DNA methylation regulates hypothalamic gene expression linking parental diet during pregnancy to the offspring's risk of obesity in Psammomys obesus.

BACKGROUND/OBJECTIVE: The rising incidence of obesity is a major public health issue worldwide. Recent human and animal studies suggest that parental diet can influence fetal development and is implicated with risk of obesity and type 2 diabetes in offspring. The hypothalamus is central to body energy homoeostasis and appetite by controlling endocrine signals. We hypothesise that offspring susceptibility to obesity is programmed in the hypothalamus in utero and mediated by changes to DNA methylation, which persist to adulthood. We investigated hypothalamic genome-wide DNA methylation in Psammomys obesus diet during pregnancy to the offspring's risk of obesity. METHODS: Using methyl-CpG binding domain capture and deep sequencing (MBD-seq), we examined the hypothalamus of offspring exposed to a low-fat diet and standard chow diet during the gestation and lactation period. RESULTS: Offspring exposed to a low-fat parental diet were more obese and had increased circulating insulin and glucose levels. Methylome profiling identified 1447 genomic regions of differential methylation between offspring of parents fed a low-fat diet compared with parents on standard chow diet. Pathway analysis shows novel DNA methylation changes of hypothalamic genes associated with neurological function, nutrient sensing, appetite and energy balance. Differential DNA methylation corresponded to changes in hypothalamic gene expression of Tas1r1 and Abcc8 in the offspring exposed to low-fat parental diet. CONCLUSION: Subject to parental low-fat diet, we observe DNA methylation changes of genes associated with obesity in offspring.

The effect of daily usage of Listerine Cool Mint mouthwash on the oropharyngeal microbiome: a substudy of the PReGo trial.

Introduction. ListerineÒ is a bactericidal mouthwash widely used to prevent oral health problems such as dental plaque and gingivitis. However, whether it promotes or undermines a healthy oral microbiome is unclear.Hypothesis/Gap Statement. We hypothesized that the daily use of Listerine Cool Mint would have a significant impact on the oropharyngeal microbiome.Aim. We aimed to assess if daily usage of Listerine Cool Mint influenced the composition of the pharyngeal microbiome.Methodology. The current microbiome substudy is part of the Preventing Resistance in Gonorrhoea trial. This was a double-blind single-centre, crossover, randomized controlled trial of antibacterial versus placebo mouthwash to reduce the incidence of gonorrhoea/chlamydia/syphilis in men who have sex with men (MSM) taking HIV pre-exposure prophylaxis (PrEP). Fifty-nine MSM taking HIV PrEP were enrolled. In this crossover trial, participants received 3 months of daily Listerine followed by 3 months of placebo mouthwash or vice versa. Oropharyngeal swabs were taken at baseline and after 3 months use of each mouthwash. DNA was extracted for shotgun metagenomic sequencing (Illumina Inc.). Non-host reads were taxonomically classified with MiniKraken and Bracken. The alpha and beta diversity indices were compared between baseline and after each mouthwash use. Differentially abundant bacterial taxa were identified using ANOVA-like differential expression analysis.Results. Streptococcus was the most abundant genus in most samples (n = 103, 61.7 %) with a median relative abundance of 31.5% (IQR 20.6-44.8), followed by Prevotella [13.5% (IQR 4.8-22.6)] and Veillonella [10.0% (IQR 4.0-16.8)]. Compared to baseline, the composition of the oral microbiome at the genus level (beta diversity) was significantly different after 3 months of Listerine (P = 0.006, pseudo-F = 2.29) or placebo (P = 0.003, pseudo-F = 2.49, permutational multivariate analysis of variance) use. Fusobacterium nucleatum and Streptococcus anginosus were significantly more abundant after Listerine use compared to baseline.Conclusion. Listerine use was associated with an increased abundance of common oral opportunistic bacteria previously reported to be enriched in periodontal diseases, oesophageal and colorectal cancer, and systemic diseases. These findings suggest that the regular use of Listerine mouthwash should be carefully considered.

Is chronic hepatitis B being undertreated in the United States?

Chronic infection with the hepatitis B virus (HBV) is a major risk factor for development of end-stage liver disease, including cirrhosis, liver failure and primary liver cancer. There are now seven antiviral agents approved by the United States Food and Drug Administration (FDA) for the management of chronic HBV infection. Despite the fact that there are between 1.4 and 2 million chronic HBV infections in the United States, fewer than 50,000 people per year receive prescriptions for HBV antiviral medications. This report discusses possible explanations for the disparity between the number of people who are chronically infected and the number of people who receive treatment. Explanations for this incongruence include the potentially large number of infected persons who are unscreened and thus remain undiagnosed, and lack of access, including insurance, education and referral to appropriate medical care, particularly for disproportionately infected populations.

Altered mRNA expression due to acute mesenteric ischaemia in a porcine model.

INTRODUCTION: Messenger RNA (mRNA) changes in the small intestine in response to acute mesenteric ischaemia (AMI) could offer novel diagnostic possibilities, but have not been described. The aim was to characterize the mRNA response to experimental AMI. MATERIALS AND METHODS: Twelve pigs underwent catheterisation of the superior mesenteric artery with injection of polivinylalcohol embolisation particles or sodium chloride. Laparotomy and intestinal tissue sampling were performed. Microarray analysis was performed using the GeneChip(®) whole porcine genome array. RESULTS: Seven down-regulated cellular pathways were associated with protein, lipid and carbohydrate metabolism. Seventeen up-regulated pathways were associated with inflammatory and immunological activity, regulation of extracellular matrix and decreased cellular proliferation. Thrombospondin (THS), monocyte chemoattractant protein 1(MCP-1) and gap junction alpha 1(GJA-1) were consistently up-regulated in all embolised pigs. Genes encoding earlier proposed biomarkers for AMI were up-regulated, such as lactate dehydrogenase and creatine kinase, or down-regulated, such as intestinal fatty acid binding protein and glutathione S-transferase. CONCLUSION: This study describes the intestinal tissue response on a gene expression level to AMI. THS, MCP-1 and GJA-1 were consistently up-regulated by ischaemia, whereas earlier proposed biomarkers for AMI were not. Gene expression may not be directly linked to the use of the corresponding proteins as potential clinical biomarkers.

Treatment of chronic hepadnavirus infection in a woodchuck animal model with an inhibitor of protein folding and trafficking.

A novel strategy for anti-viral intervention of hepatitis B virus (HBV) through the disruption of the proper folding and transport of the hepadnavirus glycoproteins is described. Laboratory reared woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated with N-nonyl-deoxynojirimycin (N-nonyl-DNJ), an inhibitor of the endoplasmic reticulum (ER) alpha-glucosidases. The woodchucks experienced significant dose dependent decreases in enveloped WHV, resulting in undetectable amounts in some cases. The reduction in viremia correlated with the levels of hyperglucosylated glycan in the serum of treated animals. This correlation supports the mechanism of action associated with the drug and highlights the extreme sensitivity of the virus to this type of glycan inhibitor. At N-nonyl-DNJ concentrations that prevented WHV secretion, the glycosylation of most serum glycoproteins appeared unaffected, suggesting great selectivity for this class of therapeutics. Indeed, this may account for the low toxicity of the compound over the treatment period. We provide the first evidence that glucosidase inhibitors can be used in vivo to alter specific steps in the N-linked glycosylation pathway and that this inhibition has anti-viral effects.

Use, re-use or discard? Quantitatively defined variance in the functional integrity of N95 respirators following vaporized hydrogen peroxide decontamination during the COVID-19 pandemic.

BACKGROUND: Coronavirus disease 2019 has stretched the ability of many institutions to supply needed personal protective equipment, especially N95 respirators. N95 decontamination and re-use programmes provide one potential solution to this problem. Unfortunately, a comprehensive evaluation of the effects of decontamination on the fit of various N95 models using a quantitative fit test (QNFT) approach is lacking. AIMS: To investigate the effects of up to eight rounds of vaporized hydrogen peroxide (VHP) decontamination on the fit of N95 respirators currently in use in a hospital setting, and to examine if N95 respirators worn by one user can adapt to the face shape of a second user with no compromise to fit following VHP decontamination. METHODS: The PortaCount Pro+ Respirator Fit Tester Model 8038 was used to quantitatively define functional integrity, measured by fit, of N95 respirators following decontamination with VHP. FINDINGS: There was an observable downward trend in the functional integrity of Halyard Fluidshield 46727 N95 respirators throughout eight cycles of decontamination with VHP. Functional integrity of 3M 1870 N95 respirators was reduced significantly after the respirator was worn, decontaminated with VHP, and then quantitatively fit tested on a second user. Furthermore, inconsistencies between qualitative fit test and QNFT results were uncovered that may have strong implications on the fit testing method used by institutions. CONCLUSIONS: The data revealed variability in the functional integrity of different N95 models after VHP decontamination, and exposed potential limitations of N95 decontamination and re-use programmes.

Decontaminating N95 respirators during the COVID-19 pandemic: simple and practical approaches to increase decontamination capacity, speed, safety and ease of use.

BACKGROUND: The COVID-19 pandemic has caused a severe shortage of personal protective equipment (PPE), especially N95 respirators. Efficient, effective and economically feasible methods for large-scale PPE decontamination are urgently needed. AIMS: (1) to develop protocols for effectively decontaminating PPE using vaporized hydrogen peroxide (VHP); (2) to develop novel approaches that decrease set-up and take-down time while also increasing decontamination capacity; (3) to test decontamination efficiency for N95 respirators heavily contaminated by make-up or moisturizers. METHODS: We converted a decommissioned Biosafety Level 3 laboratory into a facility that could be used to decontaminate N95 respirators. N95 respirators were hung on metal racks, stacked in piles, placed in paper bags or covered with make-up or moisturizer. A VHP® VICTORY™ unit from STERIS was used to inject VHP into the facility. Biological and chemical indicators were used to validate the decontamination process. FINDINGS: N95 respirators individually hung on metal racks were successfully decontaminated using VHP. N95 respirators were also successfully decontaminated when placed in closed paper bags or if stacked in piles of up to six. Stacking reduced the time needed to arrange N95 respirators for decontamination by approximately two-thirds while almost tripling facility capacity. Make-up and moisturizer creams did not interfere with the decontamination process. CONCLUSIONS: Respirator stacking can reduce the hands-on time and increase decontamination capacity. When personalization is needed, respirators can be decontaminated in labelled paper bags. Make up or moisturizers do not appear to interfere with VHP decontamination.

Impact of MDCT with intravenous contrast on the survival in patients with acute superior mesenteric artery occlusion.

Acute thromboembolic occlusion in the superior mesenteric artery (SMA) is a condition with high mortality and morbidity. Multi-detector computerised tomography with intravenous contrast enhancement (MDCTiv) may improve diagnostic accuracy and survival. Patients with acute SMA occlusion were identified between 2004 and 2008 at Malmö University Hospital, Sweden. Medical records were analysed. Each MDCTiv was re-evaluated. A total of 67 patients were identified with SMA occlusion, of which 36 were examined with MDCTiv and ten with plain MDCT without intravenous contrast. In all, 24 (67%) of the 36 patients were correctly diagnosed by MDCTiv at first evaluation. Clinical suspicion of intestinal ischemia followed by a distinct inquiry for intestinal ischemia was associated with trend for a higher rate of correct radiological diagnosis, 18 of 23 (78%), at first evaluation (0.06) but without affecting in-hospital survival (p = 0.27). At re-evaluation, SMA occlusion was found in all cases with MDCTiv, whereas intestinal findings were present in half. In-hospital mortality rate was 42% for patients who underwent MDCTiv, which was significantly lower compared to 90% for the ten patients examined with plain MDCT (p = 0.007) and 71% for patients not examined with MDCTiv or plain MDCT (p = 0.031). Patients that underwent plain MDCT had higher levels of creatinine compared to those examined with MDCTiv (p = 0.005). Patients who underwent intestinal revascularisation, endovascular or open, had higher survival rate (p = 0.001). Examination with MDCTiv in patients with acute SMA occlusion was associated with survival benefit. Hence, MDCTiv seems to be the method of choice in the workup phase. Radiologists should routinely describe the mesenteric vessels in patients with acute abdomen even when the diagnosis is not asked for. Patients with high creatinine levels are at risk to be examined without intravenous contrast, and survival in these patients is poor.

Mutants of PC12 cells with altered cyclic AMP responses.

PC12 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 microM 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(beta, gamma-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, Ns. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PC12 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PC12 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.

The Pancreas Can Take the Cold: Lower Waitlist Times Through Importation.

BACKGROUND: Our center has used a strategy of pancreas importation owing to long regional waitlist times. Here we assess the clinical outcomes and financial considerations of this strategy. METHODS: This was a retrospective observational cohort study of patients who received a pancreas transplant at Montefiore Medical Center (MMC) from 2014 to 2017 (n = 28). Clinical parameters, including hemoglobin A1c and complications, were analyzed. The cohort was compared with United Network for Organ Sharing (UNOS) Region 9 with the use of the UNOS/Organ Procurement and Transplantation Network database. Cost analysis of length of stay (LOS), standard acquisition (SAC) fees, and transportation was performed with the use of internal financial data. RESULTS: Pancreas importation resulted in significantly shorter simultaneous pancreas kidney transplant waitlist times compared with Region 9: 518 days vs 1001 days (P = .038). In addition, postoperative complications and 1-year HbA1c did not differ between groups: local 6.30% vs import 6.17% (P = .87). Patients receiving local pancreata stayed an average of 9.2 days compared with 11 days for the import group (P = .36). As such, pancreas importation was associated with higher mean charges ($445,968) compared with local pancreas recipients ($325,470). CONCLUSIONS: Long waitlist times in Region 9 have encouraged our center's adoption of pancreas importation to address the needs of our patient population. This practice has resulted in a reduction of waitlist times by an average of 483 days. Understandably, centers have long been wary of importation owing to perceived risk in clinical outcomes. In our single-center experience, we have demonstrated equivalent postoperative glucose control and graft survival. Importantly, there does appear to be increased costs associated with importation, which are mainly driven by LOS. Curiously, importation from regions with lower SAC fees has the potential to offset costs related to transportation expenses. Notwithstanding these findings, pancreas importation does have the potential to lessen the financial societal burden through reduction in waitlist times.

Improving diet, activity and wellness in adults at risk of diabetes: randomized controlled trial.

OBJECTIVE: The purpose of this analysis is to examine the effect of an algorithm-driven online diabetes prevention program on changes in eating habits, physical activity and wellness/productivity factors. METHODS: The intervention, Alive-PD, used small-step individually tailored goal setting and other features to promote changes in diet and physical activity. A 6-month randomized controlled trial was conducted among patients from a healthcare delivery system who had confirmed prediabetes (n =339). Change in weight and glycemic markers were measured in the clinic. Changes in physical activity, diet and wellness/productivity factors were self-reported. Mean age was 55 (s.d. 8.9) years, mean body mass index was 31 (s.d. 4.4) kg m(-2), 68% were white and 69% were male. RESULTS: The intervention group increased fruit/vegetable consumption by 3.71 (95% confidence interval (CI) 2.73, 4.70) times per week (effect size 0.62), and decreased refined carbohydrates by 3.77 (95% CI 3.10, 4.44) times per week both significantly (P<0.001) greater changes than in the control group. The intervention group also reported a significantly greater increase in physical activity than in the control group, effect size 0.49, P<0.001. In addition, the intervention group reported a significant increase in self-rated health, in confidence in ability to make dietary changes and in ability to accomplish tasks, and a decrease in fatigue, compared with the control group. These changes paralleled the significant treatment effects on glycemic markers and weight. CONCLUSIONS: In addition to promoting improvements in weight and glycemic markers, the Alive-PD program appears to improve eating habits and physical activity, behaviors important not just for diabetes prevention but for those with diagnosed diabetes or obesity. The improvements in wellness/productivity may derive from the diet and activity improvements, and from the satisfaction and self-efficacy of achieving goals.

Alpha-glucosidase inhibitors as potential broad based anti-viral agents.

N-Linked oligosaccharides play many roles in the fate and functions of glycoproteins. One function is to assist in the folding of proteins by mediating interactions of the lectin-like chaperone proteins calnexin and calreticulin with nascent glycoproteins. These interactions can be prevented by inhibitors of the alpha-glucosidases and this causes some proteins to be misfolded and retained within the endoplasmic reticulum. In human immunodeficiency virus (HIV) and hepatitis B virus (HBV) the misfolding of key viral envelope glycoproteins interferes with the viral life cycle. It has been demonstrated in an animal model of chronic HBV that glucosidase inhibitors can alter glycosylation and have anti-viral activity. As the mechanism of action of alpha-glucosidase inhibitors is the induction of misfolded or otherwise defective viral glycoproteins, such inhibitors may be useful therapeutics for many viruses, especially those which bud from the endoplasmic reticulum (where protein folding takes place). For example bovine viral diarrhea virus, a pestivirus akin to hepatitis C virus, is also extremely sensitive to glucosidase inhibition.

Preclinical activity of trans-indazolium[tetrachlorobisindazoleruthenate(III)] (NSC 666158; IndCR; KP 1019) against tumour colony-forming units and haematopoietic progenitor cells.

Trans-indazolium[tetrachlorobisindazoleruthenate(III)] (KP 1019) is a new heavy metal complex with promising activity against tumour cell lines and in animal models. We studied the antineoplastic effects of KP 1019 (final concentrations: 1, 10, 100 micrograms/ml) on in vitro proliferation of clonogenic cells from freshly explanted human tumours in a capillary soft agar cloning system, and compared the activity of KP 1019 with conventional antineoplastic agents. 53 of 75 specimens (71%) showed adequate growth in controls. KP 1019 inhibited tumour colony formation in a concentration-dependent manner in both short- (1 h) and long-term (21 d) exposure experiments. KP 1019 at 100 micrograms/ml with 1 h exposure was as active as bleomycin, cisplatin, doxorubicin, etoposide, 5-fluorouracil, methotrexate, mitomycin-C and vinblastine, with only paclitaxel more active than KP 1019 (P = 0.002). The antitumour activity of KP 1019 was more pronounced after long-term exposure, indicating the potential schedule dependency of KP 1019. Activity was observed against non-small cell lung, breast and renal cancer. We conclude that if appropriate plasma levels can be achieved in patients, KP 1019 may have significant clinical activity against a variety of different tumour types.

Preclinical activity of taxotere (RP 56976, NSC 628503) against freshly explanted clonogenic human tumour cells: comparison with taxol and conventional antineoplastic agents.

Taxotere (TER) and taxol (TA) are new antitumour agents currently undergoing clinical evaluation. We studied the antineoplastic effects of these agents (final concentrations: 4.0, 0.4, 0.04 mumol/l) on the in vitro proliferation of clonogenic cells from freshly explanted human tumours using a capillary soft agar cloning system. We also compared the activity of these new compounds to conventional antineoplastic agents (bleomycin, cisplatin, dacarbazine, doxorubicin, etoposide, 5-fluorouracil, vinblastine, interferon-alpha 2). Using a 21-28-day continuous drug exposure, 54/81 specimens (67%) were evaluable for comparisons, and using a 1-h drug exposure followed by 21-28 days incubation, 50/80 specimens (63%) were similarly evaluable. With both schedules, TA and TER showed concentration-related antitumour activity. At 0.4 mumol/l, median colony survival was 0.61 x control (range 0.09-0.96) for TA and 0.51 x control (0.15-0.81) for TER in the 1-h incubation (P = 0.0002). Median colony formation was also reduced significantly more by TER as compared to TA in the long-term incubation schedule. Statistical analysis indicated that TER but not TA was significantly more active than cisplatin (P = 0.02), doxorubicin (P = 0.01), 5-fluorouracil (P = 0.01) and interferon-alpha 2 (P = 0.01). We conclude that TER and TA are more active against in vitro tumour colony formation from freshly explanted human tumours. TER appears to be slightly more active than taxol and promises to be active against tumours resistant to conventional antineoplastics.

A phage-linked immunoabsorbant system for the detection of pathologically relevant antigens.

This report describes a novel system for the immunological detection of immobilized antigen. The detection of herpes simplex virus (HSV) antigen was used as an example. Bacteriophage M13, containing the E. coli lac Z gene, was used as the "reporter" molecule in an immunoassay which is otherwise analogous to the enzyme-linked immunoabsorbant assay (ELISA). Briefly, HSV infected cells were incubated with a mouse monoclonal antibody specific for HSV antigen, followed by rabbit anti-mouse serum and mouse anti-M13 serum. Immune complexes were incubated with viable M13 phage. M13 binding was due to the presence of M13 antibodies, whose presence ultimately depended on the binding of monoclonal antibody to HSV. Phage was recovered by elution in pH = 11. Recovered phage was used to infect E. coli. M13 was quantitated by either plaque assay or by an assay for phage-induced beta-galactosidase activity in appropriate E. coli strains. The amount of M13 recovered was proportional to the number of HSV infected cells probed. Therefore, M13 served as a "bio-amplifiable tag" to antibody, as enzymes do in the ELISA. Since M13 is viable, its signal can be amplified by infection of susceptible bacteria, and the promise for an enormously sensitive immunoassay exists. The sensitivity of the assay described here is compared to the ELISA in the detection of HSV infection cells, as an example of the novel assay's potential. Significantly, the novel assay was more sensitive than the ELISA when samples were tested under identical circumstances. This technique is called the phage-linked immunoabsorbant assay (PHALISA), by analogy to the ELISA.

Herpes simplex virus type 1 alpha gene containing plasmids can inhibit expression regulated from an alpha promoter in CV-1 but not HeLa cells.

Transfection of plasmids containing the herpes simplex virus type 1 (HSV-1) alpha gene 27 has been observed to inhibit gene expression from a virus alpha promoter in monkey (CV-1) but not human (HeLa) cells in a transient gene expression system. DNA mediated gene transfer to CV-1 and HeLa cells of the bacterial gene for chloramphenicol acetyl transferase (CAT) linked to the promoter regulatory domain from the HSV-1 alpha 4 gene results in production of substantial levels of CAT enzyme. Cotransfection of equal mole amounts of an alpha 27 containing plasmid with an alpha 4-CAT construct to CV-1 cells results in a greater than 85% inhibition of CAT activity. No significant inhibition of CAT activity was observed when transfection was done in HeLa cells, with the same concentrations tested. Intact alpha 27 structural genes were necessary to achieve inhibition since subgenomic fragments and restriction enzyme digested alpha 27 genes were not effective inhibitors. Cotransfection of alpha 27 genes to CV-1 cells also prevented alpha 0 as well as alpha 4 from mediating their trans-stimulation of the HSV thymidine kinase (tk) regulated CAT gene, B-CAT. This suggests that the alpha 27 gene product may down-regulate gene expression from alpha promoters.

Limited proteolysis induces woodchuck hepatitis virus infectivity for human HepG2 cells.

Previous work from our laboratory has shown that digestion of hepatitis B virus (HBV) with V8 protease rendered the virus infectious for human hepatoblastoma cell line (HepG2). It was hypothesized that the cleavage exposes a 16 amino acid region that includes a consensus 'fusion' motif necessary to mediate infectivity. Since woodchuck hepatitis virus (WHV) and HBV possess significant homology in this region of their envelope proteins, including the V8 protease cleavage site, the possibility that WHV infectivity for HepG2 cells could be induced by V8 digestion was explored. WHV isolated from the serum of chronically infected woodchucks, digested with V8 protease, was shown to loose its preS domain. V8 digested WHV eluted from gel filtration columns with a size similar to that of undigested virus, suggesting that digestion with V8 protease did not cause significant changes in virion size. The amount of progeny virus secreted into the culture medium following infection of HepG2 cells with V8 digested WHV reached 2.5 pg/ml, after 8 days. Moreover, WHV DNA replicative intermediates could be detected in the cells following infection with protease digested, but not undigested, viruses. These data suggest that protease modification of WHV, a non-human virus, induced infectivity for human tissue culture cells. These results are consistent with the hypothesis that exposure of an amino acid region of the envelope polypeptide that contains a consensus fusion motif is important in Hepadnavirus entry.

A proteomic approach for the discovery of early detection markers of hepatocellular carcinoma.

Individuals chronically infected with hepatitis B or C virus (HBV, HCV) are at high risk for the development of hepatocellular carcinoma (HCC), with disease progression occurring relentlessly over many years. The diagnosis of HCC usually occurs at late stages in the disease when there are few effective treatment options and the prognosis for patients with HCC is very poor. The long latency period, together with clearly identified at risk populations, provide opportunities for earlier detection that will allow more timely and effective treatment of this devastating cancer. We are using a proteomic approach to test the hypothesis that changes in the amount of certain serum polypeptides, or changes in their post-translational modifications, can be used to predict the onset of HCC. Advances in the standardization of two dimensional gel electrophoresis (2DE) coupled with computerized image analysis now permit the reproducible resolution of thousands of polypeptides per run. Serum polypeptides from individuals at different stages in the disease continuum are being resolved by 2DE to identify those that change with disease progression. Polypeptides found by this method can be further characterized by mass spectrometry. In addition, the potential for changes in the glycan structure of certain polypeptides to serve as a marker for disease progression can be explored. The proteomic approach is expected to liberate us from the need to "cherry pick" or guess the best biomarkers and let the data tell us which are the best indicators of disease. Information may also be gleaned about the pathobiology of the disease process.

Hepatitis B virus MHBs antigen is selectively sensitive to glucosidase-mediated processing in the endoplasmic reticulum.

Previous studies have shown that hepatitis B virus (HBV) secretion from HepG 2.2.15 cells is prevented by inhibitors of the endoplasmic reticulum (ER) glucosidase under conditions where secretion of cellular glycoproteins are not detectably affected. The 2.2.15 cells are derived from HepG2 and contain intact dimers of the viral genome. They produce and secrete infectious HBV. The secretion of the viral envelope polypeptide, MHBs, was selectively and quantitatively reduced from 2.2.15 cells in which glucosidase was inhibited, whereas the envelope polypeptide, SHBs, was relatively insensitive, being as resistant as were most host glycoproteins. Because 2.2.15 cells express all HBV ORFs, it seemed possible that the sensitivity of MHBs secretion involved its interaction with the viral nucleocapsid or other viral gene products. The work reported here showed that MHBs secretion from HepG2 cells transfected with a plasmid that expresses only the MHBs polypeptide was as sensitive to glucosidase inhibitors as it was from 2.2.15 cells. These data show that the sensitivity of the MHBs polypeptide secretion to glucosidase inhibitors is entirely encrypted within its structural gene. The reasons the MHBs polypeptide, but not SHBs, is so sensitive to glucosidase processing are discussed.

A miniature gamma camera.

We have described a mobile miniature-gamma-camera system for use in electrical trauma units and have presented images and imaging characteristics of a prototype system. The system has as its principal component a miniature gamma camera based on a PSPMT. The camera is 92 mm x 92 mm x 190 mm in size, weighs 5 kg, has a 48 mm x 48 mm field of view, and has an intrinsic resolution of approximately 3 mm FWHM and 6 mm FWTM. It is expected that devices of this type will be useful as imaging tools in electrical trauma units and laboratories where imaging studies regarding uptake mechanisms of radiopharmaceuticals for assessing tissue viability are carried out.