Kupffer cell diversity maintains liver function in alcohol-associated liver disease.

BACKGROUND AND AIMS: Liver macrophages are heterogeneous and play an important role in alcohol-associated liver disease (ALD) but there is limited understanding of the functions of specific macrophage subsets in the disease. We used a Western diet alcohol (WDA) mouse model of ALD to examine the hepatic myeloid cell compartment by single cell RNAseq and targeted KC ablation to understand the diversity and function of liver macrophages in ALD. APPROACH AND RESULTS: In the WDA liver, KCs and infiltrating monocytes/macrophages each represented about 50% of the myeloid pool. Five major KC clusters all expressed genes associated with receptor-mediated endocytosis and lipid metabolism, but most were predicted to be noninflammatory and antifibrotic with 1 minor KC cluster having a proinflammatory and extracellular matrix degradation gene signature. Infiltrating monocyte/macrophage clusters, in contrast, were predicted to be proinflammatory and profibrotic. In vivo, diphtheria toxin-based selective KC ablation during alcohol exposure resulted in a liver failure phenotype with increases in PT/INR and bilirubin, loss of differentiated hepatocyte gene expression, and an increase in expression of hepatocyte progenitor markers such as EpCAM, CK7, and Igf2bp3. Gene set enrichment analysis of whole-liver RNAseq from the KC-ablated WDA mice showed a similar pattern as seen in human alcoholic hepatitis. CONCLUSIONS: In this ALD model, KCs are anti-inflammatory and are critical for the maintenance of hepatocyte differentiation. Infiltrating monocytes/macrophages are largely proinflammatory and contribute more to liver fibrosis. Future targeting of specific macrophage subsets may provide new approaches to the treatment of liver failure and fibrosis in ALD.

Curcumin-Encapsulated Nanomicelles Improve Cellular Uptake and Cytotoxicity in Cisplatin-Resistant Human Oral Cancer Cells.

Oral cancer has a high mortality rate, which is mostly determined by the stage of the disease at the time of admission. Around half of all patients with oral cancer report with advanced illness. Hitherto, chemotherapy is preferred to treat oral cancer, but the emergence of resistance to anti-cancer drugs is likely to occur after a sequence of treatments. Curcumin is renowned for its anticancer potential but its marred water solubility and poor bioavailability limit its use in treating multidrug-resistant cancers. As part of this investigation, we prepared and characterized Curcumin nanomicelles (CUR-NMs) using DSPE-PEG-2000 and evaluated the anticancer properties of cisplatin-resistant cancer cell lines. The prepared CUR-NMs were sphere-shaped and unilamellar in structure, with a size of 32.60 ± 4.2 nm. CUR-NMs exhibited high entrapment efficiency (82.2%), entrapment content (147.96 µg/mL), and a mean zeta potential of -17.5ζ which is considered moderately stable. The cellular uptake and cytotoxicity studies revealed that CUR-NMs had significantly higher cytotoxicity and cellular uptake in cisplatin drug-resistant oral cancer cell lines and parental oral cancer cells compared to plain curcumin (CUR). The DAPI and FACS analysis corroborated a high percentage of apoptotic cells with CUR-NMs (31.14%) compared to neat CUR (19.72%) treatment. Conclusively, CUR-NMs can potentially be used as an alternative carrier system to improve the therapeutic effects of curcumin in the treatment of cisplatin-resistant human oral cancer.

In vitro differentiation of human pancreatic duct-derived PANC-1 cells into ß-cell phenotype using Tinospora cordifolia.

Type 1 diabetes mellitus is an autoimmune disorder leading to loss of beta cells. There is a dire need to inhibit apoptosis and induce regeneration of new beta cells. There are plants in the Indian medicine system having the potential for rejuvenation. In the present study, we have attempted to evaluate the capacity of aqueous extract of Tinospora cordifolia to regenerate beta cells from PANC-1 ductal cells. After differentiation, the characterization of ß-cell phenotype was carried out using dithizone and Gomori's staining and further confirmed by mRNA expression study of insulin, Pdx-1, and carbonic anhydrase-9. Insulin production was estimated with ELISA. Aqueous extract of Tinospora cordifolia at 15 µg/ml concentration can effectively induce differentiation of PANC-1 cells into beta cells. The morphological observations showed brownish-colored dithizone and purple-colored Gomori's staining. The ß-cells demonstrated significant mRNA expression of insulin and Pdx-1 and downregulation of carbonic anhydrase-9. The functionality of beta cells was demonstrated by 1.5-fold increase in insulin secretion in response to high glucose. Tinospora cordifolia has potential to differentiate PANC-1 ductal cells into functional beta cells and can be a lead towards non-invasive treatment of type 1 diabetes mellitus.

Defects in energy metabolism are associated with functional exhaustion of bone marrow mesenchymal stem cells in cirrhosis.

OBJECTIVES: Cellular and functional exhaustion of bone marrow mesenchymal stem cells (BM-MSC) is significantly associated with the loss of HSCs and hepatic osteodystrophy in cirrhosis. The molecular mechanisms underlying the dysfunction of BM-MSCs are not well understood. We investigated the underlying mechanisms of cellular and functional exhaustion of BM-MSCs in cirrhosis. METHODS: The MSCs were isolated retrospectively from bone marrow of decompensated alcoholic cirrhosis patients {(Trial registration: ClinicalTrials.gov NCT01902511) (n=10; MELD=16.2±2.3; CTP=8.7±2.3)} and age and gender-matched healthy controls (n=8). Global gene expression profile of healthy bone marrow MSCs (hBM-MSCs) and cirrhosis patients BM-MSCs (cBM-MSCs) were done by mRNA sequencing. XFe24-bioanalyzer analyzed the bioenergetic potential of cells. Level of different cytokines and growth factors in BM-plasma and MSCs secretome were analyzed by Luminex-based bead array. RESULTS: Analysis of differentially expressed genes showed significant (P<0.01) up-regulation of genes associated with ubiquitination and catabolism of proteins; TNF signaling, insulin resistance, and down-regulation of genes associated with DNA repair, protein processing, cell cycle, and mitochondrial respiration in cBM-MSCs in comparison to hBM-MSCs. Compared to hBM-MSCs, cBM-MSCs showed a significant defect in glycolysis due to insulin resistance and poor glucose uptake (P=0.002). This led to compromised self-renewal capacity and cellular loss of MSCs in cirrhosis. cBM-MSCs also showed a significant impairment in Oxidative phosphorylation (OXPHOS) due to mitochondrial dysfunction leading to defects in the osteogenic differentiation with early aging and senescence. CONCLUSION: Compromised energy metabolism due to inflammatory and metabolic stress-induced insulin resistance underlies the cellular and functional exhaustion of BM-MSCs in cirrhosis.

Syndromic approach to SARS-CoV-2 detection using QIAstat-Dx SARS-CoV-2 panel from clinical samples.

The QIAstat-Dx SARS-CoV-2 panel is a multiplex cartridge based assay based on real time PCR which can detect 17 respiratory viruses, including the novel coronavirus SARS-CoV-2. A syndromic approach is the need of the hour for COVID-19 diagnostics among patients presenting with respiratory symptoms. The present study was done to evaluate 120 archived respiratory clinical specimens for SARS-CoV-2 on the SARS-CoV-2 panel. Further, 27 specimens were tested for other respiratory viruses, in comparison with the BioFire RP1.7 platform. The sensitivity and specificity for SARS-CoV-2 on SARS panel was found to be 90.00 % and 100 % respectively, indicating good diagnostic accuracy. The positive predictive value was found to be 100 %, negative predictive value was found to be 99.93 % and accuracy was 99.93 %. Detection of other respiratory viruses observed a concordance of 77.7 %. Despite advantages of speed, minimal expertise and accurate results; significant costs and discrepancies at Ct >35 remain important limitations of the SARS panel.

Performance evaluation of automated chemiluminescence immunoassay based antigen detection - Moving towards more reliable ways to predict SARS-CoV-2 infection.

Real-time reverse transcription- polymerase chain reaction (RT-PCR) has been the most reliable armoury for the diagnosis of COVID-19, considered to be the reference standard but fails to reproduce the correct predictability about the infectivity of the disease every time. Antigen detection however puts foothold in this aspect even though lacks in sensitivity, especially conventional Rapid Antigen Tests (RATs). Recently developed Chemiluminescence Immunoassay (CLIA) based antigen detection tests are promising and displayed better sensitivity. In the current study we have evaluated VITROS® SARS-CoV-2 Ag Test CLIA Kit, which was tested on 148 patient's samples attended to a tertiary care centre for testing of SARS-CoV-2. The performance of the kit was evaluated in comparison to RT-PCR and RAT and found to be a good test for antigen detection, best within the first few days of infection. The test has shown sensitivity of 94.3 % and specificity of 100 % in samples with corresponding Ct values of ≤25 by RT-PCR, which corresponds to high viral load and can predict ability of spreading the disease by the patients. With the results being semiquantitative along with improved sensitivity it can replace RATs for antigen detection for screening, provided good laboratory set up is included under consideration.

Multi-omics analysis of respiratory specimen characterizes baseline molecular determinants associated with SARS-CoV-2 outcome.

Rapid diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection still remains a major challenge. A multi-omic approach was adopted to analyze the respiratory specimens of 20 SARS-CoV-2-positive, 20 negative and 15 H1N1 pdm 2009 positive cases. Increased basal level of MX1 (MX dynamin-like GTPase 1) and WARS (tryptophan-tRNA ligase) correlated with SARS-CoV-2 infection and its outcome. These markers were further validated in 200 suspects. MX1>30pg/ml and WARS>25ng/ml segregated virus positives [AUC = 94% CI: (0.91-0.97)] and severe patients [AUC>0.85%]. Our results documented significant increase in immune activation; metabolic reprograming and decrease in oxygen transport, wound healing and others linked proteins and metabolites in patients with coronavirus disease 2019 (COVID-19). Multi-omics profiling correlated with viremia and segregated asymptomatic patients with COVID-19. Additionally, we identified increased respiratory pathogens (Burkholderiales, Klebsiella pneumonia) and decreased lactobacillus salivarius (FDR<0.05) in COVID-19 specimens. In conclusion, increased basal MX1 and WARS levels correlates with SARS-CoV-2 infection and could aid in the identification of patient's predisposed to higher severity.

A Recombinant Fragment of Human Surfactant Protein D Binds Spike Protein and Inhibits Infectivity and Replication of SARS-CoV-2 in Clinical Samples.

Coronavirus disease (COVID-19) is an acute infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human SP-D (surfactant protein D) is known to interact with the spike protein of SARS-CoV, but its immune surveillance against SARS-CoV-2 is not known. The current study aimed to examine the potential of a recombinant fragment of human SP-D (rfhSP-D) as an inhibitor of replication and infection of SARS-CoV-2. The interaction of rfhSP-D with the spike protein of SARS-CoV-2 and human ACE-2 (angiotensin-converting enzyme 2) receptor was predicted via docking analysis. The inhibition of interaction between the spike protein and ACE-2 by rfhSP-D was confirmed using direct and indirect ELISA. The effect of rfhSP-D on replication and infectivity of SARS-CoV-2 from clinical samples was assessed by measuring the expression of RdRp gene of the virus using quantitative PCR. In silico interaction studies indicated that three amino acid residues in the receptor-binding domain of spike protein of SARS-CoV-2 were commonly involved in interacting with rfhSP-D and ACE-2. Studies using clinical samples of SARS-CoV-2-positive cases (asymptomatic, n = 7; symptomatic, n = 8) and negative control samples (n = 15) demonstrated that treatment with 1.67 µM rfhSP-D inhibited viral replication by ∼5.5-fold and was more efficient than remdesivir (100 µM) in Vero cells. An approximately two-fold reduction in viral infectivity was also observed after treatment with 1.67 µM rfhSP-D. These results conclusively demonstrate that the rfhSP-D mediated calcium independent interaction between the receptor-binding domain of the S1 subunit of the SARS-CoV-2 spike protein and human ACE-2, its host cell receptor, and significantly reduced SARS-CoV-2 infection and replication in vitro.

Vaccine Breakthrough Infections by SARS-CoV-2 Variants after ChAdOx1 nCoV-19 Vaccination in Healthcare Workers.

This study elucidated the clinical, humoral immune response and genomic analysis of vaccine breakthrough (VBT) infections after ChAdOx1 nCoV-19/Covishield vaccine in healthcare workers (HCWs). Amongst 1858 HCWs, 1639 had received either two doses (1346) or a single dose (293) of ChAdOx1 nCoV-19 vaccine. SARS-CoV-2 IgG antibodies and neutralizing antibodies were measured in the vaccinated group and the development of SARS-CoV-2 infection was monitored.Forty-six RT-PCR positive samples from the 203 positive samples were subjected to whole genome sequencing (WGS). Of the 203 (10.92%) infected HCWs, 21.46% (47/219) were non-vaccinated, which was significantly more than 9.52% (156/1639) who were vaccinated and infection was higher in doctors and nurses. Unvaccinated HCWs had 1.57 times higher risk compared to partially vaccinated HCWs and 2.49 times higher risk than those who were fully vaccinated.The partially vaccinated were at higher risk than the fully vaccinated (RR 1.58). Antibody non-response was seen in 3.44% (4/116), low antibody levels in 15.51% (18/116) and medium levels were found in 81.03% (94/116). Fully vaccinated HCWs had a higher antibody response at day 42 than those who were partially vaccinated (8.96 + 4.00 vs. 7.17 + 3.82). Whole genome sequencing of 46 samples revealed that the Delta variant (B.1.617.2) was predominant (69.5%). HCWs who had received two doses of vaccine showed better protection from mild, moderate, or severe infection, with a higher humoral immune response than those who had received a single dose. The genomic analysis revealed the predominance of the Delta variant (B.1.617.2) in the VBT infections.

Baseline Plasma Metabotype Correlates With Direct-Acting Antiviral Therapy Nonresponse for HCV in HIV-HCV Coinfected Patients.

Introduction: With the advent of direct-acting antiviral (DAA) therapy for HCV, the cure is achieved at similar rates among HIV-HCV coinfected patients as in HCV mono-infected patients. The present study evaluates host plasma metabolites as putative indicators in predicting the treatment response in baseline HIV-HCV patients. Methods: Non-cirrhotic HIV-HCV (N = 43) coinfected patients were treated with sofosbuvir and daclatasvir for 12 weeks. Plasma metabolite profiling of pre- and post-therapy was analyzed in 20/43 patients. Of the 20 selected, 10 (50%) attained the sustained viral response [(SVR) (responders)] as defined by the absence of HCV RNA at 12 weeks after the treatment, and 10 (50%) did not attain the cure for HCV (nonresponders). Results: A total of 563 features were annotated (metabolomic/spectral databases). Before therapy, 39 metabolites differentiated (FC ±1.5, p < 0.05) nonresponders from responders. Of these, 20 upregulated and 19 downregulated were associated with tryptophan metabolism, nicotinamide metabolism, and others. Post therapy, 62 plasma metabolites (12 upregulated and 50 downregulated, FC±1.5, p < 0.05) differentiated nonresponders from responders and highlighted a significant increase in the steroid and histidine metabolism and significant decrease in tryptophan metabolism and ascorbate and pyruvate metabolism in the nonresponders. Based on random forest and multivariate linear regression analysis, the baseline level of N-acetylspermidine (FC > 2, AUC = 0.940, Bfactor = -0.267) and 2-acetolactate (FC > 2, AUC = 0.880, Bfactor = -0.713) significantly differentiated between nonresponders from responders in HIV-HCV coinfected patients and was able to predict the failure of treatment response. Conclusion: Increased baseline levels of N-acetylspermidine and 2-acetolactate levels are associated with the likeliness of failure to attain the cure for HCV in HIV-HCV coinfected patients.

Identification of miRNAs associated with dendritic cell dysfunction during acute and chronic hepatitis B virus infection.

The uptake or expression of hepatitis B virus (HBV) proteins by dendritic cells (DCs) is considered important for disease outcome. Differential expression of microRNA (miRNA) may have a role in viral persistence and hepatocellular injury. The miRNA expression was investigated by microarray in DCs from different stages of HBV infection and liver disease namely, immune active (IA; n = 20); low replicative (LR; n = 20); HBeAg negative (n = 20); acute viral hepatitis (AVH, n = 20) and healthy controls (n = 20). miRNA levels were analyzed by unsupervised hierarchical clustering and principal component analyses and validated by quantitative polymerase Chain Reaction (qPCR). The miRNA-messenger RNA (mRNA)regulatory networks identified 19 miRNAs and 12 target gene interactions in major histocompatibility complex and other immune pathways. miR-2278, miR-615-3p, and miR-3681-3p were downregulated in the IA group compared to healthy control, miR-152-3p and miR-3613-3p in the LR group compared to IA group and miR-152-3p and miR-503-3p in HBe negative compared to LR group. However, miR-7-1-1-3p, miR-192-5p, miR-195-5p, and miR-32-5p in LR, miR-342-3p, and miR-940 in HBe negative, and miR-34a-5p, miR-130b-3p, miR-221-3p, miR-320a, miR-324-5p, and miR-484 in AVH were upregulated. Further, qPCR confirmed changes in miRNA levels and their target genes associated with antigen processing and presentation. Thus, a deregulated network of miRNAs-mRNAs in DCs seems responsible for an impaired immune response during HBV pathogenesis.

In vitro model using cytokine cocktail to evaluate apoptosis in Min6 pancreatic beta cells.

INTRODUCTION: Development of therapy options for treatment of type 1 diabetes mellitus is hampered by non-availability of appropriate experimental models that can exactly mimic the in vivo situation. Apoptosis of beta cells by T cells and cytokine action leads to loss of beta cells. We propose a simple and elegant model using cytokine cocktail of TNF-α, IFN-γ and IL-1ß, the major cytokines responsible for apoptosis in Min6 beta cell line. METHODS: A cocktail of TNF-α, IFN-γ and IL-1ß was used to induce apoptosis in Min6 beta cell line. Apoptosis was assessed by flow cytometry using CytoFLEX (Beckman Coulter). The destruction of beta cells is through production of nitric oxide (NO), oxidative stress and change in mitochondrial membrane permeability. NO was measured using Griess reagent. Oxidative stress was assessed using 2',7'-dichlorofluorescein diacetate, a cell-permeable fluorogenic dye and mitochondrial membrane potential was determined on the basis of retention of rhodamine 123 using flow cytometer. RESULTS AND DISCUSSION: Very low concentration of the cocktail viz. TNF-α 25 ng/ml, IFN-γ 25 ng/ml and IL-1ß 50 ng/ml has demonstrated effective early and late apoptosis in as short a time period as 6 h. The experimental model used demonstrated 1.5 fold higher production of NO, 1.2 fold increased oxidative stress and lower mitochondrial membrane potential as compared to the positive control used. Hence the above model can be easily used for assessment and screening of drugs that can prevent apoptosis of beta cells and stop progression of type 1 diabetes.

Cellular and functional loss of liver endothelial cells correlates with poor hepatocyte regeneration in acute-on-chronic liver failure.

BACKGROUND AND AIM: Acute hepatic insult triggers regeneration. If acute-on-chronic liver failure (ACLF) patients have a poorer regenerative response than acute liver failure (ALF) patients, and if so, the mechanisms underlying this, are not well understood. METHODS: We investigated the status of hepatocyte proliferation, hepatic progenitor cell (HPC) mediated regeneration, non-parenchymal cells (through immunohistochemistery), cytokines and growth factors (cytokine bead array) in liver and peripheral blood of ACLF (n = 29) and ALF (n = 17) patients. Liver endothelial cells, mesenchymal cells and Kupffer cells were isolated from explant livers and analysis of regenerative factors was done by qRT-PCR. RESULTS: Unlike ALF, the ACLF livers showed decreased hepatocyte proliferation (p < 0.001) and profound ductular-reaction with increased CK19 + hepatocytes (p < 0.0001). However, only decrease in Ki67+ hepatocytes was associated with 28 day mortality in ACLF (p < 0.001; HR = 0.78; 95% CI 0.69-0.88). In both groups, increase in plasma hepatocyte growth factor (HGF) (OR = 21.87 p = 0.002;), macrophage colony stimulating factor (MCSF) (OR = 21.73; p = 0.002) and stromal derived factor (SDF1)(OR = 10.2; p = 0.001) were associated with hepatocyte proliferation and decreased (> fivefolds) levels were associated with poor hepatocyte regeneration in ACLF patients. ACLF livers showed decrease in endothelial cells (p < 0.01) and expression of regenerative angiocrine factors C-X-C chemokine receptor type 7 (CXCR7), Inhibitor of DNA Binding 1(IDI) and HGF compared to ALF. In co-culture, while ALF liver mesenchymal stromal cells (LMSCs) induced the expression of CXCR7, IDI and HGF in human umbilical cord endothelial cells (HUVECs), the ACLF LMSCs were defective and showed decreased production of SDF-1, HGF and MCSF compared to ALF. CONCLUSIONS: Decrease in hepatic endothelial cells and their regenerative angiocrine functions indicated by defective CXCR7-ID1 dependent HGF expression underlie the poor hepatocyte proliferation in ACLF compared to ALF patients. A robust hepatocyte self-replication is lacking in the livers of ACLF patients and is associated with poor survival.

Acceleration of protein glycation by oxidative stress and comparative role of antioxidant and protein glycation inhibitor.

Hyperglycemia in diabetes causes protein glycation that leads to oxidative stress, release of cytokines, and establishment of secondary complications such as neuropathy, retinopathy, and nephropathy. Several other metabolic disorders, stress, and inflammation generate free radicals and oxidative stress. It is essential to study whether oxidative stress independently enhances protein glycation leading to rapid establishment of secondary complications. Oxidative stress was experimentally induced using rotenone and Fenton reagent for in vivo and in vitro studies, respectively. Results showed significant increase in the rate of modification of BSA in the form of fructosamine and protein-bound carbonyls in the presence of fenton reagent. Circular dichroism studies revealed gross structural changes in the reduction of alpha helix structure and decreased protein surface charge was confirmed by zeta potential studies. Use of rotenone demonstrated enhanced AGE formation, ROS generation, and liver and kidney tissue glycation through fluorescence measurement. Similar findings were also observed in cell culture studies. Use of aminoguanidine, a protein glycation inhibitor, demonstrated reduction in these changes; however, a combination of aminoguanidine along with vitamin E demonstrated better amelioration. Thus, oxidative stress accelerates the process of protein glycation causing gross structural changes and tissue glycation in insulin-independent tissues. Use of antioxidants and protein glycation inhibitors in combination are more effective in preventing such changes and could be an effective therapeutic option for preventing establishment of secondary complications of diabetes.

Early cirrhosis and a preserved bone marrow niche favour regenerative response to growth factors in decompensated cirrhosis.

BACKGROUND: Exogenous growth factor-mobilized bone marrow (BM) stem cells have shown a differential response in the management of decompensated cirrhosis (DC). This study was designed to evaluate potential clinical benefit of adding Erythropoietin (EPO) in granulocyte-colony stimulating factor (G-CSF)-mobilized stem cell therapy, possible mechanisms of regeneration and predictive factors of regenerative response. METHODS: Sixty consecutive DC patients received either G-CSF with EPO (Group A; n = 30) or G-CSF and placebo (Group B; n = 30) for 2 months and were carefully followed up for 1 year. Baseline and post-treatment liver biopsy, BM biopsy and BM aspirate were analysed for fibro-inflammatory and regenerative response and BM hematopoietic reservoir. RESULTS: Addition of EPO to G-CSF showed a significant improvement in Child-Pugh score (P = 0.03) and MELD score (P = 0.003) as compared to G-CSF alone, with reduction in mortality (16.6% vs 36.7%, P = 0.09). The combination arm also demonstrated a decreased incidence of acute kidney injury (P < 0.001), encephalopathy (P = 0.005) and refilling of ascites (P = 0.03). Compared to monotherapy, it increased CD163+ macrophages (P = 0.013), Ki67+ index (P < 0.001) with decrease in α-SMA levels (P < 0.001) in liver tissue. The response was better with grade 1 and 2 than with grade 3 ascites; Child B cirrhosis and MELD < 16. Non-responders had lower hematopoietic stem cells (HSCs) at baseline. On multivariate analysis, the liver disease severity (MELD < 16) and a relatively preserved BM (BM-HSCs > 0.4) predicted therapeutic response (AUROC = 0.82). CONCLUSIONS: Early DC (MELD < 16) patients with mild-moderate ascites and those with a healthy cellular baseline BM respond better to growth factor therapy. Addition of EPO to G-CSF provides better regenerative response than G-CSF monotherapy.

Early metabolic changes in the gut leads to higher expression of intestinal alpha glucosidase and thereby causes enhanced postprandial spikes.

AIMS: Prediabetes manifests several years earlier, before it progresses to diabetes. It is essential to track the earliest metabolic changes occurring in the prediabetic state and to understand the precise mechanism of how diabetes is initiated. MAIN METHODS: Alpha glucosidase was isolated from rat intestine and assayed using maltose as substrate. In vitro glycation of the enzyme was studied using varying fructose content through measurement of fructosamine, general and specific fluorescence. In vivo experiments were carried out through feed of 4 g fructose per day. Protein expression was studied using western blot and mRNA expression using RT-PCR method. KEY FINDINGS: Fructose inhibits alpha glucosidase to the extent of 48.97% in 4 h at 2.5 M concentration. In vivo studies demonstrated an inhibition of 56.96% in three days. Activity was found to rise by seven days and normalized by 10 days. Protein expression was found to increase by 10.56 fold and SI mRNA by 41.84 fold on 10 days of fructose feed. Long term fructose feed for 60 days demonstrated increase in alpha glucosidase activity by 2.12 fold and increase in postprandial glucose spike. SIGNIFICANCE: Glycation of alpha glucosidase causes inhibition of the enzyme activity leading to compensation through higher protein expression. Long term fructose feed leads to more than two fold increase in enzyme activity causing postprandial spikes and ultimately manifesting as diabetes mellitus.

Global microRNA expression profiling in the liver biopsies of hepatitis B virus-infected patients suggests specific microRNA signatures for viral persistence and hepatocellular injury.

Hepatitis B virus (HBV) can manipulate the microRNA (miRNA) regulatory networks in infected cells to create a permissive environment for viral replication, cellular injury, disease onset, and its progression. The aim of the present study was to understand the miRNA networks and their target genes in the liver of hepatitis B patients involved in HBV replication, liver injury, and liver fibrosis. We investigated differentially expressed miRNAs by microarray in liver biopsy samples from different stages of HBV infection and liver disease (immune-tolerant [n = 8], acute viral hepatitis [n = 8], no fibrosis [n = 16], early [F1+F2, n = 19] or late [F3+F4, n = 14] fibrosis, and healthy controls [n = 7]). miRNA expression levels were analyzed by unsupervised principal component analysis and hierarchical clustering. Analysis of miRNA-mRNA regulatory networks identified 17 miRNAs and 18 target gene interactions with four distinct nodes, each representing a stage-specific gene regulation during disease progression. The immune-tolerant group showed elevated miR-199a-5p, miR-221-3p, and Let-7a-3p levels, which could target genes involved in innate immune response and viral replication. In the acute viral hepatitis group, miR-125b-5p and miR-3613-3p were up, whereas miR-940 was down, which might affect cell proliferation through the signal transducer and activator of transcription 3 pathway. In early fibrosis, miR-34b-3p, miR-1224-3p, and miR-1227-3p were up, while miR-499a-5p was down, which together possibly mediate chronic inflammation. In advanced fibrosis, miR-1, miR-10b-5p, miR-96-5p, miR-133b, and miR-671-5p were up, while miR-20b-5p and miR-455-3p were down, possibly allowing chronic disease progression. Interestingly, only 8 of 17 liver-specific miRNAs exhibited a similar expression pattern in patient sera. CONCLUSION: miRNA signatures identified in this study corroborate previous findings and provide fresh insight into the understanding of HBV-associated liver diseases which may be helpful in developing early-stage disease diagnostics and targeted therapeutics. (Hepatology 2018;67:1695-1709).

Bone marrow stem cells and their niche components are adversely affected in advanced cirrhosis of the liver.

UNLABELLED: Bone marrow (BM) is a reservoir for immune and hematopoietic cells and critical for tissue repair and regeneration. All of these functions are severely altered in cirrhosis. We investigated the cellular and functional state of BM in cirrhosis patients. We studied the histological, cellular, and molecular changes in BM of cirrhosis patients (n = 168) and controls (n = 44). Hematopoietic stem cells (HSCs) and associated niche cells, mesenchymal stem cells, Schwann cells, neural fibers, and endothelial cells were evaluated by immunohistochemistry. Cytokines and growth factors were analyzed in peripheral blood and BM plasma. Cirrhotic BM showed an inverse correlation between cluster of differentiation 34+HSCs and Model of End-Stage Liver Disease (ρ = -0.582, P < 0.001) and Child's scores (P < 0.038). BMs of cirrhosis patients with higher Model of End-Stage Liver Disease (>15) showed significantly decreased HSCs, mesenchymal stem cells, Schwann cells, and neural fibers; increased interleukin-1ß (P = 0.004), tumor necrosis factor-α (P = 0.040), and interferon-γ (P = 0.03); and decreased oncostatin M (P = 0.04), stem cell factor (P = 0.05), and stromal cell-derived factor 1 (P = 0.03) compared to those with lower Model of End-Stage Liver Disease scores (≤15). The cluster of differentiation 34+ cell population was a predictor for the development of sepsis (P < 0.001), and per unit loss increased the probability of sepsis by 16%. Cirrhosis patients with fewer HSCs had lower hemoglobin (P = 0.05) and platelet counts (P = 0.05) and showed early graft dysfunction. CONCLUSIONS: Increasing severity of cirrhosis causes derangement of the hematopoietic niche and loss of HSCs, contributing to the hematological and immunological dysfunctions and reduced potential for regeneration; restoring BM functions could provide new therapeutic options in cirrhosis. (Hepatology 2016;64:1273-1288).

Analysis of hepatitis B virus genotype changes in patients with chronic hepatitis B infection on tenofovir therapy.

Antiviral therapy for chronic hepatitis B (CHB) is often required for prolonged periods. We investigated the instance of one HBV genotype switching to another during tenofovir therapy. Of the 67 patients, genotype A was present in 6 (8.9%), D in 43 (65.6%), C in 1 (1.5%), and mixed in 17 (23.8%) patients. Genotype changes were detected in 51 (76.1%) patients on therapy during a follow-up of 192 (range 52-312) weeks. Inter-genotype changes were seen in 17 (33.3%) and intra-genotype in 28 (55%) and both inter- and intra-genotype in 6 of 51 (11.7%) patients. The distribution of genotypes in patients achieving complete virological response was genotype D, 32/43 (74.4%); genotype A, 6/6 (100%); and mixed genotypes, 13/17 (76.47%). The cumulative time of genotype switch among genotype A was 12 months (range 6-18), in genotype D, 12 months (range 6-48), and mixed genotype, 18 months (range 6-24). The type of inter-genotype switch most frequently detected among genotype A1 was from A1 to D1 5/6 (83.3%), followed by mixed to genotype D3 7/13 (54%) and among intra-genotype changes, from D1 to D3 in 14/20 (70%). Pretreatment HBV genotype was the only factor predicting inter-genotype switches with genotype A or mixed genotypes more likely to undergo inter-genotype switches as compared to genotype D patients (OR 66.6 [13.6-327.0, P < 0.001]). Compared to genotype D, genotype A, and mixed genotypes are more inclined to switch while on tenofovir therapy. Genotypes tend to switch and select to a particular type possibly due to constant antiviral drug pressure. J. Med. Virol. 88:1364-1375, 2016. © 2016 Wiley Periodicals, Inc.

Key elements of the RNAi pathway are regulated by hepatitis B virus replication and HBx acts as a viral suppressor of RNA silencing.

The host-mediated RNAi pathways restrict replication of viruses in plant, invertebrate and vertebrate systems. However, comparatively little is known about the interplay between RNAi and various viral infections in mammalian hosts. We show in the present study that the siRNA-mediated silencing of Drosha, Dicer and Ago2 [argonaute RISC (RNA-induced silencing complex) catalytic component 2] transcripts in Huh7 cells resulted in elevated levels of HBV (hepatitis B virus)-specific RNAs and, conversely, we observed a decrease in mRNA and protein levels of same RNAi components in HepG2 cells infected with HBV. Similar reductions were also detectable in CHB (chronic hepatitis B) patients. Analysis of CHB liver biopsy samples, with high serum HBV DNA load (>log108 IU/ml), revealed a reduced mRNA and protein levels of Drosha, Dicer and Ago2. The low expression levels of key RNAi pathway components in CHB patient samples as well as hepatic cells established a link between HBV replication and RNAi components. The HBV proteins were also examined for RSS (RNA-silencing suppressor) properties. Using GFP-based reversion of silencing assays, in the present study we found that HBx is an RSS protein. Through a series of deletions and substitution mutants, we found that the full-length HBx protein is required for optimum RSS activity. The in vitro dicing assays revealed that the HBx protein inhibited the human Dicer-mediated processing of dsRNAs into siRNAs. Together, our results suggest that the HBx protein might function as RSS to manipulate host RNAi defence, in particular by abrogating the function of Dicer. The present study may have implications in the development of newer strategies to combat HBV infection.