Life cycle contributions of copper from vessel painting and maintenance activities

Copper-based epoxy and ablative antifouling painted panels were exposed in natural seawater to evaluate environmental loading parameters. In situ loading factors including initial exposure, passive leaching, and surface refreshment were measured utilizing two protocols developed by the US Navy: the dome method and the in-water hull cleaning sampling method. Cleaning techniques investigated included a soft-pile carpet and a medium duty 3M^? pad for fouling removal. Results show that the passive leach rates of copper peaked three days after both initial deployment and cleaning events (CEs), followed by a rapid decrease over about 15 days and a slow approach to asymptotic levels on approximately day 30. Additionally, copper was more bioavailable during a CE in comparison to the passive leaching that immediately followed. A paint life cycle model quantifying annual copper loading estimates for each paint and cleaning method based on a three-year cycle of painting, episodic cleaning, and passive leaching is presented.     

Dynamics underlying hydroxylation selectivity of cytochrome P450cam

Structural heterogeneity and the dynamics of the complexes of enzymes with substrates can determine the selectivity of catalysis; however, fully characterizing how remains challenging as heterogeneity and dynamics can vary at the spatial level of an amino acid residue and involve rapid timescales. We demonstrate the nascent approach of site-specific two-dimensional infrared (IR) spectroscopy to investigate the archetypical cytochrome P450, P450cam, to better delineate the mechanism of the lower regioselectivity of hydroxylation of the substrate norcamphor in comparison to the native substrate camphor. Specific locations are targeted throughout the enzyme by selectively introducing cyano groups that have frequencies in a spectrally isolated region of the protein IR spectrum as local vibrational probes. Linear and two-dimensional IR spectroscopy were applied to measure the heterogeneity and dynamics at each probe and investigate how they differentiate camphor and norcamphor recognition. The IR data indicate that the norcamphor complex does not fully induce a large-scale conformational change to a closed state of the enzyme adopted in the camphor complex. Additionally, a probe directed at the bound substrate experiences rapidly interconverting states in the norcamphor complex that explain the hydroxylation product distribution. Altogether, the study reveals large- and small-scale structural heterogeneity and dynamics that could contribute to selectivity of a cytochrome P450 and illustrates the approach of site-selective IR spectroscopy to elucidate protein dynamics.     

Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

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Specific potassium ion interactions facilitate homocysteine binding to betaine‐homocysteine S‐methyltransferase

Betaine‐homocysteine S‐methyltransferase (BHMT) is a zinc‐dependent methyltransferase that uses betaine as the methyl donor for the remethylation of homocysteine to form methionine. This reaction supports S‐adenosylmethionine biosynthesis, which is required for hundreds of methylation reactions in humans. Herein we report that BHMT is activated by potassium ions with an apparent K_M for K⁺ of about 100 µM. The presence of potassium ions lowers the apparent K_M of the enzyme for homocysteine, but it does not affect the apparent K_M for betaine or the apparent k_cat for either substrate. We employed molecular dynamics (MD) simulations to theoretically predict and protein crystallography to experimentally localize the binding site(s) for potassium ion(s). Simulations predicted that K⁺ ion would interact with residues Asp26 and/or Glu159. Our crystal structure of BHMT bound to homocysteine confirms these sites of interaction and reveals further contacts between K⁺ ion and BHMT residues Gly27, Gln72, Gln247, and Gly298. The potassium binding residues in BHMT partially overlap with the previously identified DGG (Asp26‐Gly27‐Gly28) fingerprint in the Pfam 02574 group of methyltransferases. Subsequent biochemical characterization of several site‐specific BHMT mutants confirmed the results obtained by the MD simulations and crystallographic data. Together, the data herein indicate that the role of potassium ions in BHMT is structural and that potassium ion facilitates the specific binding of homocysteine to the active site of the enzyme. Proteins 2014; 82:2552–2564. © 2014 Wiley Periodicals, Inc.     

Continued Tumor Reduction of Metastatic Pheochromocytoma/Paraganglioma Harboring Succinate Dehydrogenase Subunit B Mutations with Cyclical Chemotherapy

Patients harboring germline mutations in the succinate dehydrogenase complex subunit B (SDHB) gene present with pheochromocytomas and paragangliomas (PPGL) that are more likely malignant and clinically aggressive. The combination chemotherapy cyclophosphamide, vincristine, and dacarbazine (CVD) was retrospectively evaluated in patients with SDHB-associated metastatic PPGL.Query Twelve metastatic PPGL patients harboring SDHB mutations/polymorphisms with undetectable SDHB immunostaining were treated with CVD. CVD therapy consisted of 750 mg/m² cyclophosphamide with 1.4 mg/m² vincristine on day 1 and 600 mg/m² dacarbazine on days 1 and 2, every 21–28 days. Treatment outcome was determined by RECIST criteria as well as determination of response duration and progression-free and overall survivals. A median of 20.5 cycles (range 4–41) was administered. All patients had tumor reduction (12–100% by RECIST). Complete response was seen in two patients, while partial response was observed in 8. The median number of cycles to response was 5.5. Median duration of response was 478 days, with progression-free and overall survivals of 930 and 1190 days, respectively. Serial [¹⁸F]-fluorodeoxyglucose positron emission tomography and computed tomography imaging demonstrated continued incremental reduction in maximal standardized uptake values (SUV_max) values in 26/30 lesions. During treatment administration, the median SUV decreased from?> 25 to?< 6, indicating the efficacy of chemotherapy over a prolonged period of time. Prolonged therapy results in continued incremental tumor reduction, and is consistent with persistent drug sensitivity. CVD chemotherapy is recommended to be considered part of the initial management in patients with metastatic SDHB-related PPGL.     

Rural Livelihood Variation and its Effects on Child Growth in Timor-Leste

Economic development introduces opportunities for subsistence households to diversify income sources. Timor-Leste is undergoing this transition; however, little is known about the patterns of household strategies and the effects of rural development on child wellbeing. We derive strategies from 190 households in two rural Timor-Leste communities and examine the links between resource strategies and child growth using linear mixed modeling. Children’s z-height, z-weight, and z-BMI are well below international standards. We find agriculture remains predominantly subsistence-based, with some reliance on cash flow from government pensions and salaries. Households with stable income sources are better able to accumulate wealth, and children living in salaried households have better z-height. However, child growth is best predicted by individual-level factors rather than household ecology. Substantial variation in household strategy and little association of strategy with growth indicates there is no ‘best’ strategy in this transitioning environment.     

Factors associated with a low prevalence of exclusive breastfeeding during hospital stay in urban and semi-rural areas of southern Vietnam

Background

There is a paucity of data regarding risk factors associated with suboptimal breastfeeding practices in urbanized areas of low-middle income countries (LMICs).

Methods

Through a large prospective birth cohort, which enrolled 6706 infants in Vietnam between 2009 and 2013, we investigated the practice of exclusive breastfeeding during hospital stay in urban and semi-rural populations and aimed to identify factors associated with suboptimal breastfeeding practices. Univariate and multivariable logistic regression were performed to determine factors associated with not exclusive breastfeeding during hospital stay.

Results

Of 6076 mothers, 33% (2187) breastfed their infant exclusively before hospital discharge; 9% (364/4248) in urban and 74% (1823/2458) in semi-rural areas. Exclusive breastfeeding up to 4 months was recorded in 15% (959/6210) of participants; this declined to <?1% (56/6093) at 6 months. Delivery by Caesarean section (Odds Ratio [OR] 0.07; 95% Confidence Interval [CI] 0.04, 0.11 and OR 0.05; 95% CI 0.03, 0.08) and neonatal complications (OR 0.2; 95% CI 0.07, 0.47 and OR 0.25; 95% CI 0.14, 0.46) were common and highly significant risk factors associated with a lack of exclusive breastfeeding during hospital stay in urban and semi-rural settings, respectively.

Conclusions

To our knowledge, this is the first large-scale investigation aimed at identifying factors associated with exclusive breastfeeding during hospital stay in Vietnam. Breastfeeding promotion strategies should prioritize common risk factors in hospital, such as Caesarean section and neonatal complications, and other location specific factors associated with socioeconomics.

     

The inference of phased haplotypes for the immunoglobulin H chain V region gene loci by analysis of VDJ gene rearrangements

The existence of many highly similar genes in the lymphocyte receptor gene loci makes them difficult to investigate, and the determination of phased "haplotypes" has been particularly problematic. However, V(D)J gene rearrangements provide an opportunity to infer the association of Ig genes along the chromosomes. The chromosomal distribution of H chain genes in an Ig genotype can be inferred through analysis of VDJ rearrangements in individuals who are heterozygous at points within the IGH locus. We analyzed VDJ rearrangements from 44 individuals for whom sufficient unique rearrangements were available to allow comprehensive genotyping. Nine individuals were identified who were heterozygous at the IGHJ6 locus and for whom sufficient suitable VDJ rearrangements were available to allow comprehensive haplotyping. Each of the 18 resulting IGHV│IGHD│IGHJ haplotypes was unique. Apparent deletion polymorphisms were seen that involved as many as four contiguous, functional IGHV genes. Two deletion polymorphisms involving multiple contiguous IGHD genes were also inferred. Three previously unidentified gene duplications were detected, where two sequences recognized as allelic variants of a single gene were both inferred to be on a single chromosome. Phased genomic data brings clarity to the study of the contribution of each gene to the available repertoire of rearranged VDJ genes. Analysis of rearrangement frequencies suggests that particular genes may have substantially different yet predictable propensities for rearrangement within different haplotypes. Together with data highlighting the extent of haplotypic variation within the population, this suggests that there may be substantial variability in the available Ab repertoires of different individuals.     

Identification of the Niemann-Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. With ～170 million individuals infected and current interferon-based treatment having toxic side effects and marginal efficacy, more effective antivirals are crucially needed. Although HCV protease inhibitors were just approved by the US Food and Drug Administration (FDA), optimal HCV therapy, analogous to HIV therapy, will probably require a combination of antivirals targeting multiple aspects of the viral lifecycle. Viral entry represents a potential multifaceted target for antiviral intervention; however, to date, FDA-approved inhibitors of HCV cell entry are unavailable. Here we show that the cellular Niemann-Pick C1-like 1 (NPC1L1) cholesterol uptake receptor is an HCV entry factor amendable to therapeutic intervention. Specifically, NPC1L1 expression is necessary for HCV infection, as silencing or antibody-mediated blocking of NPC1L1 impairs cell culture-derived HCV (HCVcc) infection initiation. In addition, the clinically available FDA-approved NPC1L1 antagonist ezetimibe potently blocks HCV uptake in vitro via a virion cholesterol-dependent step before virion-cell membrane fusion. Moreover, ezetimibe inhibits infection by all major HCV genotypes in vitro and in vivo delays the establishment of HCV genotype 1b infection in mice with human liver grafts. Thus, we have not only identified NPC1L1 as an HCV cell entry factor but also discovered a new antiviral target and potential therapeutic agent.     

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)_n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.