Proteome profiling, biochemical and histological analysis of diclofenac-induced liver toxicity in Yersinia enterocolitica and Lactobacillus fermentum fed rat model: a comparative analysis.

Diclofenac is a hepatotoxic non-steroidal anti-inflammatory drug (NSAID) that affects liver histology and its protein expression levels. Here, we studied the effect of diclofenac on rat liver when co-administrated with either Yersinia enterocolitica strain 8081 serotype O:8 biovar 1B (D*Y) or Lactobacillus fermentum strain 9338 (D*L). Spectroscopic analysis of stool samples showed biotransformation of diclofenac. When compared with each other, D*Y rats lack peaks at 1709 and 1198 cm-1, while D*L rats lack peaks at 1411 cm-1. However, when compared to control, both groups lack peaks at 1379 and 1170 cm-1. Assessment of serum biomarkers of hepatotoxicity indicated significantly altered activities of AST (D*Y: 185.65 ± 8.575 vs Control: 61.9 ± 2.607, D*L: 247.5 ± 5.717 vs Control: 61.9 ± 2.607), ALT (D*Y: 229.8 ± 6.920 vs Control: 70.7 ± 3.109, D*L: 123.75 ± 6.068 vs Control: 70.7 ± 3.109), and ALP (D*Y: 276.4 ± 18.154 vs Control: 320.6 ± 9.829, D*L: 298.5 ± 12.336 vs Control: 320.6 ± 9.829) in IU/L. The analysis of histological alterations showed hepatic sinusoidal dilation with vein congestion and cell infiltration exclusively in D*Y rats along with other histological changes that are common to both test groups, thereby suggesting more pronounced alterations in D*Y rats. Further, LC-MS/MS based label-free quantitation of proteins from liver tissues revealed 74.75% up-regulated, 25.25% down-regulated in D*Y rats and 51.16% up-regulated, 48.84% down-regulated in D*L experiments. The proteomics-identified proteins majorly belonged to metabolism, apoptosis, stress response and redox homeostasis, and detoxification and antioxidant defence that demonstrated the potential damage of rat liver, more pronounced in D*Y rats. Altogether the results are in favor that the administration of lactobacilli somewhat protected the rat hepatic cells against the diclofenac-induced toxicity.

Bridging the Evidence and Practice Gap in Chronic Kidney Disease: A System Thinking Approach to Population Health.

Chronic kidney disease (CKD) is common, costly, and life-limiting, requiring dialysis and transplantation in advanced stages. Although effective guideline-based therapy exists, the asymptomatic nature of CKD together with low health literacy, adverse social determinants of health, unmet behavioral health needs, and primary care providers' (PCP) limited understanding of CKD result in defects in screening and diagnosis. Care is fragmented between PCPs and specialty nephrologists, with limited time, expertise, and resources to address systemic gaps. In this article, the authors define how they classified defects in care and report the current numbers of patients exposed to these defects, both nationally and in their health system Accountable Care Organization. They describe use of the health system's three-pillar leadership model (believing, belonging, and building) to empower providers to transform CKD care. Believing entailed engaging individuals to believe defects in CKD care could be eliminated and were a collective responsibility. Belonging fostered the creation of learning communities that broke down silos and encouraged open communication and collaboration between PCPs and nephrologists. Building involved constructing a fractal management infrastructure with transparent reporting and shared accountability, which would enable success in innovation and transformation. The result is proactive and relational CKD care organized around the patient's needs in University Hospitals Systems of Excellence. Systems of excellence combine multiple domains of expertise to promote best practice guidelines and integrate care throughout the system. The authors further describe a preliminary pilot of the CKD System of Excellence in primary care.

Chronic kidney disease associated cardiomyopathy: recent advances and future perspectives.

PURPOSE OF REVIEW: Cardiomyopathy in chronic kidney disease (CKD) is a complex condition with multiple triggers and poor prognosis. This review provides an overview of recent advances in CKD-associated cardiomyopathy, with a focus on pathophysiology, newly discovered biomarkers and potential therapeutic targets. RECENT FINDINGS: CKD is associated with a specific pattern of myocardial hypertrophy and fibrosis, resulting in diastolic and systolic dysfunction, and often triggered by nonatherosclerotic processes. Novel biomarkers, including amino-terminal type III procollagen peptide (PIIINP), carboxy-terminal type I procollagen peptide (PICP), FGF23, marinobufagenin, and several miRNAs, show promise for early detection and risk stratification. Treatment options for CKD-associated cardiomyopathy are limited. Sodium glucose cotransporter-2 inhibitors have been shown to reduce left ventricle hypertrophy and improve ejection fraction in individuals with diabetes and mild CKD, and are currently under investigation for more advanced stages of CKD. In hemodialysis patients calcimimetic etelcalcetide resulted in a significant reduction in left ventricular mass. SUMMARY: CKD-associated cardiomyopathy is a common and severe complication in CKD. The identification of novel biomarkers may lead to future therapeutic targets. Randomized clinical trials in individuals with more advanced CKD would be well posed to expand treatment options for this debilitating condition.

Culturing the unculturables: strategies, challenges, and opportunities for gut microbiome study.

Metagenome sequencing techniques revolutionized the field of gut microbiome study. However, it is equipped with experimental and computational biases, which affect the downstream analysis results. Also, live microbial strains are needed for a better understanding of host-microbial crosstalks and for designing next-generation treatment therapies based on probiotic strains and postbiotic molecules. Conventional culturing methodologies are insufficient to get the dark gut matter on the plate; therefore, there is an urgent need to propose novel culturing methods that can fill the limitations of metagenomics. The current work aims to provide a consolidated evaluation of the available methods for host-microbe interaction with an emphasis on in vitro culturing of gut microbes using organoids, gut on a chip, and gut bioreactor. Further, the knowledge of microbial crosstalk in the gut helps us to identify core microbiota, and key metabolites that will aid in designing culturing media and co-culturing systems for gut microbiome study. After the deeper mining of the current culturing methods, we recommend that 3D-printed intestinal cells in a multistage continuous flow reactor equipped with an extended organoid system might be a good practical choice for gut microbiota-based studies.

Lepidopteran insects: emerging model organisms to study infection by enteropathogens.

The in vivo analysis of a pathogen is a critical step in gaining greater knowledge of pathogen biology and host-pathogen interactions. In the last two decades, there has been a notable rise in the number of studies on developing insects as a model for studying pathogens, which provides various benefits, such as ethical acceptability, relatively short life cycle, and cost-effective care and maintenance relative to routinely used rodent infection models. Furthermore, lepidopteran insects provide many advantages, such as easy handling and tissue extraction due to their large size relative to other invertebrate models, like Caenorhabditis elegans. Additionally, insects have an innate immune system that is highly analogous to vertebrates. In the present review, we discuss the components of the insect's larval immune system, which strengthens its usage as an alternative host, and present an updated overview of the research findings involving lepidopteran insects (Galleria mellonella, Manduca sexta, Bombyx mori, and Helicoverpa armigera) as infection models to study the virulence by enteropathogens due to the homology between insect and vertebrate gut.

Proteomic, biochemical, histopathological, and elevated plus maze analysis reveals the gut damaging role of ketoprofen with Yersinia enterocolitica and altered behavior in Wistar rats.

The long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) is known to damage the intestinal epithelial cells (IECs) that play numerous important roles, including nutrient absorption and barrier protection. In the current study, we determined the effect of ketoprofen on the rat gut when administered with Yersinia enterocolitica. On performing the label-free quantitation of the rat gut proteins, the expression of 494 proteins out of 1628 proteins was altered, which has a profound effect on NF-kB signaling pathway, immune system, dysbiosis, and gut injury. Further, the biochemical [enhanced malondialdehyde (MDA) & hepatic enzyme activities and reduced serotonin & antioxidants levels i.e., catalase (CAT) and superoxide dismutase (SOD)] and histopathological analysis suggested the significant damage in treated rats, compared to control rats. Lastly, the elevated plus maze (EPM) study confirmed high levels of anxiety in treated rats in comparison to the control group. Altogether, results suggest that the co-administration of ketoprofen with Y. enterocolitica damages gut, alters hepatic enzyme activities, and affects behavioral responses in the treated rats.

Balancing reactive oxygen species generation by rebooting gut microbiota.

Reactive oxygen species (ROS; free radical form O2 •- , superoxide radical; OH• , hydroxyl radical; ROO• , peroxyl; RO• , alkoxyl and non-radical form 1 O2 , singlet oxygen; H2 O2 , hydrogen peroxide) are inevitable companions of aerobic life with crucial role in gut health. But, overwhelming production of ROS can cause serious damage to biomolecules. In this review, we have discussed several sources of ROS production that can be beneficial or dangerous to the human gut. Micro-organisms, organelles and enzymes play crucial role in ROS generation, where NOX1 is the main intestinal enzyme, which produce ROS in the intestine epithelial cells. Previous studies have reported that probiotics play significant role in gut homeostasis by checking the ROS generation, maintaining the antioxidant level, immune system and barrier protection. With current knowledge, we have critically analysed the available literature and presented the outcome in the form of bubble maps to suggest that the probiotics help in controlling the ROS-specific intestinal diseases, such as inflammatory bowel disease (IBD) and colon cancer. Finally, it has been concluded that rebooting of the gut microbiota with probiotics, postbiotics or faecal microbiota transplantation (FMT) can have crucial implications in the structuring of gut communities for the personalized management of the gastrointestinal (GI) diseases.

Yersinia enterocolitica and Lactobacillus fermentum induces differential cellular and behavioral responses during diclofenac biotransformation in rat gut.

Non-steroidal anti-inflammatory drugs (NSAIDs) can induce small-intestinal injuries through inhibition of prostaglandin synthesis. Gut has an important role in building and maintaining the barriers to avoid the luminal gut microbiota from invading the host, and cytoskeleton plays a crucial role in the maintenance of cellular barrier. The recent advances suggest a bi-directional interaction between the drugs and gut microbiota, where gut microbes can metabolize the drugs, and in response drugs can alter the composition of gut microbiota. In the present study, we evaluated the effect of diclofenac on rat gut, when co-administrated with either Yersinia enterocolitica strain 8081 (an enteropathogen) or Lactobacillus fermentum strain 9338 (a probiotic). The LC-MS/MS based label-free quantitation of rat gut proteins revealed 51.38% up-regulated, 48.62% down-regulated in diclofenac-Y. enterocolitica strain 8081 (D*Y), and 74.31% up-regulated, 25.69% down-regulated in diclofenac-L. fermentum strain 9338 (D*L) experiments. The identified proteins belonged to cytoskeleton, metabolism, heme biosynthesis and binding, stress response, apoptosis and redox homeostasis, immune and inflammatory response, and detoxification and antioxidant defence. Further, the histopathological and biochemical analysis indicated more pronounced histological alterations and oxidative stress (enhanced malonaldehyde and altered antioxidant levels) in D*Y rats than D*L rats, compared to control rats. Elevated plus maze (EPM) test performed to determine the behavioral changes, suggested increased anxiety in D*Y rats than D*L rats, compared to control rats. These results together suggest the differential role of either bacterium in biotransformation of diclofenac, and inflammatory and cellular redox response.

Infected insect gut reveals differentially expressed proteins for cellular redox, metal resistance and secretion system in Yersinia enterocolitica-Helicoverpa armigera pathogenic model.

OBJECTIVE: Mouse infection models are frequently used to study the host-pathogen interaction studies. However, due to several constraints, there is an urgent need for a simple, rapid, easy to handle, inexpensive, and ethically acceptable in vivo model system for studying the virulence of enteropathogens. Thus, the present study was performed to develop the larvae of Helicoverpa armigera as a rapid-inexpensive in vivo model system to evaluate the effect of Yersinia enterocolitica strain 8081 on its midgut via a label-free proteomic approach. RESULTS: Helicoverpa armigera larvae fed with Yersinia enterocolitica strain 8081 manifested significant reduction in body weight and damage in midgut. On performing label-free proteomic study, secretory systems, putative hemolysin, and two-component system emerged as the main pathogenic proteins. Further, proteome comparison between control and Yersinia added diet-fed (YADF) insects revealed altered cytoskeletal proteins in response to increased melanization (via a prophenoloxidase cascade) and free radical generation. In concurrence, FTIR-spectroscopy, and histopathological and biochemical analysis confirmed gut damage in YADF insects. Finally, the proteome data suggests that the mechanism of infection and the host response in Y. enterocolitica-H. armigera system mimics Yersinia-mammalian gut interactions. CONCLUSIONS: All data from current study collectively suggest that H. armigera larva can be considered as a potential in vivo model system for studying the enteropathogenic infection by Y. enterocolitica strain 8081.

Inflammatory bowel disease: tri-directional relationship between microbiota, immune system and intestinal epithelium.

Human gut microbiota contributes to host nutrition and metabolism, sustains intestinal cell proliferation and differentiation, and modulates host immune system. The alterations in their composition lead to severe gut disorders, including inflammatory bowel disease (IBD) or inflammatory bowel syndrome (IBS). IBD including ulcerative colitis (UC) and Crohn's disease (CD) are gamut of chronic inflammatory disorders of gut, mediated by complex interrelations among genetic, environmental, and internal factors. IBD has debateable aetiology, however in recent years, exploring the central role of a tri-directional relationship between gut microbiota, mucosal immune system, and intestinal epithelium in pathogenesis is getting the most attention. Increasing incidences and early onset explains the exponential rise in IBD burden on health-care systems. Industrialization, hypersensitivity to allergens, lifestyle, hygiene hypothesis, loss of intestinal worms, and gut microbial composition, explains this shifted rise. Hitherto, the interventions modulating gut microbiota composition, microfluidics-based in vitro gastrointestinal models, non-allergic functional foods, nutraceuticals, and faecal microbiota transplantation (FMT) from healthy donors are some of the futuristic approaches for the disease management.

Bioevaluation and molecular docking analysis of novel phenylpropanoid derivatives as potent food preservative and anti-microbials.

Novel derivatives were synthesized using natural scaffold, like phenylpropanoids C6-C3 backbone to reduce unfavorable browning of food due to tyrosinase and oxidative spoilage. Most of the compounds displayed mushroom tyrosinase inhibition better than kojic acid. Compound CE48 exhibited better anti-tyrosinase (IC50-29.64 µM) and antioxidant (EC50-12.67 µM) activity than the reference compounds, kojic acid (IC50-50.30 µM) and ascorbic acid (EC50-14.55 µM), respectively. Compounds SAM30, SE78, 11F, and CE48 showed better anti-B. subtilis, anti-S. aureus, and anti-A. niger activity, respectively, compared to their parents. Molecular docking studies between inhibitors and mushroom tyrosinase corroborated the experimental reports, except SAM30 (glide score - 8.117) and SE78 (glide score - 6.151). In silico absorption, distribution, metabolism, excretion/toxicity (ADME/T) and toxicological studies of these newly synthesized compounds exhibited acceptable pharmacokinetic and safety profiles, like good aqueous solubility (- 3.34 to - 7.57), low human oral absorption (e.g., SAM30, SE78, FAM34), low gut-blood barrier permeability [36.67-209.88 nm/s in Cancer coli-2 (Caco-2) cells] and [19.45-91.51 nm/s in Madin-Darby Canine Kidney (MDCK) cells], low blood-brain barrier penetration, non-mutagenicity, and non-carcinogenicity. Interestingly, the synthesized compounds also possessed multifunctional properties, like microbial growth inhibitor, free radicals scavenger, and it also prevented browning of raw fruits and vegetables by inhibiting tyrosinase enzyme. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02636-0.

Stabilization-destabilization and redox properties of laccases from medicinal mushroom Ganoderma lucidum and human pathogen Yersinia enterocolitica.

Laccases or benzenediol oxygen oxidoreductases (EC 1.10.3.2) are polyphenol multicopper oxidases that are known for their structural and functional diversity in various life forms. In the present study, the molecular and physico-chemical properties (redox-potential and secondary structures) of fungal laccase isozymes (FLIs) isolated from a medicinal mushroom Ganoderma lucidum were analyzed and compared with those of the recombinant bacterial laccases (rLac) obtained from different Yersinia enterocolitica strains. It was revealed that the FLIs contained His-Cys-His as the most conserved residue in its domain I Cu site, while the fourth and fifth residues were variable (Ile, Leu, or Phe). Evidently, the cyclic voltammetric measurements of Glac L2 at Type 1 Cu site revealed greater E° for ABTS/ABTS+ (0.312 V) and ABTS+/ABTS2+ (0.773 V) compared to the E° of rLac. Furthermore, circular dichroism-based conformational analysis revealed structural stability of the FLIs at acidic pH (3.0) and low temperature (<30 °C), while the isozymes were destabilized at neutral pH (7.0) and high-temperature conditions (>70 °C). The zymographic studies further confirmed the functional inactivation of FLIs at high temperatures (≥70 °C), predominantly due to domain unfolding. These findings provide novel insight into the evolution of the catalytic efficiency and redox properties of the FLIs, contributing to the existing knowledge regarding stress responses, metabolite production, and the biotechnological utilization of metabolites.

Immunological co-ordination between gut and lungs in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into a major pandemic called coronavirus disease 2019 (COVID-19) that has created unprecedented global health emergencies, and emerged as a serious threat due to its strong ability for human-to-human transmission. The reports indicate the ability of SARS-CoV-2 to affect almost any organ due to the presence of a receptor known as angiotensin converting enzyme 2 (ACE2) across the body. ACE2 receptor is majorly expressed in the brush border of gut enterocytes along with the ciliated cells and alveolar epithelial type II cells in the lungs. The amino acid transport function of ACE2 has been linked to gut microbial ecology in gastrointestinal (GI) tract, thereby suggesting that COVID-19 may, to some level, be linked to the enteric microbiota. The significant number of COVID-19 patients shows extra-pulmonary symptoms in the GI tract. Many subsequent studies revealed viral RNA of SARS-CoV-2 in fecal samples of COVID-19 patients. This presents a new challenge in the diagnosis and control of COVID-19 infection with a caution for proper sanitation and hygiene. Here, we aim to discuss the immunological co-ordination between gut and lungs that facilitates SARS-CoV-2 to infect and multiply in the inflammatory bowel disease (IBD) and non-IBD patients.

Proteomic analysis reveals the damaging role of low redox laccase from Yersinia enterocolitica strain 8081 in the midgut of Helicoverpa armigera.

OBJECTIVE: Earlier, we have found that the enteropathogenic Yersinia enterocolitica have evolved the survival mechanisms that regulate the expression of laccase-encoding genes in the gut. The present study aims to characterize the purified recombinant laccase from Y. enterocolitica strain 8081 biovar 1B and understand its effect on the midgut of cotton bollworm, Helicoverpa armigera (Hübner) larvae. RESULTS: The recombinant laccase protein showed high purity fold and low molecular mass (~ 43 kDa). H. armigera larvae fed with laccase protein showed a significant decrease in body weight and damage in the midgut. Further, transmission electron microscopy (TEM) studies revealed the negative effect of laccase protein on trachea, malpighian tubules, and villi of the insect. The proteome comparison between control and laccase-fed larvae of cotton bollworm showed significant expression of proteolytic enzymes, oxidoreductases, cytoskeletal proteins, ribosomal proteins; and proteins for citrate (TCA cycle) cycle, glycolysis, stress response, cell redox homeostasis, xenobiotic degradation, and insect defence. Moreover, it also resulted in the reduction of antioxidants, increased melanization (insect innate immune response), and enhanced free radical generation. CONCLUSIONS: All these data collectively suggest that H. armigera (Hübner) larvae can be used to study the effect of microbes and their metabolites on the host physiology, anatomy, and survival.

Molecular modeling and MD-simulation studies: Fast and reliable tool to study the role of low-redox bacterial laccases in the decolorization of various commercial dyes.

Synthetic dyes are toxic and carcinogenic in nature, which also causes environmental pollution. The present study was aimed to decolorize various commercial dyes using purified recombinant bacterial laccases. Laccase gene from Yersinia enterocolitica strain 8081 (yacK), Y. enterocolitica strain 7 (yacK) and Bacillus pumilus DSKK1 was cloned in vector pET28a and overproduced in host Escherichia coli BL21. The high yield of recombinant laccase protein resulted in the formation of inclusion bodies, which were further solubilized, refolded, and purified. The purified recombinant laccases were alkali-tolerant and thermostable, with pH optima at 7-8, temperature optima at 60-70 °C and low redox potential. For in silico studies, laccase protein models of B. pumilus DSKK1, Y. enterocolitica strain 7 and Y. enterocolitica strain 8081 were docked with commercial dyes. This is the first and foremost study where the stability of docked complexes of pathogenic and non-pathogenic microorganism has been explored via molecular dynamics (MD) simulations using Gromacs version 4.5.5 with the gromos96 43a force field. Finally, the in silico results were validated experimentally and it was found that purified laccases from B. pumilus DSKK1 and Y. enterocolitica strain 7 efficiently decolorized rose bengal (90.4%), malachite green (77.7%), and congo red (74.5%) dyes.