An Approach to Track and Analyze the Trend of Antimicrobial Resistance Using Python: A Pilot Study for Anand, Gujarat, India-May 2022-August 2023.

The present work deals with the analysis and monitoring of bacterial resistance in using Python for the state of Gujarat, India, where occurrences of drug-resistant bacteria are prevalent. This will provide an insight into the portfolio of drug-resistant bacteria reported, which can be used to track resistance behavior and to suggest a treatment regime for the particular bacteria. The present analysis has been done using Python on Jupyter Notebook as the integrated development environment and its data analysis libraries such as Pandas, Seaborn, and Matplotlib. The data have been loaded from excel file using Pandas and cleaned to transform features into required format. Seaborn and Matplotlib have been used to create data visualizations and represent the data inexplicable manner using graphs, plots, and tables. This program can be used to study disaster epidemiology, tracking, analyzing, and surveillance of antimicrobial resistance with a proper system integration approach.

The immune response to systemically administered endotoxin in the murine intestinal microcirculation under pentobarbital versus isoflurane anesthesia.

BACKGROUND: Pentobarbital and isoflurane are commonly used veterinary anesthetics. Due to the dangers of overdose by repeat-bolus regimen of pentobarbital, isoflurane has been recommended. However, literature suggests isoflurane-induced inhibition of cytokine and adhesion molecule release, impacting leukocyte adhesion. OBJECTIVE: This study aims to characterize the impacts of pentobarbital versus isoflurane on leukocyte interactions within the intestinal microcirculation with and without endotoxin challenge. METHODS: Female BALB/c mice were subjected to pentobarbital or isoflurane (N = 20) and challenged with endotoxin or saline by intraperitoneal injection. The mice were kept under anesthesia for 2 hours. Fluorochromes, rhodamine-6 G and fluorescein isothiocyanate, were injected intravenously. To visualize leukocyte adhesion within the intestinal microcirculation, laparotomy and intravital microscopy was performed. Leukocyte rolling and adhesion was quantified offline in a blinded fashion. RESULTS: Within collecting venules, leukocyte rolling and adhesion showed no significant differences between pentobarbital and isoflurane anesthesia under basal conditions. Endotoxin challenge caused a similar response in both anesthetic groups. Within postcapillary venules, no statistical differences between the two anesthetics were found for adhering leukocytes under basal conditions or following endotoxin challenge either. However, leukocyte rolling after LPS-challenge was significantly decreased in postcapillary venules during isoflurane anesthesia compared to pentobarbital anesthesia. CONCLUSIONS: Isoflurane anesthesia showed only minor differences in the immune response to endotoxin within the intestinal microcirculation compared to pentobarbital anesthesia. Due to the superior safety profile of volatile anesthetics, immunological studies may choose isoflurane over pentobarbital as the veterinary anesthetic of choice.

Cannabidiol Reduces Systemic Immune Activation in Experimental Acute Lung Injury.

Background: The underlying pathomechanism of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is the immune response to inflammation or infection within the pulmonary microcirculation. Systemic spread of pathogens, activated immune cells, and inflammatory mediators contributes significantly to mortality in patients with ARDS. Objective: The endogenous cannabinoid system is a major modulator of the immune response during inflammation and infection. Phytocannabinoids, such as cannabidiol (CBD), have shown promising anti-inflammatory effects in several pathologies. The overall objective of this study was to evaluate the effects of CBD on local and systemic inflammation in endotoxin-induced ALI in mice. Materials and Methods: ALI was induced by pulmonary endotoxin challenge. Four groups of male C57BL/6 mice were randomized in this study: control, ALI, ALI with CBD treatment, and control with CBD treatment. Analyses of local and systemic cytokine levels, lung histology, and leukocyte activation as visualized by intravital microscopy of the intestinal and pulmonary microcirculation were performed 6 h following intranasal endotoxin administration. Results: Pulmonary endotoxin challenge induced significant inflammation evidenced by local and systemic cytokine and chemokine release, lung histopathology, and leukocyte adhesion. Intraperitoneal CBD treatment resulted in a significant decrease in systemic inflammation as shown by reduced leukocyte adhesion in the intestinal microcirculation and reduced plasma cytokine and chemokine levels. Pulmonary chemokine levels were decreased, while pulmonary cytokine levels were unchanged. Surprisingly, the ALI score was slightly increased by CBD treatment in a manner driven by enhanced neutrophil infiltration of the alveoli. Conclusion: In this model of experimental ALI, CBD administration was associated with reduced systemic inflammation and heterogeneous effects on pulmonary inflammation. Future studies should explore the mechanisms involved as they relate to neutrophil infiltration and proinflammatory mediator production within the lungs.

Selective CB2 Receptor Agonist, HU-308, Reduces Systemic Inflammation in Endotoxin Model of Pneumonia-Induced Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) and sepsis are risk factors contributing to mortality in patients with pneumonia. In ARDS, also termed acute lung injury (ALI), pulmonary immune responses lead to excessive pro-inflammatory cytokine release and aberrant alveolar neutrophil infiltration. Systemic spread of cytokines is associated with systemic complications including sepsis, multi-organ failure, and death. Thus, dampening pro-inflammatory cytokine release is a viable strategy to improve outcome. Activation of cannabinoid type II receptor (CB2) has been shown to reduce cytokine release in various in vivo and in vitro studies. Herein, we investigated the effect of HU-308, a specific CB2 agonist, on systemic and pulmonary inflammation in a model of pneumonia-induced ALI. C57Bl/6 mice received intranasal endotoxin or saline, followed by intravenous HU-308, dexamethasone, or vehicle. ALI was scored by histology and plasma levels of select inflammatory mediators were assessed by Luminex assay. Intravital microscopy (IVM) was performed to assess leukocyte adhesion and capillary perfusion in intestinal and pulmonary microcirculation. HU-308 and dexamethasone attenuated LPS-induced cytokine release and intestinal microcirculatory impairment. HU-308 modestly reduced ALI score, while dexamethasone abolished it. These results suggest administration of HU-308 can reduce systemic inflammation without suppressing pulmonary immune response in pneumonia-induced ALI and systemic inflammation.

Antiviral and Anti-Inflammatory Plant-Derived Bioactive Compounds and Their Potential Use in the Treatment of COVID-19-Related Pathologies.

The highly contagious coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been declared a global pandemic and public health emergency as it has taken the lives of over 5.7 million in more than 180 different countries. This disease is characterized by respiratory tract symptoms, such as dry cough and shortness of breath, as well as other symptoms, including fever, chills, and fatigue. COVID-19 is also characterized by the excessive release of cytokines causing inflammatory injury to the lungs and other organs. It is advised to undergo precautionary measures, such as vaccination, social distancing, use of masks, hygiene, and a healthy diet. This review is aimed at summarizing the pathophysiology of COVID-19 and potential biologically active compounds (bioactive) found in plants and plant food. We conclude that many plant food bioactive compounds exhibit antiviral and anti-inflammatory properties and support in attenuating organ damage due to reduced cytokine release and improving the recovery process from COVID-19 infection.

Loss of function of transcription factor EB remodels lipid metabolism and cell death pathways in the cardiomyocyte.

Glucolipotoxicity following nutrient overload causes cardiomyocyte injury by inhibiting TFEB and suppressing lysosomal function. We ascertained whether in addition to the amount, the type of fatty acids (FAs) and duration of FA exposure regulate TFEB action and dictate cardiomyocyte viability. Saturated FA, palmitate, but not polyunsaturated FAs decreased TFEB content in a concentration- and time-dependent manner in cardiomyocytes. Hearts from high-fat high-sucrose diet-fed mice exhibited a temporal decline in nuclear TFEB content with marked elevation of diacylglycerol and triacylglycerol, suggesting that lipid deposition and TFEB loss are concomitant molecular events. Next, we examined the identity of signaling and metabolic pathways engaged by the loss of TFEB action in the cardiomyocyte. Transcriptome analysis in murine cardiomyocytes with targeted deletion of myocyte TFEB (TFEB-/-) revealed enrichment of differentially expressed genes (DEG) representing pathways of nutrient metabolism, DNA damage and repair, cell death and cardiac function. Strikingly, genes involved in macroautophagy, mitophagy and lysosome function constituted a small portion of DEGs in TFEB-/- cardiomyocytes. In myoblasts and/or myocytes, nutrient overload-induced lipid droplet accumulation and caspase-3 activation were exacerbated by silencing TFEB or attenuated by overexpressing constitutively active TFEB. The effect of TFEB overexpression were persistent in the presence of Atg7 loss-of-function, signifying that the effect of TFEB in the myocyte is independent of changes in the macroautophagy pathway. In the cardiomyocyte, the non-canonical effect of TFEB to reprogram energy metabolism is more evident than the canonical action of TFEB on lysosomal autophagy. Loss of TFEB function perturbs metabolic pathways in the cardiomyocyte and renders the heart prematurely susceptible to nutrient overload-induced injury.

Lysosomal Biology and Function: Modern View of Cellular Debris Bin.

Lysosomes are the main proteolytic compartments of mammalian cells comprising of a battery of hydrolases. Lysosomes dispose and recycle extracellular or intracellular macromolecules by fusing with endosomes or autophagosomes through specific waste clearance processes such as chaperone-mediated autophagy or microautophagy. The proteolytic end product is transported out of lysosomes via transporters or vesicular membrane trafficking. Recent studies have demonstrated lysosomes as a signaling node which sense, adapt and respond to changes in substrate metabolism to maintain cellular function. Lysosomal dysfunction not only influence pathways mediating membrane trafficking that culminate in the lysosome but also govern metabolic and signaling processes regulating protein sorting and targeting. In this review, we describe the current knowledge of lysosome in influencing sorting and nutrient signaling. We further present a mechanistic overview of intra-lysosomal processes, along with extra-lysosomal processes, governing lysosomal fusion and fission, exocytosis, positioning and membrane contact site formation. This review compiles existing knowledge in the field of lysosomal biology by describing various lysosomal events necessary to maintain cellular homeostasis facilitating development of therapies maintaining lysosomal function.

Autotaxin-LPA signaling contributes to obesity-induced insulin resistance in muscle and impairs mitochondrial metabolism.

Autotaxin (ATX) is an adipokine that generates the bioactive lipid, lysophosphatidic acid (LPA). ATX-LPA signaling has been implicated in diet-induced obesity and systemic insulin resistance. However, it remains unclear whether the ATX-LPA pathway influences insulin function and energy metabolism in target tissues, particularly skeletal muscle, the major site of insulin-stimulated glucose disposal. The objective of this study was to test whether the ATX-LPA pathway impacts tissue insulin signaling and mitochondrial metabolism in skeletal muscle during obesity. Male mice with heterozygous ATX deficiency (ATX+/-) were protected from obesity, systemic insulin resistance, and cardiomyocyte dysfunction following high-fat high-sucrose (HFHS) feeding. HFHS-fed ATX+/- mice also had improved insulin-stimulated AKT phosphorylation in white adipose tissue, liver, heart, and skeletal muscle. Preserved insulin-stimulated glucose transport in muscle from HFHS-fed ATX+/- mice was associated with improved mitochondrial pyruvate oxidation in the absence of changes in fat oxidation and ectopic lipid accumulation. Similarly, incubation with LPA decreased insulin-stimulated AKT phosphorylation and mitochondrial energy metabolism in C2C12 myotubes at baseline and following palmitate-induced insulin resistance. Taken together, our results suggest that the ATX-LPA pathway contributes to obesity-induced insulin resistance in metabolically relevant tissues. Our data also suggest that LPA directly impairs skeletal muscle insulin signaling and mitochondrial function.

Lysophosphatidic acid receptor mRNA levels in heart and white adipose tissue are associated with obesity in mice and humans.

BACKGROUND: Lysophosphatidic acid (LPA) receptor signaling has been implicated in cardiovascular and obesity-related metabolic disease. However, the distribution and regulation of LPA receptors in the myocardium and adipose tissue remain unclear. OBJECTIVES: This study aimed to characterize the mRNA expression of LPA receptors (LPA1-6) in the murine and human myocardium and adipose tissue, and its regulation in response to obesity. METHODS: LPA receptor mRNA levels were determined by qPCR in i) heart ventricles, isolated cardiomyocytes, and perigonadal adipose tissue from chow or high fat-high sucrose (HFHS)-fed male C57BL/6 mice, ii) 3T3-L1 adipocytes and HL-1 cardiomyocytes under conditions mimicking gluco/lipotoxicity, and iii) human atrial and subcutaneous adipose tissue from non-obese, pre-obese, and obese cardiac surgery patients. RESULTS: LPA1-6 were expressed in myocardium and white adipose tissue from mice and humans, except for LPA3, which was undetectable in murine adipocytes and human adipose tissue. Obesity was associated with increased LPA4, LPA5 and/or LPA6 levels in mice ventricles and cardiomyocytes, HL-1 cells exposed to high palmitate, and human atrial tissue. LPA4 and LPA5 mRNA levels in human atrial tissue correlated with measures of obesity. LPA5 mRNA levels were increased in HFHS-fed mice and insulin resistant adipocytes, yet were reduced in adipose tissue from obese patients. LPA4, LPA5, and LPA6 mRNA levels in human adipose tissue were negatively associated with measures of obesity and cardiac surgery outcomes. This study suggests that obesity leads to marked changes in LPA receptor expression in the murine and human heart and white adipose tissue that may alter LPA receptor signaling during obesity.

Validation of optimal reference genes for quantitative real time PCR in muscle and adipose tissue for obesity and diabetes research.

The global incidence of obesity has led to an increasing need for understanding the molecular mechanisms that drive this epidemic and its comorbidities. Quantitative real-time RT-PCR (RT-qPCR) is the most reliable and widely used method for gene expression analysis. The selection of suitable reference genes (RGs) is critical for obtaining accurate gene expression information. The current study aimed to identify optimal RGs to perform quantitative transcriptomic analysis based on RT-qPCR for obesity and diabetes research, employing in vitro and mouse models, and human tissue samples. Using the ReFinder program we evaluated the stability of a total of 15 RGs. The impact of choosing the most suitable RGs versus less suitable RGs on RT-qPCR results was assessed. Optimal RGs differed between tissue and cell type, species, and experimental conditions. By employing different sets of RGs to normalize the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), we show that sub-optimal RGs can markedly alter the PGC1α gene expression profile. Our study demonstrates the importance of validating RGs prior to normalizing transcriptional expression levels of target genes and identifies optimal RG pairs for reliable RT-qPCR normalization in cells and in human and murine muscle and adipose tissue for obesity/diabetes research.

Autophagic dysregulation in doxorubicin cardiomyopathy.

Doxorubicin (DOX)-induced cardiotoxicity has been a well-known phenomenon to clinicians and scientists for decades; however, molecular mechanisms underlying DOX cardiotoxicity are still being uncovered. Although the majority of prior research have implicated nuclear and mitochondrial events to be an important etiological aspects of DOX cardiomyopathy, recent discoveries in autophagy have highlighted the renewed interest in the role of lysosome in DOX cardiomyopathy. Indeed, dysregulation of lysosomal autophagy is observed in pre-clinical models of DOX cardiotoxicity. In this review, we provide a comprehensive overview on mechanisms describing regulation of the autophagy pathway by DOX and its influence on cardiotoxic outcomes. We have put specific emphasis on experimental models, dosing and treatment duration with DOX, and methods to monitor autophagy, all of which contribute to inconsistencies observed in the literature. We have clarified processes by which DOX dysregulates macroautophagy in the heart by primarily focusing on the contribution of LC3, p62, Beclin, mTOR and AMPK pathways. We have also highlighted the impact of DOX on mitochondrial reactive oxygen species (ROS) and its contribution to the process of mitophagy. We have presented mechanisms by which DOX compromises lysosomal acidification, integrity and chaperone-mediated autophagy through its effect on lysosome-associated and resident proteins such as LAMP, vATPase, Hsp90, Hsc70 and cathepsins. Furthermore, we have discussed novel pathways in DOX cardiotoxicity, the most prominent being DOX-induced loss of TFEB, a member of the MITF family of transcription factors, which governs lysosomal biogenesis and function. This review summarizes that in the myocardium, DOX dysregulates autophagy by impairing transcriptional factors regulating lysosomal function, thereby, precipitating proteotoxicity, mitochondrial dysfunction and cell death, thus rendering the heart susceptible to cardiomyopathic failure.

Glucolipotoxicity diminishes cardiomyocyte TFEB and inhibits lysosomal autophagy during obesity and diabetes.

Impaired cardiac metabolism in the obese and diabetic heart leads to glucolipotoxicity and ensuing cardiomyopathy. Glucolipotoxicity causes cardiomyocyte injury by increasing energy insufficiency, impairing proteasomal-mediated protein degradation and inducing apoptosis. Proteasome-evading proteins are degraded by autophagy in the lysosome, whose metabolism and function are regulated by master regulator transcription factor EB (TFEB). Limited studies have examined the impact of glucolipotoxicity on intra-lysosomal signaling proteins and their regulators. By utilizing a mouse model of diet-induced obesity, type-1 diabetes (Akita) and ex-vivo model of glucolipotoxicity (H9C2 cells and NRCM, neonatal rat cardiomyocyte), we examined whether glucolipotoxicity negatively targets TFEB and lysosomal proteins to dysregulate autophagy and cause cardiac injury. Despite differential effects of obesity and diabetes on LC3B-II, expression of proteins facilitating autophagosomal clearance such as TFEB, LAMP-2A, Hsc70 and Hsp90 were decreased in the obese and diabetic heart. In-vivo data was recapitulated in H9C2 and NRCM cells, which exhibited impaired autophagic flux and reduced TFEB content when exposed to a glucolipotoxic milieu. Notably, overloading myocytes with a saturated fatty acid (palmitate) but not an unsaturated fatty acid (oleate) depleted cellular TFEB and suppressed autophagy, suggesting a fatty acid specific regulation of TFEB and autophagy in the cardiomyocyte. The effect of glucolipotoxicity to reduce TFEB content was also confirmed in heart tissue from patients with Class-I obesity. Therefore, during glucolipotoxicity, suppression of lysosomal autophagy was associated with reduced lysosomal content, decreased cathepsin-B activity and diminished cellular TFEB content likely rendering myocytes susceptible to cardiac injury.

Doxorubicin impairs cardiomyocyte viability by suppressing transcription factor EB expression and disrupting autophagy.

Doxorubicin (DOX) is an effective anti-cancer agent. However, DOX treatment increases patient susceptibility to dilated cardiomyopathy. DOX predisposes cardiomyocytes to insult by suppressing mitochondrial energy metabolism, altering calcium flux, and disrupting proteolysis and proteostasis. Prior studies have assessed the role of macroautophagy in DOX cardiotoxicity; however, limited studies have examined whether DOX mediates cardiac injury through dysfunctions in inter- and/or intra-lysosomal signaling events. Lysosomal signaling and function is governed by transcription factor EB (TFEB). In the present study, we hypothesized that DOX caused myocyte injury by impairing lysosomal function and signaling through negative regulation of TFEB. Indeed, we found that DOX repressed cellular TFEB expression, which was associated with impaired cathepsin proteolytic activity across in vivo, ex vivo, and in vitro models of DOX cardiotoxicity. Furthermore, we observed that loss of TFEB was associated with reduction in macroautophagy protein expression, inhibition of autophagic flux, impairments in lysosomal cathepsin B activity, and activation of cell death. Restoration and/or activation of TFEB in DOX-treated cardiomyocytes prevented DOX-induced suppression of cathepsin B activity, reduced DOX-mediated reactive oxygen species (ROS) overproduction, attenuated activation of caspase-3, and improved cellular viability. Collectively, loss of TFEB inhibits lysosomal autophagy, rendering cardiomyocytes susceptible to DOX-induced proteotoxicity and injury. Our data reveal a novel mechanism wherein DOX primes cardiomyocytes for cell death by depleting cellular TFEB.

Cardioprotective effect of Azadirachta indica A. Juss. on isoprenaline induced myocardial infarction in rats.

The present study was designed to evaluate the cardioprotective potential of aqueous leaf extract of Azadirachta indica A. Juss. (AI) on the basis of haemodynamic, biochemical and histopathological parameters in isoprenaline induced myocardial infarction in rats and to compare with vitamin E, a known cardioprotective antioxidant. A significant (p<0.01) decrease in mean arterial blood pressure (MAP), systolic arterial blood pressure (SAP), diastolic arterial blood pressure (DAP) and increase in heart rate (HR) were observed in isoprenaline control group. Isoprenaline showed significant decrease in the level of cardiac marker enzymes [Lactate dehydrogenase (LDH) and Serum Glutamate Oxalotransaminase (SGOT)] in the heart homogenate with a corresponding increase in their level in serum. In vitamin E control group significant (p<0.05) increase in LDH in heart homogenate and decrease of SGOT and LDH in serum was observed. In isoprenaline control group, significant (p<0.01) increase in total cholesterol and triglycerides levels while decrease in high-density lipoproteins (HDL) was observed. On histopathological examination, myocardial damage in isoprenaline control group further confirmed cardiotoxic effect of isoprenaline. Our data showed that AI (250, 500 and 1000 mg/kg, p.o.) and vitamin E (100 mg/kg, p.o.) significantly restores most of the haemodynamic, biochemical and histopathalogical parameters. Finally we concluded that AI leaf extract exerts equipotent cardioprotective activity in the experimental model of isoprenalin induced myocardial necrosis in rats as compared to vitamin E, a known cardioprotective antioxidant.