Bisphenol A Impairs Hepatic Glucose Sensing in C57BL/6 Male Mice

Aims/Hypothesis

Glucose sensing (eg. glucokinase activity) becomes impaired in the development of type 2 diabetes, the etiology of which is unclear. Estrogen can stimulate glucokinase activity, whereas the pervasive environmental pollutant bisphenol A (BPA) can inhibit estrogen action, hence we aimed to determine the effect of BPA on glucokinase activity directly.

Methods

To evaluate a potential acute effect on hepatic glucokinase activity, BPA in water (n = 5) vs. water alone (n = 5) was administered at the EPA’s purported “safe dose” (50 µg/kg) by gavage to lean 6-month old male C57BL/6 mice. Two hours later, animals were euthanized and hepatic glucokinase activity measured over glucose levels from 1–20 mmol/l in liver homogenate. To determine the effect of chronic BPA exposure on hepatic glucokinase activity, lean 6-month old male C57BL/6 mice were provided with water (n = 15) or water with 1.75 mM BPA (∼50 µg/kg/day; n = 14) for 2 weeks. Following the 2-week exposure, animals were euthanized and glucokinase activity measured as above.

Results

Hepatic glucokinase activity was signficantly suppressed after 2 hours in animals given an oral BPA bolus compared to those who received only water (p = 0.002–0.029 at glucose 5–20 mmol/l; overall treatment effect p<0.001). Exposure to BPA over 2 weeks also suppressed hepatic glucokinase activity in exposed vs. unexposed mice (overall treatment effect, p = 0.003). In both experiments, the Hill coefficient was higher and Vmax lower in mice treated with BPA.

Conclusions/Interpretation

Both acute and chronic exposure to BPA significantly impair hepatic glucokinase activity and function. These findings identify a potential mechanism for how BPA may increase risk for diabetes.     

Cell-Type-Specific Activation of the Oligoadenylate Synthetase–RNase L Pathway by a Murine Coronavirus

Previous studies have demonstrated that the murine coronavirus mouse hepatitis virus (MHV) nonstructural protein 2 (ns2) is a 2′,5′-phosphodiesterase that inhibits activation of the interferon-induced oligoadenylate synthetase (OAS)-RNase L pathway. Enzymatically active ns2 is required for efficient MHV replication in macrophages, as well as for the induction of hepatitis in C57BL/6 mice. In contrast, following intranasal or intracranial inoculation, efficient replication of MHV in the brain is not dependent on an enzymatically active ns2. The replication of wild-type MHV strain A59 (A59) and a mutant with an inactive phosphodiesterase (ns2-H126R) was assessed in primary hepatocytes and primary central nervous system (CNS) cell types—neurons, astrocytes, and oligodendrocytes. A59 and ns2-H126R replicated with similar kinetics in all cell types tested, except macrophages and microglia. RNase L activity, as assessed by rRNA cleavage, was induced by ns2-H126R, but not by A59, and only in macrophages and microglia. Activation of RNase L correlated with the induction of type I interferon and the consequent high levels of OAS mRNA induced in these cell types. Pretreatment of nonmyeloid cells with interferon restricted A59 and ns2-H126R to the same extent and failed to activate RNase L following infection, despite induction of OAS expression. However, rRNA degradation was induced by treatment of astrocytes or oligodendrocytes with poly(I·C). Thus, RNase L activation during MHV infection is cell type specific and correlates with relatively high levels of expression of OAS genes, which are necessary but not sufficient for induction of an effective RNase L antiviral response.     

Mass spectrometry images acylcarnitines,phosphatidylcholines, and sphingomyelin in MDA-MB-231 breast tumor models

The lipid compositions of different breast tumor microenvironments are largely unknown due to limitations in lipid imaging techniques. Imaging lipid distributions would enhance our understanding of processes occurring inside growing tumors, such as cancer cell proliferation, invasion, and metastasis. Recent developments in MALDI mass spectrometry imaging (MSI) enable rapid and specific detection of lipids directly from thin tissue sections. In this study, we performed multimodal imaging of acylcarnitines, phosphatidylcholines (PC), a lysophosphatidylcholine (LPC), and a sphingomyelin (SM) from different microenvironments of breast tumor xenograft models, which carried tdTomato red fluorescent protein as a hypoxia-response element-driven reporter gene. The MSI molecular lipid images revealed spatially heterogeneous lipid distributions within tumor tissue. Four of the most-abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1), and PC(18:0/18:1), were localized in viable tumor regions, whereas LPC(16:0/0:0) was detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoylcarnitine, stearoylcarnitine, PC(16:0/22:1), and SM(d18:1/16:0) sodium adduct, which colocalized primarily with hypoxic tumor regions. For the first time, we have applied a multimodal imaging approach that has combined optical imaging and MALDI-MSI with ion mobility separation to spatially localize and structurally identify acylcarnitines and a variety of lipid species present in breast tumor xenograft models.     

Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.     

Fecal Shedding of Zoonotic Food-Borne Pathogens by Wild Rodents in a Major Agricultural Region of the Central California Coast

Recent outbreaks of food-borne illness associated with the consumption of produce have increased concern over wildlife reservoirs of food-borne pathogens. Wild rodents are ubiquitous, and those living close to agricultural farms may pose a food safety risk should they shed zoonotic microorganisms in their feces near or on agricultural commodities. Fecal samples from wild rodents trapped on 13 agricultural farms (9 produce, 3 cow-calf operations, and 1 beef cattle feedlot) in Monterey and San Benito Counties, CA, were screened to determine the prevalence and risk factors for shedding of several food-borne pathogens. Deer mice (Peromyscus maniculatus) were the most abundant rodent species trapped (72.5%). Cryptosporidium species (26.0%) and Giardia species (24.2%) were the predominant isolates from rodent feces, followed by Salmonella enterica serovars (2.9%) and Escherichia coli O157:H7 (0.2%). Rodent trap success was significantly associated with detection of Salmonella in rodent feces, while farm type was associated with fecal shedding of Cryptosporidium and Giardia. Seasonal shedding patterns were evident, with rodents trapped during the spring and summer months being significantly less likely to be shedding Cryptosporidium oocysts than those trapped during autumn. Higher rodent species diversity tended to correlate with lower fecal microbial prevalence, and most spatiotemporal pathogen clusters involved deer mice. Rodents in the study area posed a minimal risk as environmental reservoirs of E. coli O157:H7, but they may play a role in environmental dissemination of Salmonella and protozoa. Rodent control efforts that potentially reduce biodiversity may increase pathogen shedding, possibly through promotion of intraspecific microbial transmission.     

Hibernation patterns of Turkish hamsters: influence of sex and ambient temperature

Turkish hamsters (Mesocricetus brandti) are a model organism for studies of hibernation, yet a detailed account of their torpor characteristics has not been undertaken. This study employed continuous telemetric monitoring of body temperature (T _b) in hibernating male and female Turkish hamsters at ambient temperatures (T _as) of 5 and 13 °C to precisely characterize torpor bout depth, duration, and frequency, as well as rates of entry into and arousal from torpor. Hamsters generated brief intervals of short (<12 h), shallow test bouts (T _b > 20 °C), followed by deep torpor bouts lasting 4–6 days at T _a = 5 °C and 2–3 days at T _a = 13 °C. Females at T _a = 5 °C had longer bouts than males, but maintained higher torpor T _b; there were no sex differences at T _a = 13 °C. Neither body mass loss nor food intake differed between the two T _as. Hamsters entered torpor primarily during the scotophase (subjective night), but timing of arousals was highly variable. Hamsters at both T _as generated short, shallow torpor bouts between deep bouts, suggesting that this species may be capable of both hibernation and daily torpor.     

Biosynthesis of ethylene glycol in Escherichia coli

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L^?1 of EG from 40.0 g?L^?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.     

Design and synthesis of tricyclic cores for kinase inhibition

Interest in therapeutic kinase inhibitors continues to grow beyond success in oncology. To date, ATP-mimetic kinase inhibitors have focused primarily on monocyclic and bicyclic heterocyclic cores. We sought to expand on the repertoire of potential cores for kinase inhibition by exploring tricyclic variants of classical bicyclic hinge binding motifs such as pyrrolopyridine and pyrrolopyrazine. Herein we describe the syntheses of eight alternative tricyclic cores as well as in vitro screening results for representative kinases of potential therapeutic interest.     

Anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors with sub-micromolar potency in the cell-based replicon assay

Optimization efforts on the anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors 1 and 2 resulted in the identification of multiple structural elements that contributed to improved cell culture potency. The additive effect of these elements resulted in compound 46, an inhibitor with enzymatic (IC₅₀) and cell culture (EC₅₀) potencies of less than 100 nanomolar.