SUMMARY Ames dwarf (Prop1df, df/df) mice are characterized by growth hormone (GH), prolactin and thyrotropin deficiency, remarkable extension of longevity and increased insulin sensitivity with low levels of fasting insulin and glucose. Plasma levels of anti-inflammatory adiponectin are increased in df/df mice while pro-inflammatory IL-6 was decreased in plasma and epididymal fat. This represents an important shift in the balance between pro- and anti-inflammatory adipokines in adipose tissue, which was not exposed to GH signals during development or adult life. To determine the role of adipose tissue in the control of insulin signaling in these long living mutants, we examined the effects of surgical removal of visceral (epididymal and perinephric) adipose tissue. Comparison of the results obtained in df/df mice and their normal (N) siblings indicated different effects of visceral fat removal (VFR) on insulin sensitivity and glucose tolerance. The analysis of the expression of genes related to insulin signaling indicated that VFR improved insulin action in skeletal muscle in N mice. Interestingly, this surgical intervention did not improve insulin signaling in df/df mice skeletal muscle but caused suppression of the signal in subcutaneous fat. We conclude that altered profile of adipokines secreted by visceral fat of Ames dwarf mice may act as a key contributor to increased insulin sensitivity and extended longevity of these animals.

The molecular events that drive Hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system. Here we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We first describe a primary ex vivo HBV-infection model derived from healthy donor liver organoids after challenge with recombinant virus or HBV-infected patient serum. HBV infected organoids produced cccDNA, HBeAg, expressed intracellular HBV RNA and proteins, and produced infectious HBV. This ex vivo HBV infected primary differentiated hepatocyte organoid platform was amenable to drug screening for both anti-HBV activity as well as for drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor NTCP after lentiviral transduction were not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. We also generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription. Finally, we generated HBV-infected patient-derived liver organoids from non-tumor cirrhotic tissue of explants from liver transplant patients. Interestingly, transcriptomic analysis of patient-derived liver organoids indicated the presence of an aberrant early cancer gene signature, which clustered with the HCC cohort on the TCGA LIHC dataset and away from healthy liver tissue, and may provide invaluable novel biomarkers for the development of HCC and surveillance in HBV infected patients.

Post-Golgi trafficking of mature VLDL (very-low-density lipoprotein) is crucial in maintaining normal TAG (triacylglycerol) homoeostasis of hepatocytes; however, the mechanism that regulates the exit of mature VLDL from the TGN (trans-Golgi network) is not known. We developed an in vitro TGN-budding assay that allowed us to examine the formation of secretory vesicles from the TGN in primary rat hepatocytes. We isolated TAG-rich PG-VTVs (post-TGN VLDL transport vesicles) using a continuous sucrose density gradient. PG-VTVs were distributed in low-density fractions, whereas protein transport vesicles were present in relatively higher-density fractions of the same sucrose gradient. EM revealed large intact PG-VTVs ranging 300–350 nm in size. The biogenesis of PG-VTVs from the TGN required cytosol, ATP, GTP hydrolysis and incubation at 37 °C. PG-VTVs concentrated the VLDL proteins: apolipoproteins apoB100, apoAIV, apoAI and apoE, but did not contain either albumin or transferrin. Proteinase K treatment did not degrade VLDL core proteins, suggesting that PG-VTVs were sealed. PG-VTVs were able to fuse with and deliver VLDL to the PM (plasma membrane) in a vectorial manner. We conclude that we have identified a new TGN-derived vesicle, the PG-VTV, which specifically transports mature VLDL from the TGN to the PM.

The emergence of novel pathogenic strains with increased antibacterial resistance patterns poses a significant threat to the management of infectious diseases. In this study, we aimed at utilizing the subtractive genomic approach to identify novel drug targets against Salmonella enterica subsp. enterica serovar Poona strain ATCC BAA-1673. We employed in silico bioinformatics tools to subtract the strain-specific paralogous and host-specific homologous sequences from the bacterial proteome. The sorted proteome was further refined to identify the essential genes in the pathogenic bacterium using the database of essential genes (DEG). We carried out metabolic pathway and subcellular location analysis of the essential proteins of the pathogen to elucidate the involvement of these proteins in important cellular processes. We found 52 unique essential proteins in the target proteome that could be utilized as novel targets to design newer drugs. Further, we investigated these proteins in the DrugBank databases and 11 of the unique essential proteins showed druggability according to the FDA approved drug bank databases with diverse broad-spectrum property. Molecular docking analyses of the novel druggable targets with the drugs were carried out by AutoDock Vina option based on scoring functions. The results showed promising candidates for novel drugs against Salmonella infections.

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OBJECTIVE: Group B streptococcus (GBS) is the leading cause of neonatal sepsis in the US. As such, rectovaginal culture for all women between 35-37 weeks gestation is recommended to screen for those colonized with the pathogen. Early onset disease in neonates can be prevented by intrapartum antibiotic prophylaxis (IAP), but missed screening opportunities and false negative maternal cultures result in preventable neonatal disease. We sought to evaluate the cost-effectiveness of universal IAP in reducing neonatal morbidity and mortality related to GBS. STUDY DESIGN: A decision-analytic model was created using TreeA-gePro; probabilities, costs, and utilities were derived from the literature. We compared universal screening with rectovaginal culture by 37 weeks gestation to no screening and IAP given to all women at delivery after 37 weeks. This model was applied to a theoretical cohort of 1,600,000 women with no documented penicillin allergy based on the estimated number of nulliparous, term-births annually in the United States. The cost-effectiveness threshold was set to $100,000 per quality adjusted life year (QALY). Univariate sensitivity analyses were used to evaluate the robustness of the results. RESULTS: In our theoretical cohort of 1.6 million women, we found that IAP given to all laboring women, no matter their GBS status, was cost effective when compared to IAP given to GBS positive women based on screening culture. Universal IAP results in 803 fewer cases of neonatal sepsis, 29 fewer cases of meningitis, 13 fewer cases of neurodevelopmental delay, and 24 fewer neonatal deaths, despite an increase in cases of maternal anaphylaxis, maternal death from anaphylaxis, and increased cost overall. The universal antibiotics strategy resulted in 1,134 additional QALYs and an incremental cost effectiveness ratio of $22,300/QALY. Univariate sensitivity analysis demonstrated that universal IAP was cost-effective when the cost of culture was between $16.75 and $48.25, and was cost-saving for culture cost above $48.25. CONCLUSION: Our findings suggest that the current standard of care for prenatal screening of GBS may not be cost-effective compared to prophylactically treating all women in pregnancy with penicillin. Further research is necessary to determine if a change in diagnostic test or screening strategy would improve maternal and neonatal outcomes.