A designed four-alpha-helix bundle that binds the volatile general anesthetic halothane with high affinity

The structural features of volatile anesthetic binding sites on proteins are being examined with the use of a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. Previous work has suggested that introducing a cavity into the hydrophobic core improves anesthetic binding affinity. The more polarizable methionine side chain was substituted for a leucine, in an attempt to enhance the dispersion forces between the ligand and the protein. The resulting bundle variant has an improved affinity (K(d) = 0.20 +/- 0.01 mM) for halothane binding, compared with the leucine-containing bundle (K(d) = 0.69 +/- 0.06 mM). Photoaffinity labeling with (14)C-halothane reveals preferential labeling of the W15 residue in both peptides, supporting the view that fluorescence quenching by bound anesthetic reports both the binding energetics and the location of the ligand in the hydrophobic core. The rates of amide hydrogen exchange were similar for the two bundles, suggesting that differences in binding affinity were not due to changes in protein stability. Binding of halothane to both four-alpha-helix bundle proteins stabilized the native folded conformations. Molecular dynamics simulations of the bundles illustrate the existence of the hydrophobic core, containing both W15 residues. These results suggest that in addition to packing defects, enhanced dispersion forces may be important in providing higher affinity anesthetic binding sites. Alternatively, the effect of the methionine substitution on halothane binding energetics may reflect either improved access to the binding site or allosteric optimization of the dimensions of the binding pocket. Finally, preferential stabilization of folded protein conformations may represent a fundamental mechanism of inhaled anesthetic action.     

Circadian misalignment and weekend alcohol use in late adolescent drinkers: preliminary evidence

Alcohol use accelerates during late adolescence, predicting the development of alcohol use disorders (AUDs) and other negative outcomes. Identifying modifiable risk factors for alcohol use during this time could lead to novel prevention approaches. Burgeoning evidence suggests that sleep and circadian factors are cross-sectionally and longitudinally linked to alcohol use and problems, but more proximal relationships have been understudied. Circadian misalignment, in particular, is hypothesized to increase the risk for AUDs, but almost no published studies have included a biological measure of misalignment. In the present study, we aimed to extend existing research by assessing the relationship between adolescent circadian misalignment and alcohol use on a proximal timeframe (over two weeks) and by including three complementary measures of circadian alignment. We studied 36 healthy late (18–22 years old, 22 females) alcohol drinkers (reporting ≥1, standard drink per week over the past 30 days) over 14 days. Throughout the study, participants reported prior day’s alcohol use and prior night’s sleep each morning via smartphone and a secure, browser-based interface. Circadian phase was assessed via the dim light melatonin onset (DLMO) in the laboratory on two occasions (Thursday and Sunday nights) in counterbalanced order. The three measures of circadian alignment included DLMO-midsleep interval, “classic” social jet lag (weekday-weekend difference in midsleep), and “objective” social jet lag (weekday-weekend difference in DLMO). Multivariate imputation by chained equations was used to impute missing data, and Poisson regression models were used to assess associations between circadian alignment variables and weekend alcohol use. Covariates included sex, age, Thursday alcohol use, and Thursday sleep characteristics. As predicted, greater misalignment was associated with greater weekend alcohol use for two of the three alignment measures (shorter DLMO-midsleep intervals and larger weekday-weekend differences in midsleep), while larger weekday-weekend differences in DLMO were associated with less alcohol use. Notably, in contrast to expectations, the distribution of weekday-weekend differences in DLMO was nearly equally distributed between individuals advancing over the weekend and those delaying over the weekend. This unexpected finding plausibly reflects the fact that college students are not subject to the same systematically earlier weekday schedules observed in high school students and working adults. These preliminary findings support the need for larger, more definitive studies investigating the proximal relationships between circadian alignment and alcohol use among late adolescents.     

Correlation of cellulase gene expression and cellulolytic activity throughout the gut of the termite Reticulitermes flavipes

Termites have developed cellulose digestion capabilities that allow them to obtain energy and nutrition from nutritionally poor food sources, such as lignocellulosic plant material and residues derived from it (e.g., wood and humus). Lower termites, which are equipped with both endogenous (i.e., of termite origin) and symbiotic cellulases, feed primarily on wood and wood-related materials. This study investigated cellulase gene diversity, structure, and activity in the lower termite, Reticulitermes flavipes (Kollar). We initially used a metagenomics approach to identify four genes encoding one endogenous and three symbiotic cellulases, which we refer to as Cell-1, -2, -3 and -4. These four genes encode proteins that share significant sequence similarity with known endoglucanases, exoglucanases and xylanases. Phylogenetic analyses further supported these inferred relationships by showing that each of the four cellulase proteins clusters tightly with respective termite, protozoan or fungal cellulases. Gene structure studies revealed that Cell-1, -3 and -4 are intron-free, while Cell-2 contains the first intron sequence to be identified from a termite symbiont cellulase. Quantitative real-time PCR (qRT-PCR) revealed that the endogenous Cell-1 gene is expressed exclusively in the salivary gland/foregut, whereas symbiotic Cell-2, -3, and -4 are highly expressed in the hindgut (where cellulolytic protists are harbored). Cellulase activity assays mapped the distribution pattern of endoglucanase, exoglucanase and xylanase activity throughout the R. flavipes digestive tract. Cellulase gene expression correlated well with the specific types of cellulolytic activities observed in each gut region (foregut+salivary gland, midgut and hindgut). These results suggest the presence of a single unified cellulose digestion system, whereby endogenous and symbiotic cellulases work sequentially and collaboratively across the entire digestive tract of R. flavipes.     

A New Glycan-Dependent CD4-Binding Site Neutralizing Antibody Exerts Pressure on HIV-1 In Vivo

The CD4 binding site (CD4bs) on the envelope glycoprotein is a major site of vulnerability that is conserved among different HIV-1 isolates. Many broadly neutralizing antibodies (bNAbs) to the CD4bs belong to the VRC01 class, sharing highly restricted origins, recognition mechanisms and viral escape pathways. We sought to isolate new anti-CD4bs bNAbs with different origins and mechanisms of action. Using a gp120 2CC core as bait, we isolated antibodies encoded by IGVH3-21 and IGVL3-1 genes with long CDRH3s that depend on the presence of the N-linked glycan at position-276 for activity. This binding mode is similar to the previously identified antibody HJ16, however the new antibodies identified herein are more potent and broad. The most potent variant, 179NC75, had a geometric mean IC₈₀ value of 0.42 μg/ml against 120 Tier-2 HIV-1 pseudoviruses in the TZM.bl assay. Although this group of CD4bs glycan-dependent antibodies can be broadly and potently neutralizing in vitro, their in vivo activity has not been tested to date. Here, we report that 179NC75 is highly active when administered to HIV-1-infected humanized mice, where it selects for escape variants that lack a glycan site at position-276. The same glycan was absent from the virus isolated from the 179NC75 donor, implying that the antibody also exerts selection pressure in humans.     

Determining Microvascular Obstruction and Infarct Size with Steady-State Free Precession Imaging Cardiac MRI

Purpose

In cardiac MRI (cMRI) injection of contrast medium may be performed prior to the acquisition of cine steady-state free precession (SSFP) imaging to speed up the protocol and avoid delay before late Gadolinium enhancement (LGE) imaging. Aim of this study was to evaluate whether a condensed clinical protocol with contrast cine SSFP imaging is able to detect early microvascular obstruction (MO) and determine the infarct size compared to the findings of LGE inversion recovery sequences.

Materials and Methods

The study complies with the Declaration of Helsinki and was performed following approval by the ethic committee of the University of Erlangen-Nuremberg. Written informed consent was obtained from every patient. 68 consecutive patients (14 females/54 males) with acute ST-elevation myocardial infarction (STEMI) treated by percutaneous coronary revascularization were included in this study. CMRI was performed 6.6±2 days after symptom onset and MO and infarct size in early contrast SSFP cine imaging were compared to LGE imaging.

Results

MO was detected in 47/68 (69%) patients on cine SSFP and in 41/68 (60%) patients on LGE imaging. In 6 patients MO was found on cine SSFP imaging but was not detectable on LGE imaging. Infarct size on cine SSFP showed a strong agreement to LGE imaging (intraclass correlation coefficient [ICC] of 0.96 for enddiastolic, p<0.001 and 0.96 for endsystolic, p<0.001 respectively). Significant interobserver agreement was found measuring enddiastolic and endsystolic infarct size on cine SSFP imaging (p<0.01).

Conclusions

In patients after STEMI infarct size and presence of MO can be detected with contrast cine SSFP imaging. This could be an option in patients who are limited in their ability to comply with the demands of a cMRI protocol.     

Hexamerin-based regulation of juvenile hormone-dependent gene expression underlies phenotypic plasticity in a social insect

Worker termites of the genus Reticulitermes are temporally-arrested juvenile forms that can terminally differentiate into adultsoldier- or reproductive-caste phenotypes. Soldier-caste differentiation is a developmental transition that is induced by high juvenile hormone (JH) titers. Recently, a status quo hexamerin mechanism was identified, which reduces JH efficacy and maximizes colony fitness via the maintenance of high worker-caste proportions. Our goal in these studies was to investigate more thoroughly the influences of the hexamerins on JH-dependent gene expression in termite workers. Our approach involved RNA interference (RNAi), bioassays and quantification of gene expression. We first investigated the expression of 17 morphogenesis-associated genes in response to RNAi-based hexamerin silencing. Hexamerin silencing resulted in significant downstream impacts on 15 out of the 17 genes, suggesting that these genes are members of a JH-responsive genomic network. Next, we compared gene-expression profiles in workers after RNAi-based hexamerin silencing to that of (i) untreated workers that were held away from the colony; and (ii) workers that were also held away from the colony, but with ectopic JH. Here, although there was no correlation between hexamerin silencing and colony-release effects, we observed a significant correlation between hexamerin silencing and JH-treatment effects. These findings provide further evidence supporting the hypothesis that the hexamerins modulate JH availability, thus limiting the impacts of JH on termite caste polyphenism. Results are discussed in a context relative to outstanding questions on termite developmental biology, particularly on regulatory gene networks that respond to JH-, colony- and environmental-cues.