We describe a protocol using minimal volumes of archival and prospective serum/plasma samples to define the RNA contents of EVs and discuss its benefits and limitations. Methods RNA-seq analysis of matched tumor biopsy, circulating EVs from breast cancer patients (EV-C, n = 26) and healthy donors (EV-H, n = 4) was performed and differentially expressed genes were validated by RT-PCR in a separate series of samples (EV-C, n = 32 and EV-H, n = 22). A total of 84 samples were studied. Results RNA-seq data from 500 μl serum samples yielded more than 17000 genes, of which 320 were DEGs (adjusted p value ≤ 0.05) between EV-C and EV-H samples. Pathways for Myc V1, reactive oxygen species, angiogenesis, allograft rejection and Interferon regulated genes were over-represented in EV-C samples. Computational deconvolution algorithms for cell signatures identified immune cells such as Th1 and memory T-cells, endothelial cells, and osteoblasts from the stromal compartment as significant. Top 6 genes were validated by qRT-PCR in all samples (n = 84) and they consistently and correctly classified cancer and healthy groups. An independent set of 374 and 640 DEGs could segregate ER positive/ER negative and non-metastatic versus metastatic samples, respectively. EVs from metastatic samples had higher variability in gene expression patterns whereas those from non-metastatic samples showed a better correlation. Conclusion By using low serum amounts successfully for EV transcriptomics, we demonstrate that a minimally invasive technique could be converted to a microinvasive format. These data lay the foundation for EV RNA based biomarker discovery for segregating breast cancers.

Previous studies have demonstrated that in certain medical conditions, fragility fractures tend to occur even at bone mineral density (BMD) levels that are in the nonosteoporotic range. This warrants the assessment of other factors beyond BMD that might confer an increased propensity to fracture. Hip structural analysis (HSA) is also performed by the DXA scanner and evaluates different variables pertaining to proximal hip geometry. Bone Strain Index (BSI) is another novel DXA-based tool that was recently developed to further assess bone health. This has been based on a finite element analysis of grey scale images of density distribution of the femoral and lumbar spine scans obtained from a DXA scanner. Preliminary studies assessing the utility of BSI in predicting fragility fractures have been promising. This review will focus on the technical details and utility of the HSA and BSI beyond conventional BMD assessment.

Introduction This study aims to valorise cheese whey waste by converting it into bioactive peptides that have several health benefits, potentially leading to the development of nutraceuticals and functional foods and also used in pharmaceutical industry. Methods The study evaluates the antidiabetic, antioxidative, and anti-inflammatory properties of fermented cheese whey with Limosilactobacillus fermentum (M4), along with the production of antioxidative and antidiabetic peptides. SDS PAGE and 2D PAGE were also performed to identify proteins by molecular weight and isoelectric point, while RP-HPLC distinguished peptide fractions. Peptide sequences from 2D gel spots were identified using RPLC/MS, and RP-HPLC analyzed 3 kDa and 10 kDa permeates. Peakview software characterized the LC/MS results, and FTIR analysis measured structural changes in bioactive peptides. Results The antioxidative and antidiabetic properties in cheese whey fermented with M4 showed a progressive growth over extended incubation periods, higher effects were observed after fermentation for 48 hours. Inhibitory activities in α-glucosidase, α-amylase & lipase were found to be 65.39%, 66.09%, and 56.74% respectively. ABTS assay was performed to measure antioxidant activity (63.39%) and the highest proteolytic activity (7.62 mg/ml) was measured at 2.5% inoculation rate for 48 hours. In SDS-PAGE, protein bands between 10 & 30 kDa were observed, whereas peptide spots within the range of 10 to 70 kDa were also visualized on the 2D PAGE. RP-HPLC was used to distinguish different fractions of a peptide. Peptide sequences from 2D gel spots were identified using RP-HPLC & RPLC/MS. Peakview software was utilized to characterize the LC/MS results. Sequences of peptides generated from α-lactalbumin and β-lactoglobulin were searched in the BIOPEP database to validate the antidiabetic and antioxidative activities of fermented cheese whey peptides. 0.50 mg/mL of fermented cheese whey significantly LPS suppressed the production of proinflammatory cytokines as well as the mediators that govern them including IL-6, IL-1β, NO, and TNF-α in RAW 264.7 cells. FTIR was used to analysis of protein secondary structure and conformational changes. Conclusion This study aims to the production of antidiabetic and antioxidative peptides from dairy waste, and cheese whey.

Upon exposure to ionizing irradiation, the MRE11–RAD50–NBS1 complex potentiates the recruitment of ATM (ataxia-telangiectasia mutated) kinase to the double-strand breaks. We show that the lack of BLM causes a decrease in the autophosphorylation of ATM in mice mammary glands, which have lost one or both copies of BLM. In isogenic human cells, the DNA damage response (DDR) pathway was dampened in the absence of BLM, which negatively affected the recruitment of DDR factors onto the chromatin, thereby indicating a direct role of BLM in augmenting DDR. Mechanistically, this was due to the BLM-dependent dissociation of inactive ATM dimers into active monomers. Fragmentation analysis of BLM followed by kinase assays revealed a 20-mer BLM peptide (91–110 aa), sufficient to enhance ATM-dependent p53 phosphorylation. ATM-mediated phosphorylation of BLM at Thr99 within BLM (91–110) peptide enhanced ATM kinase activity due to its interaction with NBS1 and causing ATM monomerization. Delivery of phosphomimetic T99E counterpart of BLM (91–110 aa) peptide led to ATM activation followed by restoration of the DDR even in the absence of ionizing irradiation (both in cells and in BLM knockout mice), indicating its role as a DDR agonist, which can be potentially used to prevent the initiation of neoplastic transformation.

Microbes and parasites have evolved several means to evade and usurp the host cellular machinery to mediate pathogenesis. Being the major microtubule-organizing center (MTOC) of the cell, the centrosome is targeted by multiple viral and nonviral pathogens to mediate their assembly and trafficking within the host cell. This review examines the consequence of such targeting to the centrosome and associated cytoskeletal machinery. We have also amassed a substantial body of evidence of viruses utilizing the cilia within airway epithelium to mediate infection and the hijacking of host cytoskeletal machinery for efficient entry, replication, and egress. While infections have been demonstrated to induce structural, functional, and numerical aberrations in centrosomes, and induce ciliary dysfunction, current literature increasingly supports the notion of a pro-viral role for these orga-nelles. Although less explored, the impact of bacterial and parasitic pathogens on these structures has also been addressed very briefly. Mechanistically, the molecular pathways responsible for these effects remain largely uncharacterized in many instances. Future research focusing on the centriolar triad comprising the centrosome, cilia, and centriolar satellites will undoubtedly provide vital insights into the tactics employed by infectious agents to subvert the host centriole and cytoskeleton-based machinery.

Scientists working on domestic, model, non-model, and wild animal species now have access to genomic tools due to the rapid advancement of sequencing technologies over the past two decades. Animal scientists and veterinary researchers are attempting to use cutting-edge genomic techniques in response to the availability of numerous annotated animal genomes to improve breeding, accurately characterize genetic traits linked to productive traits, connect genomic, proteomic, and transcriptomic data to animal disease, and also understand the impact of epigenetic changes on the expression of genes. Recent research has suggested that epigenetic effects can be passed down from generation to generation. In this chapter, we highlight the implications of these findings for animal research and livestock as well as some features of the epigenetic mechanisms that control animal phenotypic behavior. This chapter also focuses on the use of epigenomic technologies in livestock, spanning reproduction, productivity, and healthcare, to essentially address animal and veterinary science researchers.

Single Nucleotide Polymorphism (SNP) is a significant point mutation that happens often in genomes and has a wide range of applications. SNPs are the preferred marker in genetic analysis and are helpful in identifying genes linked to specific traits or diseases. The identification of functional SNPs for complex diseases or important economic traits is one of the active research areas in animals, especially in veterinary science. SNPs present in gene coding regions can alter the protein sequence; such SNPs are called missense or non-synonymous SNPs (nsSNPs). nsSNPs are important factors leading to the functional diversity of the encoded proteins. All nsSNPs are not structurally or functionally influencing, although many harmful variants can have an impact on the physiology of an organism. According to experimental investigations, one-third of nsSNP mutations are harmful therefore, the identification of deleterious nsSNPs is still a major challenge. Thus, in this post-genomic era, computational approaches are established as potential strategies for the screening of most deleterious nsSNPs and their structural and functional consequences. There are various computational tools available for the prediction of damaging or functional nsSNPs. In this chapter, we have provided systematic information about the use of such tools, for the screening of nsSNPs along with available databases. Moreover, we have provided comprehensive details about nsSNPs studied in livestock animals along with their effect on protein structural and conformational dynamics. The application of reported nsSNPs that serve as potential targets in veterinary science and livestock productivity has also been summarized.

The endonucleolytic cleavage step of the eukaryotic mRNA 3′-end processing is considered imprecise, which leads to heterogeneity of cleavage site (CS) with hitherto unknown function. Contrary to popular belief, we show that this imprecision in the cleavage is tightly regulated, resulting in the CS heterogeneity (CSH) that controls gene expression in antioxidant response. CSH centres around a primary CS, followed by several subsidiary cleavages determined by CS's positions. Globally and using reporter antioxidant mRNA, we discovered an inverse relationship between the number of CS and the gene expression, with the primary CS exhibiting the highest cleavage efficiency. Strikingly, reducing CSH and increasing primary CS usage induces gene expression. Under oxidative stress (we employ three conditions that induce antioxidant response, tBHQ, H2O2, and NaAsO2) conditions, there is a decrease in the CSH and an increase in the primary CS usage to induce antioxidant gene expression. Key oxidative stress response genes (NQO1, HMOX1, PRDX1, and CAT) also show higher CSH compared to the non-stress response genes and that the number of CSs are reduced to impart cellular response to oxidative stresses. Concomitantly, ectopic expression of one of the key antioxidant response gene (NQO1) driven by the primary CS but not from other subsidiary CSs, or reduction in CSH imparts tolerance to cellular oxidative stresses (H2O2, and NaAsO2). Genome-wide CS analysis of stress response genes also shows a similar result. Compromised CSH or CSH-mediated gene control hampers cellular response to oxidative stress. We establish that oxidative stress induces affinity/strength of cleavage complex assembly, increasing the fidelity of cleavage at the primary CS, thereby reducing CSH inducing antioxidant response. Together, our study reports a novel cleavage imprecision- or CSH-mediated anti-oxidant response mechanism that is distinct and operates downstream but in concert with the transcriptional pathway of oxidative stress induction.