Ethanol and Reactive Species Increase Basal Sequence Heterogeneity of Hepatitis C Virus and Produce Variants with Reduced Susceptibility to Antivirals

Hepatitis C virus (HCV) exhibits a high level of genetic variability, and variants with reduced susceptibility to antivirals can occur even before treatment begins. In addition, alcohol decreases efficacy of antiviral therapy and increases sequence heterogeneity of HCV RNA but how ethanol affects HCV sequence is unknown. Ethanol metabolism and HCV infection increase the level of reactive species that can alter cell metabolism, modify signaling, and potentially act as mutagen to the viral RNA. Therefore, we investigated whether ethanol and reactive species affected the basal sequence variability of HCV RNA in hepatocytes. Human hepatoma cells supporting a continuous replication of genotype 1b HCV RNA (Con1, AJ242652) were exposed to ethanol, acetaldehyde, hydrogen peroxide, or L-buthionine-S,R-sulfoximine (BSO) that decreases intracellular glutathione as seen in patients. Then, NS5A region was sequenced and compared with genotype 1b HCV sequences in the database. Ethanol and BSO elevated nucleotide and amino acid substitution rates of HCV RNA by 4–18 folds within 48 hrs which were accompanied by oxidative RNA damage. Iron chelator and glutathione ester decreased both RNA damage and mutation rates. Furthermore, infectious HCV and HCV core gene were sufficient to induce oxidative RNA damage even in the absence of ethanol or BSO. Interestingly, the dn/ds ratio and percentage of sites undergoing positive selection increased with ethanol and BSO, resulting in an increased detection of NS5A variants with reduced susceptibility to interferon alpha, cyclosporine, and ribavirin and others implicated in immune tolerance and modulation of viral replication. Therefore, alcohol is likely to synergize with virus-induced oxidative/nitrosative stress to modulate the basal mutation rate of HCV. Positive selection induced by alcohol and reactive species may contribute to antiviral resistance

Prevention of Catheter-Related Blood Stream Infection: Back to Basics?

Background: Central venous catheter (CVC)-related infections are a substantial problem in the intensive care unit (ICU). Our infection control team initiated the routine use of antiseptic-coated (chlorhexidine-silver sulfadiazine; Chx-SS) CVCs in our adult ICUs to reduce catheter-associated (CA) and catheter-related (CR) blood stream infection (BSI) as we implemented other educational and best practice standardization strategies. Prior randomized studies documented that the use of Chx-SS catheters reduces microbial colonization of the catheter compared with an uncoated standard (Std) CVC but does not reduce CR-BSI. We therefore implemented the routine use of uncoated Std CVCs in our surgical ICU (SICU) and examined the impact of this change. Hypothesis: The use of uncoated Std CVCs does not increase CR-BSI rate in an SICU. Methods: Prospective evaluation of universal use of uncoated Std CVCs, implemented November 2007 in the SICU. The incidences of CA-BSI and CR-BSI were compared during November 2006-October 2007 (universal use of Chx-SS CVCs) and November 2007-October 2008 (universal use of Std CVCs) by t-test. The definitions of the U.S. Centers for Disease Control and Prevention were used for CA-BSI and CR-BSI. Patient data were collected via a dedicated Acute Physiology and Chronic Health Evaluation (APACHE) III coordinator for the SICU. Results: Annual use of CVCs increased significantly in the last six years, from 3,543 (2001) to 5,799 (2006) total days. The APACHE III scores on day 1 increased from a mean of 54.4 in 2004 to 55.6 in 2008 (p = 0.0010; 95% confidence interval [CI] 1.29-5.13). The mean age of the patients was unchanged over this period, ranging from 58.2 to 59.6 years. The Chx-SS catheters were implemented in the SICU in 2002. Data regarding the specific incidence of CR-BSI were collected beginning at the end of 2005, with mandatory catheter tip cultures when CVCs were removed. Little difference was identified in the incidence of BSI between the interval with universal Chx-SS use and that with Std CVC use. (Total BSI 0.7 vs. 0.8 per 1,000 catheter days; CA-BSI 0.5 vs. 0.8 per 1,000 catheter days; CR-BSI 0.2 vs. 0 per 1,000 catheter days.) No difference was seen in the causative pathogens of CA-BSI or CR-BSI. Conclusion: Eliminating the universal use of Chx-SS-coated CVCs in an SICU with a low background incidence of CR-BSIs did not result in an increase in the rate of CR-BSIs. This study documents the greater importance of adherence to standardization of the processes of care related to CVC placement than of coated CVC use in the reduction of CR-BSI.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90456/1/sur-2E2009-2E082.pd

Pan traps and bee body size in unmanaged urban habitats

Pan traps are among the most popular methods employed to survey bees and changes in some functional traits, such as body size, are increasingly used to understand how bee communities and species respond to landscape changes. Herein we assess body size differences between bees captured at ground-level and elevated (70 cm) pan traps in unmanaged urban habitats in northwestern Turkey. We compare body size at the community level as well as for the sweat bee Lasioglossum malachurum (Kirby) (Halictidae: Halictini), the most abundant species. We also compare the diversity, richness and abundance of bees sampled at both heights. A total of 31 species (13 genera of three families) were captured. We did not find significant differences in the abundance nor in the species richness between heights, and Simpson's indices were similar. At the community level, average intertegular distance was significantly greater in bees collected at the elevated traps than on the ground. Intertegular distances in L. malachurum did not differ between elevated and ground-level pan traps. Our results show an effect of pan trap height on bee body size in the urban habitat surveyed, thus suggesting that assessing bee body size from samples collected with either ground-level or elevated pan traps alone might result in biased estimates of this functional trait.National Science Foundation's REU program - DBI 126332

Tetra-methoxystilbene modulates ductal growth of the developing murine mammary gland

Extensive data suggest that estradiol contributes to the development of breast cancer by acting as a mitogen and exerting direct genotoxic effects after enzymatic conversion to 4-hydroxyestradiol (4-OHE2) via cytochrome P450 1B1 (CYP1B1). The mammary gland, ovary, and uterus all express CYP1B1. Overexpression of this enzyme has been associated with an increased risk of breast cancer and blockade might reduce this carcinogenic effect. For this reason, we conducted systematic in vitro and in vivo studies of a CYP1B1 inhibitor, TMS (2,3',4,5'-tetramethoxystilbene). We found that TMS blocked the enzymatic conversion of radiolabeled estradiol to both 2-hydroxyestradiol (2-OHE2) and 4-OHE2, but did not inhibit Cyp1b1 message formation. In vivo studies using mass spectrometry showed that TMS inhibited formation of 2-OHE2 and 4-OHE2 and the resulting estrogen-DNA adducts. To examine its biologic actions in vivo, we investigated whether TMS could block the hyperplastic changes that occur in the developing breast of aromatase-transfected mice. We found that TMS induced a significant reduction of ductal structures in mice less than 6 months in age. In older mice, no reduction in breast morphology occurred. These latter studies uncovered unexpected estrogen agonistic actions of TMS at high doses, including a paradoxical stimulation of breast ductal structures and the endometrium. These studies suggest that the enzyme inhibitory properties of TMS, as well as the effects on developing breast, could implicate a role for TMS in breast cancer prevention, but only in low doses and on developing breast

Soliton pair dynamics in patterned ferromagnetic ellipses

Confinement alters the energy landscape of nanoscale magnets, leading to the appearance of unusual magnetic states, such as vortices, for example. Many basic questions concerning dynamical and interaction effects remain unanswered, and nanomagnets are convenient model systems for studying these fundamental physical phenomena. A single vortex in restricted geometry, also known as a non-localized soliton, possesses a characteristic translational excitation mode that corresponds to spiral-like motion of the vortex core around its equilibrium position. Here, we investigate, by a microwave reflection technique, the dynamics of magnetic soliton pairs confined in lithographically defined, ferromagnetic Permalloy ellipses. Through a comparison with micromagnetic simulations, the observed strong resonances in the subgigahertz frequency range can be assigned to the translational modes of vortex pairs with parallel or antiparallel core polarizations. Vortex polarizations play a negligible role in the static interaction between two vortices, but their effect dominates the dynamics.Comment: supplemental movies on http://www.nature.com/nphys/journal/v1/n3/suppinfo/nphys173_S1.htm

Dual Chromatin and Cytoskeletal Remodeling by SETD2

Posttranslational modifications (PTMs) of tubulin specify microtubules for specialized cellular functions and comprise what is termed a "tubulin code." PTMs of histones comprise an analogous "histone code," although the "readers, writers, and erasers" of the cytoskeleton and epigenome have heretofore been distinct. We show that methylation is a PTM of dynamic microtubules and that the histone methyltransferase SET-domain-containing 2 (SETD2), which is responsible for H3 lysine 36 trimethylation (H3K36me3) of histones, also methylates α-tubulin at lysine 40, the same lysine that is marked by acetylation on microtubules. Methylation of microtubules occurs during mitosis and cytokinesis and can be ablated by SETD2 deletion, which causes mitotic spindle and cytokinesis defects, micronuclei, and polyploidy. These data now identify SETD2 as a dual-function methyltransferase for both chromatin and the cytoskeleton and show a requirement for methylation in maintenance of genomic stability and the integrity of both the tubulin and histone codes

Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator

A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. In this report, we show that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of Mad2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. We propose that Mtor/Tpr functions as a spatial regulator of the SAC, which ensures the efficient recruitment of Mad2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of Mad2 in a spindle matrix helps confine the action of a diffusible “wait anaphase” signal to the vicinity of the spindle

In Vitro and In Vivo Characterization of the Alkaloid Nuciferine

RationaleThe sacred lotus (Nelumbo nucifera) contains many phytochemicals and has a history of human use. To determine which compounds may be responsible for reported psychotropic effects, we used in silico predictions of the identified phytochemicals. Nuciferine, an alkaloid component of Nelumbo nucifera and Nymphaea caerulea, had a predicted molecular profile similar to antipsychotic compounds. Our study characterizes nuciferine using in vitro and in vivo pharmacological assays.MethodsNuciferine was first characterized in silico using the similarity ensemble approach, and was followed by further characterization and validation using the Psychoactive Drug Screening Program of the National Institute of Mental Health. Nuciferine was then tested in vivo in the head-twitch response, pre-pulse inhibition, hyperlocomotor activity, and drug discrimination paradigms.ResultsNuciferine shares a receptor profile similar to aripiprazole-like antipsychotic drugs. Nuciferine was an antagonist at 5-HT2A, 5-HT2C, and 5-HT2B, an inverse agonist at 5-HT7, a partial agonist at D2, D5 and 5-HT6, an agonist at 5-HT1A and D4 receptors, and inhibited the dopamine transporter. In rodent models relevant to antipsychotic drug action, nuciferine blocked head-twitch responses and discriminative stimulus effects of a 5-HT2A agonist, substituted for clozapine discriminative stimulus, enhanced amphetamine induced locomotor activity, inhibited phencyclidine (PCP)-induced locomotor activity, and rescued PCP-induced disruption of prepulse inhibition without induction of catalepsy.ConclusionsThe molecular profile of nuciferine was similar but not identical to that shared with several approved antipsychotic drugs suggesting that nuciferine has atypical antipsychotic-like actions

Gestational Diabetes Is Characterized by Reduced Mitochondrial Protein Expression and Altered Calcium Signaling Proteins in Skeletal Muscle

The rising prevalence of gestational diabetes mellitus (GDM) affects up to 18% of pregnant women with immediate and long-term metabolic consequences for both mother and infant. Abnormal glucose uptake and lipid oxidation are hallmark features of GDM prompting us to use an exploratory proteomics approach to investigate the cellular mechanisms underlying differences in skeletal muscle metabolism between obese pregnant women with GDM (OGDM) and obese pregnant women with normal glucose tolerance (ONGT). Functional validation was performed in a second cohort of obese OGDM and ONGT pregnant women. Quantitative proteomic analysis in rectus abdominus skeletal muscle tissue collected at delivery revealed reduced protein content of mitochondrial complex I (C-I) subunits (NDUFS3, NDUFV2) and altered content of proteins involved in calcium homeostasis/signaling (calcineurin A, α1-syntrophin, annexin A4) in OGDM (n = 6) vs. ONGT (n = 6). Follow-up analyses showed reduced enzymatic activity of mitochondrial complexes C-I, C-III, and C-IV (−60–75%) in the OGDM (n = 8) compared with ONGT (n = 10) subjects, though no differences were observed for mitochondrial complex protein content. Upstream regulators of mitochondrial biogenesis and oxidative phosphorylation were not different between groups. However, AMPK phosphorylation was dramatically reduced by 75% in the OGDM women. These data suggest that GDM is associated with reduced skeletal muscle oxidative phosphorylation and disordered calcium homeostasis. These relationships deserve further attention as they may represent novel risk factors for development of GDM and may have implications on the effectiveness of physical activity interventions on both treatment strategies for GDM and for prevention of type 2 diabetes postpartum