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Fetal liver tissue collected from a nonhuman primate (NHP) baboon model of maternal nutrient reduction (MNR) at four gestational time points (90, 120, 140, and 165 days gestation [dG], term in the baboon is ∼185 dG) was used to quantify MNR effects on the fetal liver transcriptome. 28 transcripts demonstrated different expression patterns between MNR and control livers during the second half of gestation, a developmental period when the fetus undergoes rapid weight gain and fat accumulation. Differentially expressed transcripts were enriched for fatty acid oxidation and RNA splicing-related pathways. Increased RNA splicing activity in MNR was reflected in greater abundances of transcript splice variant isoforms in the MNR group. It can be hypothesized that the increase in splice variants is deployed in an effort to adapt to the poor in utero environment and ensure near-normal development and energy metabolism. This study is the first to study developmental programming across four critical gestational stages during primate fetal liver development and reveals a potentially novel cellular response mechanism mediating fetal programming in response to MNR.

Obesity in pregnant women causes fetal cardiac dysfunction and increases offspring cardiovascular disease risk, but its effect on myocardial metabolism is unknown. We hypothesized that maternal obesity alters fetal cardiac expression of metabolism-related genes and shifts offspring myocardial substrate preference from glucose towards lipids. Female mice were fed control or obesogenic diets before and during pregnancy. Fetal hearts were studied in late gestation (embryonic day (E) 18.5; term ≈ E21), and offspring were studied at 3, 6, 9 or 24 months postnatally. Maternal obesity increased heart weight and peroxisome proliferator activated receptor gamma (Pparg) expression in female and male fetuses and caused left ventricular diastolic dysfunction in the adult offspring. Cardiac dysfunction worsened progressively with age in female, but not male, offspring of obese dams, in comparison to age-matched control animals. In 6-month-old offspring, exposure to maternal obesity increased cardiac palmitoyl carnitine-supported mitochondrial respiration in males and reduced myocardial

The prefrontal cortex (PFC) has been implicated as a key brain region responsible for age-related cognitive decline. Little is known about aging-related molecular changes in PFC that may mediate these effects. To date, no studies have used untargeted discovery methods with integrated analyses to determine PFC molecular changes in healthy female primates. We quantified PFC changes associated with healthy aging in female baboons by integrating multiple omics data types (transcriptomics, proteomics, metabolomics) from samples across the adult age span. Our integrated omics approach using unbiased weighted gene co-expression network analysis (WGCNA) to integrate data and treat age as a continuous variable, revealed highly interconnected known and novel pathways associated with PFC aging. We found GABA tissue content associated with these signaling pathways, providing one potential biomarker to assess PFC changes with age. These highly coordinated pathway changes during aging may represent early steps for aging-related decline in PFC functions, such as learning and memory, and provide potential biomarkers to assess cognitive status in humans.

The human airways are complex structures with important interactions between cells, extracellular matrix (ECM) proteins and the biomechanical microenvironment. A robust, well-differentiated in vitro culture system that accurately models these interactions would provide a useful tool for studying normal and pathological airway biology. Here, we report the feasibility and analysis of a physiologically relevant air-liquid interface (ALI) 3D airway ‘organ tissue equivalent’ (OTE) model with three novel features: native pulmonary fibroblasts, solubilized lung ECM, and hydrogel substrate with tunable stiffness and porosity. We demonstrate the versatility of the OTE model by evaluating the impact of these features on human bronchial epithelial (HBE) cell phenotype. Variations of this model were analyzed during 28 days of ALI culture by evaluating epithelial confluence, trans-epithelial resistance, and epithelial phenotype via multispectral immuno-histochemistry and next-generation sequencing. Cultures that included both solubilized lung ECM and native pulmonary fibroblasts within the hydrogel substrate formed well-differentiated ALI cultures that maintained a barrier function and expressed mature epithelial markers relating to goblet, club and ciliated cells. Modulation of hydrogel stiffness did not negatively impact HBE differentiation and could be a valuable variable to alter epithelial phenotype. This study highlights the feasibility and versatility of a 3D airway OTE model to model the multiple components of the human airway 3D microenvironment.

Background Whole-exome sequencing (WES) can expedite research on genetic variation in non-human primate (NHP) models of human diseases. However, NHP-specific reagents for exome capture are not available. This study reports the use of human-specific capture reagents in WES for olive baboons, marmosets, and vervet monkeys. Methods Exome capture was carried out using the SureSelect Human All Exon V6 panel from Agilent Technologies, followed by high-throughput sequencing. Capture of protein-coding genes and detection of single nucleotide variants were evaluated. Results Exome capture and sequencing results showed that more than 97% of old world and 93% of new world monkey protein coding genes were detected. Single nucleotide variants were detected across the genomes and missense variants were found in genes associated with human diseases. Conclusions A cost-effective approach based on commercial, human-specific reagents can be used to perform WES for the discovery of genetic variants in these NHP species.

Familial adenomatous polyposis (FAP) patients develop hundreds of premalignant polyps that progress to colorectal cancer due to a germline mutation in the APC tumor suppressor. Polyps from FAP patients uniquely facilitate interrogation of the continuum of malignant transformation from histologically normal mucosa to benign and dysplastic polyps and eventual adenocarcinomas. As part of the Human Tumor Atlas Network (HTAN), we performed multi-omic profiling, including whole genome sequencing, on 135 samples from six FAP patients across the pre-malignant continuum spanning all physical regions of the large intestine, and serving as the most comprehensive FAP dataset available. Through a comparative analysis with a published FAP cohort (58 multi-region samples across 5 patients) and sporadic colorectal cancer cohort (n=57), each including benign and malignant samples, we evaluate the timing of driver acquisitions at each stage of malignant progression. Despite being separated by vast regions of histologically normal mucosa, independently evolving polyps show extensive mutation sharing, suggesting FAP polyps are polyclonal in origin. Finally, we leverage a simplified mechanistic model of embryonic colonic development demonstrating that the path to malignant transformation in FAP is consistent with polyclonal origins attributable to early mixing, perhaps as early as in-utero. Taken together, the HTAN FAP Atlas provides a novel window into the earliest stages of cancer formation and may illuminate barriers to malignant transformation and opportunities for earlier intervention. Citation Format: Ryan O. Schenck, Aziz Khan, Aaron Horning, Edward D. Esplin, Emma Monte, Si Wu, Casey Hanson, Nasim Bararpour, Stephanie Neves, Lihua Jiang, Kévin Contrepois, Hayan Lee, Tuhin K. Guha, Zheng Hu, Rozelle Laquindanum, Meredith A. Mills, Hassan Chaib, Roxanne Chiu, Ruiqi Jian, Jeannie Chan, Mathew Ellenberger, Winston R. Becker, Bahareh Bahmani, Basil Michael, Jeanne Shen, Samuel Lancaster, Uri Ladabaum, Anshul Kundaje, Teri A. Longacre, William J. Greenleaf, James M. Ford, Michael P. Snyder, Christina Curtis. The polyclonal path to malignant transformation in familial adenomatous polyposis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3497.

Obesity in pregnant women causes fetal cardiac dysfunction and increases offspring cardiovascular disease risk but its effect on myocardial metabolism is unknown. We hypothesised that maternal obesity alters fetal cardiac expression of metabolism-related genes and shifts offspring myocardial substrate preference from glucose towards lipids. Female mice were fed control or obesogenic diets before and during pregnancy. Fetal hearts were studied in late gestation (embryonic day, E18.5; term≈E21) and offspring were studied at 3, 6, 9 or 24 months postnatally. Maternal obesity increased heart weight and peroxisome proliferator activated receptor γ (Pparg) expression in female and male fetuses and caused left ventricular diastolic dysfunction in the adult offspring. Cardiac dysfunction progressively worsened with age in female, not male, offspring of obese dams, compared to age-matched controls. In 6-month-old offspring, exposure to maternal obesity increased cardiac palmitoyl carnitine-supported mitochondrial respiration in males and reduced myocardial 18F-fluorodeoxyglucose uptake in females. Cardiac Pparg expression remained higher in adult offspring of obese than control dams and correlated with contractile and metabolic function. Maternal obesity did not affect cardiac palmitoyl carnitine respiration in females or 18F-fluorodeoxyglucose uptake in males, or alter cardiac 3H-oleic acid uptake, pyruvate respiration, lipid content or fatty acid/glucose transporter abundance in offspring of either sex. The results support our hypothesis and show that maternal obesity affects offspring cardiac metabolism in a sex-dependent manner. Persistent upregulation of Pparg expression in response to overnutrition in utero may mechanistically underpin programmed cardiac impairments and contribute to cardiovascular disease risk in children of women with obesity.

BackgroundDietary high fructose (HFr) is a known metabolic disruptor contributing to development of obesity and diabetes in Western societies. Initial molecular changes from exposure to HFr on liver metabolism may be essential to understand the perturbations leading to insulin resistance and abnormalities in lipid and carbohydrate metabolism. We studied vervet monkeys (Clorocebus aethiops sabaeus) fed a HFr (n=5) or chow diet (n=5) for 6 weeks, and obtained clinical measures of liver function, blood insulin, cholesterol and triglycerides. In addition, we performed untargeted global transcriptomics, proteomics, and metabolomics analyses on liver biopsies to determine the molecular impact of a HFr diet on coordinated pathways and networks that differed by diet.ResultsWe show that integration of omics data sets improved statistical significance for some pathways and networks, and decreased significance for others, suggesting that multiple omics datasets enhance confidence in relevant pathway and network identification. Specifically, we found that sirtuin signaling and a peroxisome proliferator activated receptor alpha (PPARA) regulatory network were significantly altered in hepatic response to HFr. Integration of metabolomics and miRNAs data further strengthened our findings.ConclusionsOur integrated analysis of three types of omics data with pathway and regulatory network analysis demonstrates the usefulness of this approach for discovery of molecular networks central to a biological response. In addition, metabolites aspartic acid and docosahexaenoic acid (DHA), protein ATG3, and genesATG7, HMGCS2link sirtuin signaling and the PPARA network suggesting molecular mechanisms for altered hepatic gluconeogenesis from consumption of a HFr diet.

Baboons (genus Papio) are broadly studied in the wild and in captivity. They are widely used as a non-human primate model for biomedical studies, and the Southwest National Primate Research Center (SNPRC) at Texas Biomedical Research Institute has maintained a large captive baboon colony for more than 50 years. Unlike other model organisms though, the genomic resources for baboons are severely lacking. This has hindered the progress of studies using baboons as a model for basic biology or human disease. Here, we describe a dataset of 100 high-coverage whole-genome sequences obtained from the mixed colony of olive (P. anubis) and yellow (P. cynocephalus) baboons housed at the SNPRC. These data provide a comprehensive catalog of common genetic variation in baboons, as well as a fine-scale genetic map. We show how the data can be used to learn about ancestry and admixture, and to correct errors in the colony records. Finally, we investigated the consequences of inbreeding within the SNPRC colony and found clear evidence for increased rates of juvenile mortality and increased homozygosity of putatively deleterious alleles in inbred individuals.

Baboons (genus Papio) are broadly studied in the wild and in captivity. They are widely used as a nonhuman primate model for biomedical studies, and the Southwest National Primate Research Center (SNPRC) at Texas Biomedical Research Institute has maintained a large captive baboon colony for more than 50 yr. Unlike other model organisms, however, the genomic resources for baboons are severely lacking. This has hindered the progress of studies using baboons as a model for basic biology or human disease. Here, we describe a data set of 100 high-coverage whole-genome sequences obtained from the mixed colony of olive (P. anubis) and yellow (P. cynocephalus) baboons housed at the SNPRC. These data provide a comprehensive catalog of common genetic variation in baboons, as well as a fine-scale genetic map. We show how the data can be used to learn about ancestry and admixture and to correct errors in the colony records. Finally, we investigated the consequences of inbreeding within the SNPRC colony and found clear evidence for increased rates of infant mortality and increased homozygosity of putatively deleterious alleles in inbred individuals.

Background Serious staphylococcal infections require prolonged courses of intravenous (IV) antibiotics. Weekly IV dalbavancin is an alternative to more frequent IV antimicrobial dosing for homeless patients or persons who inject drugs (PWID), for whom creating a treatment plan can be challenging. We examined the clinical outcomes in patients who were treated with dalbavancin compared with a similar population treated with alternative antibiotics. Methods We identified 18 patients who received dalbavancin from June 1, 2015 to December 31, 2016 using pharmacy records and 89 patients receiving IV antibiotics for similar infections treated at Harborview Medical Center from January 1, 2015 to May 31, 2015, before dalbavancin was available. Medical records were reviewed, and patient demographics, length of stay (LOS), readmission, and outcomes were abstracted using REDCap, linked to the University of Washington’s Clinical Data Repository. Results Basic demographics in Table 1. The types of infections are in Figure 1. Clinical cure rates were similar between the two groups (Figure 2) although 21% and 28% of the patients were lost to follow-up in the pre and post dalbavancin period. Among the subgroup of PWID, those who received dalbavancin had higher rates of clinical cure (64.7% vs. 29.4%, P = 0.01), a trend toward decreased LOS (11.4 ± 5.8 vs. 20.2 ± 15.1 days, P = 0.04), and fewer 30-day readmissions (0% vs. 29.4%, P = 0.02) (Figure 2). Fewer PWID in the dalbavancin group were lost to follow-up (23.5% vs. 70.6%). Conclusion Patients treated with dalbavancin had similar outcomes compared with patients treated in the pre-dalbavancin time period. Among PWID, dalbavancin use led to significantly improved outcomes including a higher clinical cure rate, lower readmission rate, and shorter hospital LOS, which offset the cost of the drug. Dalbavancin is an option for the treatment of serious staphylococcal infections in selected patients. Disclosures All authors: No reported disclosures.

Bone morphogenetic protein-2 (BMP-2) regulates growth plate chondrogenesis during development and postnatal bone growth, but the control mechanisms of BMP-2 expression in growth plate chondrocytes are unknown. Here we have used both in vitro and in vivo approaches to demonstrate that transcription factor, NF-kappaB, regulates BMP-2 gene expression in chondrocytes. Two putative NF-kappaB response elements were found in the -2712/+165 region of the BMP-2 gene. Cotransfection of mutant I-kappaBalpha expression plasmids with BMP-2 promoter-luciferase reporters into TMC-23 chondrocyte cell line suppressed BMP-2 transcription. Mutations in NF-kappaB response elements in the BMP-2 gene lead to decreases in BMP-2 promoter activity. Electrophoretic mobility shift assay using nuclear extracts from TMC-23 chondrocytic cells revealed that the NF-kappaB subunits p50 and p65 bound to the NF-kappaB response elements of the BMP-2 gene. Thus, NF-kappaB may positively regulate BMP-2 gene transcription. Consistent with these findings, expression of BMP-2 mRNA was significantly reduced in growth plate chondrocytes in NF-kappaB p50/p52 dKO mice, which associated with decreased numbers of 5-bromo-2'-deoxyuridine (BrdUrd)-positive cells in the proliferating zone of growth plate in these mice. Therefore, in postnatal growth plate chondrocytes, expression of BMP-2 is regulated by NF-kappaB, which may play an important role in chondrogenesis.

The South American opossum, Monodelphis domestica, has been used as a model system to study ultraviolet (UV)-induced genetic alterations that lead to the development of melanoma. Suckling young of Monodelphis exposed to multiple doses of UVB radiation can develop benign or malignant melanomas later as adults. Point mutations predominantly at codon 61 of the N-ras gene have been found in melanomas from sun-exposed body sites in humans. To determine if similar mutations are associated with UV-induced melanoma in Monodelphis, the nucleotide sequence of a Monodelphis N-ras cDNA was determined, and the occurrence of ras mutations in melanomas from UV-irradiated opossums was investigated. Single-strand conformation polymorphism analysis revealed no mutations in either the Monodelphis N-ras or H-ras genes in any of 24 primary malignant melanoma samples analyzed in this study. The disparate association of ras mutations with melanoma in humans and Monodelphis may be explained by differences in nucleotide sequence at codon 61 of the N-ras gene as well as differences in skin architecture between the two species. These results support the contention that a mutationally activated N-ras gene contributes to the vertical growth phase, which is specific to the progression of malignant melanoma in humans.

“The role of ABCB4 in hepatobiliary diseases in humans is well documented, but its role in dietary responsiveness in humans remains to be investigated.”

M-commerce is defined as any direct or indirect transaction conducted and facilitated through a wireless telecommunication network. This research investigates how consumers in Singapore are influenced to adopt M-commerce. The research employs the Technology Acceptance Model (TAM) proposed by Davis (1989) to examine factors affecting Singaporeans– attitudes toward M-commerce. Empirical data from a survey of 309 Singaporeans reflect consumer perceived usefulness (PU) and perceived ease of use (PEOU) influence attitude toward using (AT) M-commerce. It is also found that consumer innovativeness affects their attitude toward using M-commerce, while past adoption behavior and knowledge about other technological innovations do not influence adoption behavior. Results from multiple regression analyses further reveal that demographic variables have no effects on M-commerce adoption decision.

Plasma VLDL and LDL cholesterol were markedly elevated (>40-fold) in high-responding opossums, but moderately elevated (6-fold) in low-responding opossums after they had consumed a high-cholesterol and high-fat diet for 24 wk. In both high- and low-responding opossums, plasma triglycerides were slightly elevated, threefold and twofold, respectively. Dietary challenge also induced fatty livers in high responders, but not in low responders. We studied the lipid composition, histopathological features, and gene expression patterns of the fatty livers. Free cholesterol (2-fold), esterified cholesterol (11-fold), and triglycerides (2-fold) were higher in the livers of high responders than those in low responders, whereas free fatty acid levels were similar. The fatty livers of high responders showed extensive lobular disarray by histology. Inflammatory cells and ballooned hepatocytes were also present, as were perisinusoidal fibrosis and ductular proliferation. In contrast, liver histology was normal in low responders. Hepatic gene expression revealed differences associated with the development of steatohepatitis in high responders. The accumulation of hepatic cholesterol was concomitant with upregulation of the HMGCR gene and downregulation of the CYP27A1, ABCG8, and ABCB4 genes. Genes involved in inflammation ( TNF, NFKB1, and COX2) and in oxidative stress ( CYBA and NCF1) were upregulated. Upregulation of the growth factor genes ( PDGF and TGFB1) and collagen genes ( Col1A1, Col3A1, and Col4A1) was consistent with fibrosis. Some of the histological characteristics of the fatty livers of high-responding opossums imitate those in the livers of humans with nonalcoholic steatohepatitis.

Bile salts, cholesterol and phosphatidylcholine are secreted across the canalicular membrane of hepatocytes into bile by ATP-binding cassette (ABC) transporters. Secretion of bile salts by ABCB11 is essential for bile flow and for absorption of lipids and fat-soluble vitamins. ABCG5 and ABCG8 eliminate excess cholesterol and sterols from the body by secreting them into bile. There are two mechanisms to protect the canalicular membrane from solubilization by bile salts; ABCB4 secretes phosphatidylcholine into bile to form mixed micelles with bile salts, and ATP8B1 maintains the canalicular membrane in a liquid-ordered state. Three different forms of progressive familial intrahepatic cholestasis (PFIC) disorders, PFIC1, PFIC2 and PFIC3, are caused by mutations in ATP8B1, ABCB11 and ABCB4, respectively. Sitosterolemia is caused by mutations in ABCG5 and ABCG8. This article reviews the physiological roles of these canalicular transporters, and the pathophysiological processes and clinical features associated with their mutations.

High-responding opossums are susceptible to developing hypercholesterolemia on a high-cholesterol diet, but low-responding opossums are resistant. The observation of low biliary cholesterol and low biliary phospholipids in high responders suggested that the ABCB4 gene affects response to dietary cholesterol. Two missense mutations (Arg29Gly and Ile235Leu) were found in the ABCB4 gene of high responders. High responders (ATHH strain) were bred with low responders (ATHE or ATHL strain) to produce F1 and F2 progeny in two different genetic crosses (KUSH6 and JCX) to determine the effect of ABCB4 allelic variants on plasma cholesterol concentrations after a dietary challenge. Pedigree-based genetic association analyses consistently implicated a variant in ABCB4 or a closely linked locus as a major, but not the sole, genetic contributor to variation in the plasma cholesterol response to dietary cholesterol. High responders, but not low responders, developed liver injury as indicated by elevated plasma biomarkers of liver function, probably reflecting damage to the canalicular membrane by bile salts because of impaired phospholipid secretion. Our results implicate ABCB4 as a major determinant of diet-induced hypercholesterolemia in high-responding opossums and suggest that other genes interact with ABCB4 to regulate lipemic response to dietary cholesterol.

Objective To demonstrate whether daily bathing with cloths impregnated with 2% chlorhexidine gluconate will decrease colonization of resistant bacteria and reduce the rates of health care–associated infections in critically injured patients. Design Retrospective analysis of data collected 6 months before and after institution of a chlorhexidine bathing protocol. Setting A 12-bed intensive care unit in a level I trauma center. Patients Two hundred eighty-six severely injured patients underwent daily chlorhexidine bathing during the 6-month intervention; 253 patients were bathed without chlorhexidine prior to the intervention. Interventions Daily chlorhexidine bathing. Main Outcomes Measures Rates of ventilator-associated pneumonia (VAP), bloodstream infection, and colonization with resistant organisms (methicillin-resistant Staphylococcus aureus [MRSA] or Acinetobacter species). Results Baseline patient and injury characteristics were similar between cohorts. Patients receiving chlorhexidine baths were significantly less likely to acquire a catheter-related bloodstream infection than comparators (2.1 vs 8.4 infections per 1000 catheter-days, P = .01). The incidence of VAP was not affected by chlorhexidine baths (16.9 vs 21.6 infections per 1000 ventilator-days in those with vs those without chlorhexidine baths, respectively, P = .30). However, patients who received chlorhexidine baths were less likely to develop MRSA VAP (1.6 vs 5.7 infections per 1000 ventilator-days, P = .03). The rate of colonization with MRSA (23.3 vs 69.3 per 1000 patient-days, P Acinetobacter (1.0 vs 4.6 per 1000 patient-days, P = .36) was significantly lower in the chlorhexidine group than in the comparison group. Conclusions Daily bathing of trauma patients with cloths impregnated with 2% chlorhexidine gluconate is associated with a decreased rate of colonization by MRSA and Acinetobacter and lower rates of catheter-related bloodstream infection and MRSA VAP.

High and low responding opossums (Monodelphis domestica) differ in their plasma very low density lipoprotein and low density lipoprotein (VLDL+LDL) cholesterol concentrations when they consume a high cholesterol diet, which is due in part to absorption of a higher percentage of dietary cholesterol in high responders. We compared the expression of a set of genes that influence cholesterol absorption in high and low responders fed a basal or a high cholesterol and low fat (HCLF) diet. Up-regulation of the ABCG5, ABCG8, and IBABP genes by the HCLF diet in high and low responders may reduce cholesterol absorption to maintain cholesterol homeostasis. Differences in expression of the phospholipase genes (PLA2 and PLB) and phospholipase activity were associated with differences in cholesterol absorption when opossums were fed cholesterol-enriched diets. Higher PLA2 and PLB mRNA levels and higher phospholipase activity may increase cholesterol absorption in high responders by enhancing the release of cholesterol from bile salt micelles for uptake by intestinal cells.

A 12.5-kilobase pair (kb) segment upstream of the human albumin gene was analyzed for transcription enhancing activity using transient transfection analysis, gel mobility shift assays, DNase I footprinting, and site-specific mutagenesis. Two enhancer regions were identified, one 1.7 kb upstream of the transcription initiation site (E1.7) and the other 6 kb upstream (E6). In E1.7, a nuclear protein from HuH-7 hepatoma cells binds to an AT-rich sequence, GTTACTAATTGAC. Competition gel mobility shift assays suggested that this protein is HNF-1, which regulates the promoter of the albumin gene and several other liver-specific genes. A 60-base pair E1.7 fragment carrying the AT-rich sequence stimulates a heterologous (alpha-fetoprotein) promoter in a dose-dependent manner. In E6, a HuH-7 nuclear protein binds to a GT-rich sequence, TGTTTGGC.A 27-base pair E6 fragment carrying this sequence is able to stimulate the SV40 promoter in an orientation-independent manner. An alteration of this sequence by site-specific mutagenesis resulted in the loss of transcriptional activity as well as binding to the HuH-7 nuclear protein. Competition gel mobility shift assays showed that homologous elements exist in the albumin promoter. These results show that the promoter and enhancer of the human albumin gene are regulated by two common transcription factors through two shared cis-acting elements, one AT-rich and the other GT-rich.

A large percentage of human hepatomas produce alpha-fetoprotein (AFP), but the levels of AFP expression vary greatly among hepatomas. To understand the molecular basis for this variation, we analyzed transcriptional regulatory activities associated with the 5'-flanking region of the AFP gene in two human hepatoma cell lines, HuH-7 and huH-1/cl-2, which produce a high and a low level of AFP, respectively. We found that the low level of AFP production in huH-1/cl-2 is due to the action of at least two silencer regions located between the enhancer and the promoter of the AFP gene. In contrast, no silencer activity is expressed in HuH-7. We identified 5'-CTTCATAACTAATACTT-3' to be a core sequence responsible for the negative regulatory activity. This sequence is repeated four times in a strong, distal silencer region, Sd, whereas one copy is present in a weak, proximal silencer region, Sp. The silencer reduces transcriptional initiation by blocking enhancer activation of the AFP promoter in a position-dependent manner. The silencer functions in the presence of positive transcription factors and may play a key role in developmental repression as well as variable expression of the AFP gene in hepatomas.

Partially inbred lines of laboratory opossums differ in plasma low-density lipoprotein cholesterol concentration and cholesterol absorption on a high-cholesterol diet. The aim of the present studies was to determine whether ezetimibe inhibits cholesterol absorption and eliminates the differences in plasma cholesterol and hepatic cholesterol metabolism between high and low responders on a high-cholesterol diet. Initially, we determined that the optimum dose of ezetimibe was 5 mg/(kg d) and treated 6 high- and 6 low-responding opossums with this dose (with equal numbers of controls) for 3 weeks while the opossums consumed a high-cholesterol and low-fat diet. Plasma and low-density lipoprotein cholesterol concentrations decreased significantly (P < .05) in treated but not in untreated high-responding opossums. Plasma cholesterol concentrations increased slightly (P < .05) in untreated low responders but not in treated low responders. The percentage of cholesterol absorption was significantly higher in untreated high responders than in other groups. Livers from high responders with or without treatment were significantly (P < .01) heavier than livers from low responders with or without treatment. Hepatic cholesterol concentrations in untreated high responders were significantly (P < .05) higher than those in low responders with or without treatment (P < .001). The gall bladder bile cholesterol concentrations in untreated high responders were significantly (P < .05) lower than those in other groups. A decrease in biliary cholesterol in low responders treated with ezetimibe was associated with a decrease in hepatic expression of ABCG5 and ABCG8. These studies suggest that ezetimibe decreases plasma cholesterol levels in high responders mainly by decreasing cholesterol absorption and increasing biliary cholesterol concentrations. Because ezetimibe's target is NPC1L1 and NPC1L1 is expressed in the intestine of opossums, its effect on cholesterol absorption may be mediated by inhibiting NPC1L1 function in the intestine.

Plasma very low-density lipoprotein and low-density lipoprotein (VLDL+LDL) cholesterol levels of 2 partially inbred strains of opossums (Monodelphis domestica) differ markedly when they are fed a high-cholesterol and low-fat (HCLF) diet. High-responding opossums exhibit a dramatic increase (>10-fold) in VLDL+LDL cholesterol, whereas low-responding opossums exhibit a minimal increase (

Two partially inbred strains of laboratory opossums exhibit extremely high or low levels of low-density lipoprotein (LDL) cholesterol concentrations, respectively, when challenged with a high-cholesterol and high-fat (HCHF) diet. The present studies were conducted to determine whether the catabolism or the production of LDL apolipoprotein B (apoB) is responsible for the variability in plasma LDL cholesterol and apoB concentrations. Iodinated LDL prepared from plasma of donor opossums consuming HCHF diet was injected into high- and low-responding recipients maintained on the HCHF diet. Blood was drawn at intervals beginning at 3 minutes and ending at 24 hours. At the end of the study, animals were necropsied, and livers were removed for isolation of RNA. Plasma LDL apoB was separated by sodium dodecyl sulfate–electrophoresis, and the level of radioactivity was determined. Hepatic LDL receptor and apoB mRNA levels were measured by Northern blotting. Radioactivity decay curves were plotted by using the radioactivity at each time point as percentage of the radioactivity recovered at 3 minutes. Fractional catabolic rates (FCRs) were calculated by the curve peeling technique. Steady-state production rates were calculated by multiplying the FCR values with apoB concentrations. LDL apoB FCR was slightly higher (1.63-fold) in low responders than in high responders. On the other hand, LDL apoB production was much higher (5.5-fold) in high responders than in low responders. There was no difference in hepatic mRNA levels for either the LDL receptor or apoB. The differences in LDL apoB FCR may be explained on the basis of differences in pool size between the 2 strains. Therefore, LDL apoB production is the major determinant of diet-induced hyperlipidemia in laboratory opossums. Because LDL apoB production was not associated with hepatic mRNA levels, the production of LDL apoB is regulated posttranscriptionally or posttranslationally.

Introduction: Hospital-acquired Clostridium difficile infection remains a leading source of morbidity and mortality among hospitalized patients. The purpose of this study was to explore antibiotics use as a risk factor for a cluster of C. difficile cases in trauma-surgical intensive care unit (TSICU

Age-dependent loss of androgen sensitivity of the rat liver is associated with a marked increase in dehydroepiandrosterone/hydroxysteroid sulfotransferase (rStd) activity. Sulfonated steroid hormones are known to be ineffective in binding receptor proteins. These observations suggest that intracellular androgen sulfonation can physiologically influence androgen action. We have examined the inhibitory effect of rStd on androgen action in the human prostate cancer-derived PC-3 cells transfected with the rat androgen receptor (AR) expression plasmid and two androgen-responsive promoter reporter constructs (murine mammary tumor long-terminal repeat ligated to chloramphenicol acetyltransferase (CAT) gene and rat probasin androgen response element (ARE) ligated to firefly luciferase (LUC) gene). These transfected cells were dependent on 5alpha-dihydrotestosterone (DHT) for the activation of both reporter genes and showed about a 200- and a 800-fold increase of CAT and LUC activity, respectively, at 10(-10) M DHT over the no-hormone control. Expression of the sulfonating enzyme in this cell transfection system via the rStd expression plasmid caused a dose-dependent decline in the reporter activity with approximately 90% inhibition of androgen action at a rStd:AR plasmid ratio of 100. From these results we conclude that irrespective of a high level of AR, changes in the Std expression can markedly alter the androgen sensitivity of target cells.