Type 2 diabetes mellitus in metabolic-associated fatty liver disease vs. type 2 diabetes mellitus non-alcoholic fatty liver disease: a longitudinal cohort analysis.

INTRODUCTION AND OBJECTIVES: Type 2 Diabetes Mellitus (T2DM) is comorbidity commonly presenting with fatty liver. A recently proposed definition of "metabolic associated fatty liver disease" (MAFLD) is thought to replace non-alcoholic fatty liver disease (NAFLD). Yet, despite the significant prevalence of T2DM among fatty liver, there remains limited evidence on the impact of the change in the definition of T2DM. MATERIALS AND METHODS: The current study uses data from the United States National Health and Nutrition Examination Survey (NHANES) from 1999 to 2018. Survival analysis was conducted with a cox regression and sub-distribution hazard ratio for competing risk events. RESULTS: 6727 patients had a diagnosis of T2DM. 4982 individuals with T2DM had MAFLD and 2032 were MAFLD(+)/NAFLD(-), while 2950 patients were MAFLD(+)/NAFLD(+). The new definition increased fatty liver diagnosis by 68.89%. Patients who were classified as MAFLD(+)/NAFLD(-) were at a higher risk of major adverse cardiovascular events, advanced fibrosis, all-cause and cardiovascular-related mortality compared to MAFLD(+)/NAFLD(+). In MAFLD(+)/NAFLD(-), viral hepatitis significantly increases the odds of advanced fibrosis (OR: 6.77, CI: 3.92 to 11.7, p < 0.001) and all-cause mortality (HR: 1.75, CI: 1.29 to 2.40, p < 0.001). CONCLUSIONS: The identification and treatment of NAFLD in patients with T2DM is a major concern and the premature change to MAFLD results in an over-diagnosis of fatty liver, exaggerated mortality, and morbidity in patients with T2DM. The definition of MAFLD causes further heterogeneity in fatty liver disease/NAFLD.

A comprehensive overview of CRISPR/Cas 9 technology and application thereof in drug discovery.

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas technology possesses revolutionary potential to positively affect various domains of drug discovery. It has initiated a rise in the area of genetic engineering and its advantages range from classical science to translational medicine. These genome editing systems have given a new dimension to our capabilities to alter, detect and annotate specified gene sequences. Moreover, the ease, robustness and adaptability of the CRISPR/Cas9 technology have led to its extensive utilization in research areas in such a short period of time. The applications include the development of model cell lines, understanding disease mechanisms, discovering disease targets, developing transgenic animals and plants, and transcriptional modulation. Further, the technology is rapidly growing; hence, an overlook of progressive success is crucial. This review presents the current status of the CRISPR-Cas technology in a tailor-made format from its discovery to several advancements for drug discovery alongwith future trends associated with possibilities and hurdles including ethical concerns.

Hypertension is prevalent in non-alcoholic fatty liver disease and increases all-cause and cardiovascular mortality.

Background and aims: Hypertension (HTN) is a common comorbidity in non-alcoholic fatty liver disease (NAFLD) affecting up to 40% of individuals. However, the impact of HTN and its control on outcomes in NAFLD remains unclear. Therefore, we aimed to examine the impact of HTN on survival outcomes in a longitudinal cohort of NAFLD patients. Methods: The analysis consisted of adults in the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2018 with data on socio-demographic characteristics and comorbidities. NAFLD was diagnosed with fatty liver index (FLI) and United States-FLI at a cut-off of 60 and 30, respectively in the substantial absence of alcohol use. A multivariate regression analysis was conducted to adjust for confounders. Results: A total of 45,302 adults were included, and 27.83% were identified to have NAFLD. Overall, 45.65 and 35.12% of patients with NAFLD had HTN and uncontrolled HTN, respectively. A multivariate analysis with confounders demonstrated that hypertensive NAFLD had a significantly increased risk of all-cause mortality (HR: 1.39, CI: 1.14-1.68, p < 0.01) and cardiovascular disease (CVD) mortality (HR: 1.85, CI: 1.06-3.21, p = 0.03). Untreated HTN remained to have a significantly increased risk in all-cause (HR: 1.59, CI: 1.28-1.96, p < 0.01) and CVD mortality (HR: 2.36, CI: 1.36-4.10, p < 0.01) while treated HTN had a non-significant increased risk of CVD mortality (HR: 1.51, CI: 0.87-2.63, p = 0.14) and a lower magnitude of increase in the risk of all-cause mortality (HR: 1.26, CI: 1.03-1.55, p = 0.03). Conclusion: Despite the significant burden of HTN in NAFLD, up to a fifth of patients have adequate control, and the lack thereof significantly increases the mortality risk. With the significant association of HTN in NAFLD, patients with NAFLD should be managed with a multidisciplinary team to improve longitudinal outcomes.

Erythroid lineage-specific lentiviral RNAi vectors suitable for molecular functional studies and therapeutic applications.

Numerous genes exert multifaceted roles in hematopoiesis. Therefore, we generated novel lineage-specific RNA interference (RNAi) lentiviral vectors, H23B-Ery-Lin-shRNA and H234B-Ery-Lin-shRNA, to probe the functions of these genes in erythroid cells without affecting other hematopoietic lineages. The lineage specificity of these vectors was confirmed by transducing multiple hematopoietic cells to express a fluorescent protein. Unlike the previously reported erythroid lineage RNAi vector, our vectors were designed for cloning the short hairpin RNAs (shRNAs) for any gene, and they also provide superior knockdown of the target gene expression with a single shRNA integration per cell. High-level lineage-specific downregulation of BCL11A and ZBTB7A, two well-characterized transcriptional repressors of HBG in adult erythroid cells, was achieved with substantial induction of fetal hemoglobin with a single-copy lentiviral vector integration. Transduction of primary healthy donor CD34+ cells with these vectors resulted in >80% reduction in the target protein levels and up to 40% elevation in the γ-chain levels in the differentiated erythroid cells. Xenotransplantation of the human CD34+ cells transduced with H23B-Ery-Lin-shBCL11A LV in immunocompromised mice showed ~ 60% reduction in BCL11A protein expression with ~ 40% elevation of γ-chain levels in the erythroid cells derived from the transduced CD34+ cells. Overall, the novel erythroid lineage-specific lentiviral RNAi vectors described in this study provide a high-level knockdown of target gene expression in the erythroid cells, making them suitable for their use in gene therapy for hemoglobinopathies. Additionally, the design of these vectors also makes them ideal for high-throughput RNAi screening for studying normal and pathological erythropoiesis.

Gene therapy: Comprehensive overview and therapeutic applications.

Gene therapy is the product of man's quest to eliminate diseases. Gene therapy has three facets namely, gene silencing using siRNA, shRNA and miRNA, gene replacement where the desired gene in the form of plasmids and viral vectors, are directly administered and finally gene editing based therapy where mutations are modified using specific nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short tandem repeats (CRISPR)/CRISPR-associated protein (Cas)-associated nucleases. Transfer of gene is either through transformation where under specific conditions the gene is directly taken up by the bacterial cells, transduction where a bacteriophage is used to transfer the genetic material and lastly transfection that involves forceful delivery of gene using either viral or non-viral vectors. The non-viral transfection methods are subdivided into physical, chemical and biological. The physical methods include electroporation, biolistic, microinjection, laser, elevated temperature, ultrasound and hydrodynamic gene transfer. The chemical methods utilize calcium- phosphate, DAE-dextran, liposomes and nanoparticles for transfection. The biological methods are increasingly using viruses for gene transfer, these viruses could either integrate within the genome of the host cell conferring a stable gene expression, whereas few other non-integrating viruses are episomal and their expression is diluted proportional to the cell division. So far, gene therapy has been wielded in a plethora of diseases. However, coherent and innocuous delivery of genes is among the major hurdles in the use of this promising therapy. Hence this review aims to highlight the current options available for gene transfer along with the advantages and limitations of every method.

Ameliorative effect of cerium oxide nanoparticles against Freund's complete adjuvant-induced arthritis.

Aim: To assess the mechanistic effects of cerium oxide nanoparticles (CONPs) on Freund's complete adjuvant (FCA)-induced rheumatoid arthritis in rats. Methods: CONPs were characterized and evaluated in vitro (RAW 264.7 macrophages) and in vivo (FCA-induced rheumatoid arthritis model). Results:In vitro treatment with CONPs significantly reduced lipopolysaccharide-induced oxidative stress (as evident from dichlorodihydrofluorescein diacetate staining), diminished mitochondrial stress (as observed with tetraethylbenzimidazolylcarbocyanine iodide staining) and reduced superoxide radicals. In vivo, CONPs exhibited anti-rheumatoid arthritis activity, as evident from results of paw volume, x-ray, clinical scoring, levels of cytokines (IL-17, IL-1ß, TNF-α and TGF-ß1) and histology. Conclusion: We provide preclinical proof that CONPs may be a novel futuristic nanoparticle-based approach for therapy of rheumatoid arthritis.

It's all about the spaces between cells: role of extracellular matrix in liver fibrosis.

Liver fibrosis is one of the leading complications of a variety of chronic liver disorders, including the nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, liver cirrhosis and liver failure. The progression of liver fibrosis is driven by chronic inflammation, which activates the secretory fibroblasts to the myofibroblast phenotype. These specialized liver cells are called as hepatic stellate cells (HSCs). The excessive extracellular matrix (ECM) secretion creates a large number of complications. Fibrosis is the result of imbalance between the matrix synthesizing and matrix degrading factors. The major ECM proteins include the matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases (TIMPs), lysyl oxidases (LOX), lysyl oxidase-like (LOXLs) enzymes, tenascins and others. These ECM proteins present novel avenues for the therapeutics of liver fibrosis. The current review highlights the major role played by these critical matrix proteins in liver fibrosis. Further, some of the targeted formulations used against these proteins are discussed and suggestions are provided to select the course of research for successful clinical translation of basic research findings for the amelioration of liver fibrosis.

Withaferin A reverses bile duct ligation-induced liver fibrosis by modulating extracellular matrix deposition: Role of LOXL2/Snail1, vimentin, and NFκB signaling.

Herein, we studied the effect of Withaferin A (WFA) in reversing bile duct ligation (BDL)-induced liver fibrosis. BDL was performed on C57BL/6J mice and 2 days later, WFA (1 and 3 mg/kg) was administered for 12 days. Estimation of liver enzymes and assays for lipid peroxidation, reduced glutathione, and nitrite levels were performed. Picrosirius red, Masson's trichrome, and H&E staining were performed to study histological changes. WFA proved to be a holistic intervention for the attenuation and reversal of liver fibrosis. Reduction in inflammatory stimulus and oxidative stress restored the levels of stress-related chaperone Hsp70 (p < .001 vs. BDL) in WFA treated groups. We found 3.59-fold (p < .001) and 1.37-fold (p < .01) reduction in the expression of lysyl oxidase like2 (LOXL2) and Snail1, respectively, in WFA-treated animals as compared with BDL animals. These reductions led to 1.9-fold (p < .001) elevation in levels of E-cadherin signifying the reversal of epithelial to mesenchymal transition by WFA. Further, the reduction in LOXL2 levels enhanced the susceptibility of fibrotic scar toward degradation. The picrosirius red and Masson's trichrome staining done on liver tissue sections supported the above results. We, for the first time, report the role of WFA in modulating the expression of LOXL2 and Snail1 in addition to vimentin inhibition and regulation of NFκB signaling for the treatment of liver fibrosis.

Superoxide dismutase mimetic nanoceria restrains cerulein induced acute pancreatitis.

Aim: The present study was carried out to assess the effect of nanoceria (NC) on pancreatic inflammation caused by cerulein. Methods: NC was characterized and in vitro studies were carried out in murine macrophages. The in vivo effects were tested on cerulein-induced pancreatitis. Results:In vitro treatment with NC remarkably protected macrophages from lipopolysaccharide-induced inflammation and oxidative stress as evident from the results of 2',7'-dichlorofluorescin diacetate, JC-1 and MitoSox staining. In vivo treatment with NC showed potent superoxide dismutase and catalase mimetic activity, antipancreatitis activity and improved histology. Furthermore, it reduced the expression of p65-NF-κB and acetylation of histone H3 at lysine K14, K56 and K79 residues. Conclusion: We for the first time, demonstrate that NC may be a promising candidate for the therapy of pancreatitis.

Yttrium oxide nanoparticles reduce the severity of acute pancreatitis caused by cerulein hyperstimulation.

Oxidative stress plays a major role in acute pancreatitis (AP), leading to massive macrophage infiltration. Nanoyttria (NY) possesses potent free radical scavenging activity. As reactive oxygen species and inflammation play major role in AP, we hypothesized that NY may alleviate cerulein induced AP. NY ameliorated LPS induced oxidative stress in vitro. It reduced ROS, superoxide radical generation and restored the mitochondrial membrane potential in macrophages. Interestingly, NY reduced plasma amylase and lipase levels and attenuated the mitochondrial stress and inflammatory markers. NY suppressed the recruitment of inflammatory cells around the damaged pancreatic acinar cells. Furthermore, NY intervention perturbed the course of AP via reduction of endoplasmic reticulum (ER) stress markers (BiP, IRE1 and Ero1-Lα), and molecular chaperones (Hsp27 and Hsp70). We, to the best of our knowledge, report for first time that NY can attenuate experimental AP by restoration of mitochondrial and ER homeostasis through Nrf2/NFκB pathway modulation.

Isoproterenol-induced cardiac ischemia and fibrosis: Plant-based approaches for intervention.

Heart is the most active and incumbent organ of the body, which maintains blood flow, but due to various pathological reasons, several acute and chronic cardiac complications arise out of which myocardial infarction is one of the teething problems. Isoproterenol (ISP)-induced myocardial ischemia is a classical model to screen the cardioprotective effects of various pharmacological interventions. Phytochemicals present a novel option for treating various human maladies including those of the heart. A large number of plant products and their active ingredients have been screened for efficacy in ameliorating ISP-induced myocardial ischemia including coriander, curcumin, Momordica, quercetin, and Withania somnifera. These phytochemicals constituents may play key role in preventing disease and help in cardiac remodeling. Reactive oxygen species scavenging, antiinflammatory, and modulation of various molecular pathways such as Nrf2, NFкB, p-21 activated kinase 1 (PAK1), and p-smad2/3 signaling modulation have been implicated behind the claimed protection. In this review, we have provided a focused overview on the utility of ISP-induced cardiotoxicity, myocardial ischemia, and cardiac fibrosis for preclinical research. In addition, we have also surveyed molecular mechanism of various plant-based interventions screened for cardioprotective effect in ISP-induced cardiotoxicity, and their probable mechanistic profile is summarized.