Molecular detection of enterotoxins in multidrug resistant Aeromonas from ready to eat foods in North Western Himalayas: public health significance.

Aeromonas spp. are normal inhabitants of aquatic environments and are emerging foodborne bacterial pathogens. Aeromonas spp. contamination is frequent in ready-to-eat (RTE) seafood and can also occur in products prepared from milk or meat. The study determined the enterotoxin and antimicrobial susceptibility profiles of Aeromonas spp. isolates recovered from RTE milk products (n=105), RTE meat/fish products (n=40) and drinking water (n=60) samples collected from tourist places in Himachal Pradesh, India, in northwestern Himalayas. 7.3% (16/220) samples were found contaminated with Aeromonas spp. These isolates were identified as A. hydrophila (31.3%), A. schubertii (25.0%), A. sobria (25.0%) and A. veronii (18.8%). Aeromonas spp. contamination was significantly higher (14.3%, 15/105, p=0.0001) in RTE milk products. The contamination levels for water samples were 1.7% whereas none of the tested RTE meat or fish products yielded Aeromonas spp. Among RTE milk products, contamination was significantly higher in paneer (South Asian soft cheese) (26.1%, p=0.0027) and cream (25.0%, p=0.046) based RTE foods. All isolates carried alt (361 bp), encoding a cytotonic heat-labile enterotoxin. Ampicillin resistance was 100% and high levels (>30%) of resistance were recorded for amoxicillin/clavulanic acid, amikacin, cefotaxime and ceftazidime. Six (37.5%) isolates were multi drug resistant (MDR), showing resistance to aminoglycosides, cephams and penicillins. Isolation of alt carrying MDR isolates from RTE foods indicates that Aeromonas spp. can be potential foodborne public health threat in northwestern Himalayas.

Osteogenic human MSC-derived extracellular vesicles regulate MSC activity and osteogenic differentiation and promote bone regeneration in a rat calvarial defect model.

BACKGROUND: There is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects. METHODS: EVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10 µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50 µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry. RESULTS: Although Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM. CONCLUSIONS: This study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.

Recent Developments and Future Perspectives of Purine Derivatives as a Promising Scaffold in Drug Discovery.

Numerous purine-containing compounds have undergone extensive investigation for their medical efficacy across various diseases. The swift progress in purine-based medicinal chemistry has brought to light the therapeutic capabilities of purine-derived compounds in addressing challenging medical conditions. Defined by a heterocyclic ring comprising a pyrimidine ring linked with an imidazole ring, purine exhibits a diverse array of therapeutic attributes. This review systematically addresses the multifaceted potential of purine derivatives in combating various diseases, including their roles as anticancer agents, antiviral compounds (anti-herpes, anti-HIV, and anti-influenzae), autoimmune and anti-inflammatory agents, antihyperuricemic and anti-gout solutions, antimicrobial agents, antitubercular compounds, anti-leishmanial agents, and anticonvulsants. Emphasis is placed on the remarkable progress made in developing purine-based compounds, elucidating their significant target sites. The article provides a comprehensive exploration of developments in both natural and synthetic purines, offering insights into their role in managing a diverse range of illnesses. Additionally, the discussion delves into the structure-activity relationships and biological activities of the most promising purine molecules. The intriguing capabilities revealed by these purine-based scaffolds unequivocally position them at the forefront of drug candidate development. As such, this review holds potential significance for researchers actively involved in synthesizing purine-based drug candidates, providing a roadmap for the continued advancement of this promising field.

Investigating antiarthritic potential of polyherbal emulgel.

BACKGROUND: Rheumatoid arthritis (RA) is an inflammation of joints with increased cellularity of synovial tissue. Allopathic drugs possess several adverse effects, which have led to increase in the utilization of herbal medicines. Polyherbal emulgel resolves the bioavailability issue associated with hydrophobic drugs and can be used effectively in the treatment of RA. OBJECTIVES: The present study aimed at the formulation of polyherbal emulgel, and evaluation of in vitro anti-inflammatory activity and in vivo antiarthritic activity. METHODS: Seven emulgels F-1 to F-7 were optimally formulated. In vitro anti-inflammatory activity was determined using protein denaturation method employing Diclofenac sodium as the standard. In antiarthritic study Complete Freund's Adjuvant (CFA) model was used. The various parameters were assessed, like paw volume, body weight, hematological parameters, antioxidant parameters, Rheumatic factor (RF), and histopathological study of ankle joint. RESULTS: F-4 and F-7 were found to be optimized formulations as compared to other formulations. The in vitro anti-inflammatory activity was found to be highest in F-4 with IC50 7.74 and F-7 with IC50 8.87 in comparison with Diclofenac sodium having IC50 57.0. Both formulations F-7 and F-4 showed a significant reduction in paw volume and normalization of body weights. The formulation F-7 even showed more potent antiarthritic activity than F-4 by decreasing white blood cells (WBC), lymphocytes, increasing packed cell volume (PCV), neutrophils, superoxide dismutase (SOD), catalase and decreasing malondialdehyde (MDA) levels in serum. This was further confirmed by histopathological study. CONCLUSION: As an anti-inflammatory agent, this newly developed emulgel was found to possess more therapeutic efficacy than commercially available diclofenac sodium.

Role of Bioactive Compounds, Novel Drug Delivery Systems, and Polyherbal Formulations in the Management of Rheumatoid Arthritis.

Rheumatoid Arthritis (RA) is an autoimmune disorder that generally causes joint synovial inflammation as well as gradual cartilage and degenerative changes, resulting in progressive immobility. Cartilage destruction induces synovial inflammation, including synovial cell hyperplasia, increased synovial fluid, and synovial pane development. This phenomenon causes articular cartilage damage and joint alkalosis. Traditional medicinal system exerts their effect through several cellular mechanisms, including inhibition of inflammatory mediators, oxidative stress suppression, cartilage degradation inhibition, increasing antioxidants and decreasing rheumatic biomarkers. The medicinal plants have yielded a variety of active constituents from various chemical categories, including alkaloids, triterpenoids, steroids, glycosides, volatile oils, flavonoids, lignans, coumarins, terpenes, sesquiterpene lactones, anthocyanins, and anthraquinones. This review sheds light on the utilization of medicinal plants in the treatment of RA. It explains various phytoconstituents present in medicinal plants and their mechanism of action against RA. It also briefs about the uses of polyherbal formulations (PHF), which are currently in the market and the toxicity associated with the use of medicinal plants and PHF, along with the limitations and research gaps in the field of PHF. This review paper is an attempt to understand various mechanistic approaches employed by several medicinal plants, their possible drug delivery systems and synergistic effects for curing RA with minimum side effects.

Morphological and molecular diversity in mid-late and late maturity genotypes of cauliflower.

Genetic diversity is the prerequisite for the success of crop improvement programmes. Keeping in view, the current investigation was undertaken to assess the agro-morphological and molecular diversity involving 36 diverse mid-late and late cauliflower genotypes following α-RBD design during winter season 2021-22. Six morphological descriptors predicted as polymorphic using Shannon diversity index with maximum for leaf margin (0.94). The genotypes grouped into nine clusters based on D2 analysis with four as monogenotypic and gross plant weight (32.38%) revealed maximum contribution towards the genetic diversity. Molecular diversity analysis revealed 2-7 alleles among 36 polymorphic simple sequence repeats (SSR) with average of 4.22. Primer BoESSR492 (0.77) showed maximum polymorphic information content (PIC) with mean of 0.58. SSR analysis revealed two clusters each with two subclusters with a composite pattern of genotype distribution. STRUCTURE analysis showed homogenous mixture with least amount of gene pool introgression within the genotypes. Thus, based on morphological and molecular studies, the diverse genotypes namely, DPCaCMS-1, DPCaf-W4, DPCaf-US, DPCaf-W131W, DPCaf-S121, DPCaf-18, DPCaf-13, DPCaf-29 and DPCaf-CMS5 can be utilized in hybridization to isolate potential transgressive segregants to broaden the genetic base of cauliflower or involve them to exploit heterosis.

The Ethnopharmacological Properties of Green-Engineered Metallic Nanoparticles against Metabolic Disorders.

Metabolic syndrome is a multifaceted pathophysiologic condition that is largely caused by an imbalance between caloric intake and energy expenditure. The pathogenesis of metabolic syndrome is determined by an individual's genetic/epigenetics and acquired factors. Natural compounds, notably plant extracts, have antioxidant, anti-inflammatory, and insulin-sensitizing properties and are considered to be a viable option for metabolic disorder treatment due to their low risk of side effects. However, the limited solubility, low bioavailability, and instability of these botanicals hinder their performance. These specific limitations have prompted the need for an efficient system that reduces drug degradation and loss, eliminates unwanted side effects, and boosts drug bioavailability, as well as the percentage of the drug deposited in the target areas. The quest for an enhanced (effective) drug delivery system has led to the formation of green-engineered nanoparticles, which has increased the bioavailability, biodistribution, solubility, and stability of plant-based products. The unification of plant extracts and metallic nanoparticles has helped in the development of new therapeutics against metabolic disorders such as obesity, diabetes mellitus, neurodegenerative disorders, non-alcoholic fatty liver, and cancer. The present review outlines the pathophysiology of metabolic diseases and their cures with plant-based nanomedicine.

Functionalizing Collagen Membranes with MSC-Conditioned Media Promotes Guided Bone Regeneration in Rat Calvarial Defects.

Functionalizing biomaterials with conditioned media (CM) from mesenchymal stromal cells (MSC) is a promising strategy for enhancing the outcomes of guided bone regeneration (GBR). This study aimed to evaluate the bone regenerative potential of collagen membranes (MEM) functionalized with CM from human bone marrow MSC (MEM-CM) in critical size rat calvarial defects. MEM-CM prepared via soaking (CM-SOAK) or soaking followed by lyophilization (CM-LYO) were applied to critical size rat calvarial defects. Control treatments included native MEM, MEM with rat MSC (CEL) and no treatment. New bone formation was analyzed via micro-CT (2 and 4 weeks) and histology (4 weeks). Greater radiographic new bone formation occurred at 2 weeks in the CM-LYO group vs. all other groups. After 4 weeks, only the CM-LYO group was superior to the untreated control group, whereas the CM-SOAK, CEL and native MEM groups were similar. Histologically, the regenerated tissues showed a combination of regular new bone and hybrid new bone, which formed within the membrane compartment and was characterized by the incorporation of mineralized MEM fibers. Areas of new bone formation and MEM mineralization were greatest in the CM-LYO group. Proteomic analysis of lyophilized CM revealed the enrichment of several proteins and biological processes related to bone formation. In summary, lyophilized MEM-CM enhanced new bone formation in rat calvarial defects, thus representing a novel 'off-the-shelf' strategy for GBR.

Facial cutaneous phaeohyphomycosis in an immunocompetent individual.

ABSTRACT: Fungal infection is a rare condition in immunocompetent individuals, and it is associated with high rates of morbidity and mortality. We report on a case of cutaneous phaeohyphomycosis in healthy 25-year-old man. Based on the clinical findings, the case was first thought to be cervico-facial actinomycosis, but Alternaria was identified on the culture after debridement. Simple surgical excision resulted in the complete cure without administration of systemic antifungals.

Immune-instructive copolymer scaffolds using plant-derived nanoparticles to promote bone regeneration.

BACKGROUND: Age-driven immune signals cause a state of chronic low-grade inflammation and in consequence affect bone healing and cause challenges for clinicians when repairing critical-sized bone defects in elderly patients. METHODS: Poly(L-lactide-co-ɛ-caprolactone) (PLCA) scaffolds are functionalized with plant-derived nanoparticles from potato, rhamnogalacturonan-I (RG-I), to investigate their ability to modulate inflammation in vitro in neutrophils and macrophages at gene and protein levels. The scaffolds' early and late host response at gene, protein and histological levels is tested in vivo in a subcutaneous rat model and their potential to promote bone regeneration in an aged rodent was tested in a critical-sized calvaria bone defect. Significant differences were tested using one-way ANOVA, followed by a multiple-comparison Tukey's test with a p value ≤ 0.05 considered significant. RESULTS: Gene expressions revealed PLCA scaffold functionalized with plant-derived RG-I with a relatively higher amount of galactose than arabinose (potato dearabinated (PA)) to reduce the inflammatory state stimulated by bacterial LPS in neutrophils and macrophages in vitro. LPS-stimulated neutrophils show a significantly decreased intracellular accumulation of galectin-3 in the presence of PA functionalization compared to Control (unmodified PLCA scaffolds). The in vivo gene and protein expressions revealed comparable results to in vitro. The host response is modulated towards anti-inflammatory/ healing at early and late time points at gene and protein levels. A reduced foreign body reaction and fibrous capsule formation is observed when PLCA scaffolds functionalized with PA were implanted in vivo subcutaneously. PLCA scaffolds functionalized with PA modulated the cytokine and chemokine expressions in vivo during early and late inflammatory phases. PLCA scaffolds functionalized with PA implanted in calvaria defects of aged rats downregulating pro-inflammatory gene markers while promoting osteogenic markers after 2 weeks in vivo. CONCLUSION: We have shown that PLCA scaffolds functionalized with plant-derived RG-I with a relatively higher amount of galactose play a role in the modulation of inflammatory responses both in vitro and in vivo subcutaneously and promote the initiation of bone formation in a critical-sized bone defect of an aged rodent. Our study addresses the increasing demand in bone tissue engineering for immunomodulatory 3D scaffolds that promote osteogenesis and modulate immune responses.

Extracellular Vesicles Derived from Primed Mesenchymal Stromal Cells Loaded on Biphasic Calcium Phosphate Biomaterial Exhibit Enhanced Macrophage Polarization.

Mesenchymal stromal cells (MSC) loaded on biphasic calcium phosphate biomaterial (MSC + BCP) have been used as an advanced therapy medicinal product to treat complex maxillofacial bone defects in patients. Further, MSC-derived extracellular vesicles (EVs) are established vehicles of paracrine factors, supporting inter-cellular communication between MSC and other interacting cell types, such as monocytes/macrophages. However, the information about the immunomodulatory potential of EVs derived from MSC and biomaterial constructs (MSC + BCP:EV) and inflammatory primed constructs (MSCp + BCP:EV) are scarce. Hence, we isolated and characterized EVs from these different systems, and compared their cytokine contents with plastic-adherent MSC-derived EVs (MSC:EV). When EVs from all three MSC systems were added to the primary blood-derived macrophages in vitro, significantly higher numbers of M0 (naive) macrophages shifted to M2-like (anti-inflammatory) by MSCp + BCP:EV treatment. Further, this treatment led to enhanced switching of M1 polarized macrophages to M2 polarized, and conversely, M2 to M1, as evaluated by determining the M1/M2 ratios after treatment. The enhanced macrophage modulation by MSCp + BCP:EV was attributed to their higher immunomodulatory (TNFα, IL1ß, IL5), angiogenic (VEGF), and chemokine-rich (RANTES, MCP1, MIP1ß) cytokine cargo. In conclusion, we successfully isolated and characterized EVs from MSC + BCP constructs and demonstrated that, depending upon the tissue microenvironment, these EVs contribute towards modulating the macrophage-mediated inflammation and healing responses. The study offers new insights into the use of biomaterial-induced EVs for MSC secretome delivery, as a step towards future 'cell-free' bone regenerative therapies.

Influence of Bone Substitutes on Mesenchymal Stromal Cells in an Inflammatory Microenvironment.

Bone regeneration is driven by mesenchymal stromal cells (MSCs) via their interactions with immune cells, such as macrophages (MPs). Bone substitutes, e.g., bi-calcium phosphates (BCPs), are commonly used to treat bone defects. However, little research has focused on MSC responses to BCPs in the context of inflammation. The objective of this study was to investigate whether BCPs influence MSC responses and MSC-MP interactions, at the gene and protein levels, in an inflammatory microenvironment. In setup A, human bone marrow MSCs combined with two different BCP granules (BCP 60/40 or BCP 20/80) were cultured with or without cytokine stimulation (IL1ß + TNFα) to mimic acute inflammation. In setup B, U937 cell-line-derived MPs were introduced via transwell cocultures to setup A. Monolayer MSCs with and without cytokine stimulation served as controls. After 72 h, the expressions of genes related to osteogenesis, healing, inflammation and remodeling were assessed in the MSCs via quantitative polymerase chain reactions. Additionally, MSC-secreted cytokines related to healing, inflammation and chemotaxis were assessed via multiplex immunoassays. Overall, the results indicate that, under both inflammatory and non-inflammatory conditions, the BCP granules significantly regulated the MSC gene expressions towards a pro-healing genotype but had relatively little effect on the MSC secretory profiles. In the presence of the MPs (coculture), the BCPs positively regulated both the gene expression and cytokine secretion of the MSCs. Overall, similar trends in MSC responses were observed with BCP 60/40 and BCP 20/80. In summary, within the limits of in vitro models, these findings suggest that the presence of BCP granules at a surgical site may not necessarily have a detrimental effect on MSC-mediated wound healing, even in the event of inflammation.

Systemic and local innate immune responses to surgical co-transplantation of mesenchymal stromal cells and biphasic calcium phosphate for bone regeneration.

Bone regeneration from mesenchymal stromal cells (MSC) is attributed to comprehensive immune modulation mediated by the MSC. However, the temporal and spatial regulation of these immune responses has not yet been described. The aim of the present study was to assess the local and systemic innate immune responses to implantation of biphasic calcium phosphate biomaterial (BCP) alone, or with bone marrow derived MSC (BCP+MSC), in critical-sized calvarial bone defects of Lewis rats. Four weeks after implantation, flow cytometry analysis of innate immune cells revealed increased numbers of circulating classical monocyte-macrophages (MM) and decreased non-classical MM in the BCP+MSC group. At week 8, this differential systemic MM response was associated with an increased presence of local tissue anti-inflammatory macrophages expressing CD68 and CD163 markers (M2-like). In the BCP group without MSC, NK cells increased at weeks 1 and 4, and neutrophils increased in circulation at weeks 2 and 8. At week 8, the increase in number of neutrophils in circulation was associated with decreased local tissue neutrophils, in the BCP+MSC group. Gene expression analysis of tissue biopsies from defects implanted with BCP+MSC, in comparison to BCP alone, revealed upregulated expression of early osteogenesis genes along with macrophage differentiation-related genes at weeks 1 and 8 and neutrophil chemotaxis-related genes at week 1. This study is the first to demonstrate that surgical implantation of BCP or BCP+MSC grafts differentially regulate both systemic and local tissue innate immune responses which enhance bone formation. The results provide new insights into immune mechanisms underlying MSC-mediated bone regeneration. STATEMENT OF SIGNIFICANCE: The suitability of biphasic calcium phosphate and mesenchymal stromal cell construct (BCP+MSC) transplantation is evident from their progress in clinical trials for treating challenging maxillofacial bone defects. But less is known about the overall immune response generated by this surgical process and how it later impacts the bone formation. To this end, it is crucial to understand for both clinicians and researchers, the systemic immune response to transplanting MSC in patients for ensuring both the safety and efficacy of cell therapies. In this study, we used rat calvarial bone defect model and showed that both systemic and local innate immunes responses (monocyte-macrophages and neutrophils) are favorably directed towards enhanced bone formation in BCP+MSC implanted defects, as compared to BCP alone.

Utility of Antigen-Based Rapid Diagnostic Test for Detection of SARS-CoV-2 Virus in Routine Hospital Settings.

OBJECTIVE: This study aims to evaluate the performance of an antigen-based rapid diagnostic test (RDT) for the detection of the SARS-CoV-2 virus. METHODS: A cross-sectional study was conducted on 677 patients. Two nasopharyngeal swabs and 1 oropharyngeal swab were collected from patients. The RDT was performed onsite by a commercially available immune-chromatographic assay on the nasopharyngeal swab. The nasopharyngeal and oropharyngeal swabs were examined for SARS-CoV-2 RNA by real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay. RESULTS: The overall sensitivity of the SARS-CoV-2 RDT was 34.5% and the specificity was 99.8%. The positive predictive value and negative predictive value of the test were 96.6% and 91.5%, respectively. The detection rate of RDT in RT-qPCR positive results was high (45%) for cycle threshold values <25. CONCLUSION: The utility of RDT is in diagnosing symptomatic patients and may not be particularly suited as a screening tool for patients with low viral load. The low sensitivity of RDT does not qualify its use as a single test in patients who test negative; RT-qPCR continues to be the gold standard test.

Clinical characteristic and epidemiological features of SARS CoV -2 disease patients from a COVID 19 designated Hospital in New Delhi.

SARS CoV -2 infection is rapidly evolving as a serious global pandemic. The present study describes the clinical characteristics of SARS CoV-2 infection patients. The Samples were subjected to RT - PCR or Rapid Antigen test for diagnosis of SARS CoV- 2. A cohort of 3745 patients with confirmed diagnosis of SARS CoV -2 infection in a tertiary care center in New Delhi, India were included in this study. Data was collected from offline and online medical records over a period of six months. Amongst 3745 SARS CoV -2 infected patients, 2245 (60%) were symptomatic and 1500 (40%) were asymptomatic. Most common presenting symptom was cough (49.3%) followed febrile episodes (47.1%), breathlessness (42.7%) and sore throat (35.1%). Cough along with breathlessness (24.1) was the most common combination of symptoms followed by fever with cough (22.7). The most common comorbidity found among symptomatic group was diabetes (42.5%) followed by hypertension (21.4%) and chronic kidney disease (18%). Comorbidities like diabetes mellitus, chronic diseases of lungs, heart and kidneys were found to be common in symptomatic group and this was found to be statistically significant (p<0.05). COVID-19 is an evolving disease and data from our study help in understanding the clinic-epidemiological profile of patients. This article is protected by copyright. All rights reserved.

Termite's potential in solid waste management in Himachal Pradesh: A mini review.

One of the major reasons for the degradation of Earth's setting is inappropriate disposal of solid waste. Mountains of solid waste are increasing in every country making solid waste management a challenge almost everywhere on Earth. It is vital to look for such municipal solid waste management solutions that are financially sustainable, technically possible, socially and legally acceptable and environmentally friendly. Currently vermicomposting is the only biological solid waste treatment process that uses multicellular organisms to biodegrade organic wastes. A few species of insects are capable of digesting lignin and cellulose. Of these, termites are the most numerous and play a decisive role as scavengers. Microflora which inhabit the termites' gut contribute to their waste degrading potential. Termites act as major soil ecosystem managers and are able to breakdown and recycle organic matter and composite. Although studies in the potential of termites to increase soil fertility are well-accounted for in the literature, the potential of termites for solid waste management still needs to be explored. This mini review presents the state of information on the use of termite species in solid waste degradation focused on the potential application in the Himachal Pradesh region, India. This review highlights different termite species found in Himachal Pradesh and the challenges that are needed to be conquered. The study also aims at encouraging competent authorities/researchers to work towards the improvement of the present system by further exploring the use of termites in solid waste management through suggestions and recommendations.

Bacillus cereus: public health burden associated with ready-to-eat foods in Himachal Pradesh, India.

The study determined incidence, enterotoxigenecity and antimicrobial susceptibility profiles of Bacillus cereus isolated from ready-to-eat (RTE) milk products (n = 80), RTE meat products (n = 40), beverages (n = 40) and water samples (n = 60, from food preparing and serving outlets/restaurants) collected from eight different tourist places of Himachal Pradesh. 11.4% (25/220) samples were contaminated with Bacillus and isolates were identified as B. cereus (76.0%, n = 19), B. alvei (12.0%, n = 3), B. polymyxa (8.0%, n = 2) and B. firmus (4.0%, n = 1) by conventional and molecular methods. B. cereus incidence was highest in cheese based foods (25.0%) followed by vegetable soups (16.7%), khoa based foods (14.0%), milk based beverages (10.5%), paneer based foods (8.6%), cream based foods (8.3%) and water (8.3%) samples. Multiplex polymerase chain reaction detected enterotoxigenic genes only in B. cereus isolates. nhe complex (encoding non-haemolytic enterotoxins, ABC) genes were detected only in B. cereus isolates. 57.6% (11/19), 36.8% (7/19) and 5.3% (1/19) harboured all three (nheA, nheB, nheC), two (nheB, nheC) and one (nheC) nhe gene, respectively. Among hbl complex genes (encoding haemolytic enterotoxins CAD), only hblC (36.8%, 7/19) was detected. Incidence B. cereus cytK (encoding cytotoxin enterotoxin) was 52.6% (10/19). Each B. cereus isolate harboured two or more enterotoxigenic genes. Seven isolates had at least one gene from haemolytic and non-haemolytic complexes along with cytK. High levels (> 50%) of antimicrobial resistance were recorded for penicillin, amoxicillin, ampicillin cefixime and ceftazidine in tested B. cereus isolates. Two isolates were identified as multidrug resistant isolates with resistance to ≥ 3 antibiotic classes.

Peptidoglycan reshaping by a noncanonical peptidase for helical cell shape in Campylobacter jejuni.

Assembly of the peptidoglycan is crucial in maintaining viability of bacteria and in defining bacterial cell shapes, both of which are important for existence in the ecological niche that the organism occupies. Here, eight crystal structures for a member of the cell-shape-determining class of Campylobacter jejuni, the peptidoglycan peptidase 3 (Pgp3), are reported. Characterization of the turnover chemistry of Pgp3 reveals cell wall D,D-endopeptidase and D,D-carboxypeptidase activities. Catalysis is accompanied by large conformational changes upon peptidoglycan binding, whereby a loop regulates access to the active site. Furthermore, prior hydrolysis of the crosslinked peptide stem from the saccharide backbone of the peptidoglycan on one side is a pre-requisite for its recognition and turnover by Pgp3. These analyses reveal the noncanonical nature of the transformations at the core of the events that define the morphological shape for C. jejuni as an intestinal pathogen.

Liquid-Liquid Phase Separation of Histone Proteins in Cells: Role in Chromatin Organization.

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids has emerged as an important phenomenon in membraneless intracellular organization. We demonstrate that the linker histone H1 condenses into liquid-like droplets in the nuclei of HeLa cells. The droplets, observed during the interphase of the cell cycle, are colocalized with DNA-dense regions indicative of heterochromatin. In vitro, H1 readily undergoes LLPS with both DNA and nucleosomes of varying lengths but does not phase separate in the absence of DNA. The nucleosome core particle maintains its structural integrity inside the droplets, as demonstrated by FRET. Unexpectedly, H2A also forms droplets in the presence of DNA and nucleosomes in vitro, whereas the other core histones precipitate. The phase diagram of H1 with nucleosomes is invariant to the nucleosome length at physiological salt concentration, indicating that H1 is capable of partitioning large segments of DNA into liquid-like droplets. Of the proteins tested (H1, core histones, and the heterochromatin protein HP1α), this property is unique to H1. In addition, free nucleotides promote droplet formation of H1 nucleosome in a nucleotide-dependent manner, with droplet formation being most favorable with ATP. Although LLPS of HP1α is known to contribute to the organization of heterochromatin, our results indicate that H1 also plays a role. Based on our study, we propose that H1 and DNA act as scaffolds for phase-separated heterochromatin domains.

Establishment of a novel cancer cell line derived from vulvar carcinoma associated with lichen sclerosus exhibiting a fibroblast-dependent tumorigenic potential.

Vulvar squamous cell carcinoma associated with lichen sclerosus (VLS-VSCC) are rare tumors but with higher recurrence and worse prognosis than other types of VSCC. Lack of experimental models has limited the search for better understanding of the biology and development of treatment modalities. In this study, we isolated and characterized primary cells from VSCC (n = 7) and normal vulvar tissue adjacent to tumor (n = 7). Detailed characterization of the novel spontaneously immortalized cell line, VCC1 revealed a characteristic epithelial morphology in vitro and a well-differentiated keratinizing SCC histology in vivo, closely resembling the tumor of origin. VCC1 expressed higher levels of epithelial-mesenchymal transition markers and higher clonogenic properties as compared to other established non VLS-VSCC cell lines. In vitro 3D organotypic assays and in vivo xenografts revealed a prominent role of cancer-associated fibroblasts in VCC1 invasion and tumor formation. In conclusion, VCC1 mirrored several major VLS-VSCC features and provided a robust experimental tool for further elucidation of VLS-related oncogenesis and drug testing.