Resting and exercise ventilatory chemosensitivity across the menstrual cycle

We hypothesized that resting and exercise ventilatory chemosensitivity would be augmented in women when estrogen and progesterone levels are highest during the luteal phase of the menstrual cycle. Healthy, young females (n = 10; age = 23 ± 5 yrs) were assessed across one complete cycle: during early follicular (EF), late follicular (LF), early luteal, and mid-luteal (ML) phases. We measured urinary conjugates of estrogen and progesterone daily. To compare values of ventilatory chemosensitivity and day-to-day variability of measures between sexes, males (n = 10; age = 26 ± 7 yrs) were assessed on 5 nonconsecutive days during a 1-mo period. Resting ventilation was measured and hypoxic chemosensitivity assessed using an isocapnic hypoxic ventilatory response (iHVR) test. The hypercapnic ventilatory response was assessed using the Read rebreathing protocol and modified rebreathing tests. Participants completed submaximal cycle exercise in normoxia and hypoxia. We observed a significant effect of menstrual-cycle phase on resting minute ventilation, which was elevated in the ML phase relative to the EF and LF phases. Compared with males, resting end-tidal CO(2) was reduced in females during the EF and ML phases but not in the LF phase. We found that iHVR was unaffected by menstrual-cycle phase and was not different between males and females. The sensitivity to chemical stimuli was unaffected by menstrual-cycle phase, meaning that any hormone-mediated effect is of insufficient magnitude to exceed the inherent variation in these chemosensitivity measures. The ventilatory recruitment threshold for CO(2) was generally lower in women, which is suggestive of a hormonally related lowering of the ventilatory recruitment threshold. We detected no effect of menstrual-cycle phase on submaximal exercise ventilation and found that the ventilatory response to normoxic and hypoxic exercise was quantitatively similar between males and females. This suggests that feed-forward and feed-back influences during exercise over-ride the effects of naturally occurring changes in sex hormones.     

Genomic patterns in Acropora cervicornis show extensive population structure and variable genetic diversity

Threatened Caribbean coral communities can benefit from high‐resolution genetic data used to inform management and conservation action. We use Genotyping by Sequencing (GBS) to investigate genetic patterns in the threatened coral, Acropora cervicornis, across the Florida Reef Tract (FRT) and the western Caribbean. Results show extensive population structure at regional scales and resolve previously unknown structure within the FRT. Different regions also exhibit up to threefold differences in genetic diversity (He), suggesting targeted management based on the goals and resources of each population is needed. Patterns of genetic diversity have a strong spatial component, and our results show Broward and the Lower Keys are among the most diverse populations in Florida. The genetic diversity of Caribbean staghorn coral is concentrated within populations and within individual reefs (AMOVA), highlighting the complex mosaic of population structure. This variance structure is similar over regional and local scales, which suggests that in situ nurseries are adequately capturing natural patterns of diversity, representing a resource that can replicate the average diversity of wild assemblages, serving to increase intraspecific diversity and potentially leading to improved biodiversity and ecosystem function. Results presented here can be translated into specific goals for the recovery of A. cervicornis, including active focus on low diversity areas, protection of high diversity and connectivity, and practical thresholds for responsible restoration.     

Nature of the molecular heterogeneity of human leukocyte interferon.

The existence of two components of human leukocyte interferon has been recently reported. In the present study, the nature of this molecular heterogeneity was explored by affinity chromatography on immobilized micro- and macroligands, ion-exchange chromatography, and molecular sieving. Chromatography on a series of alkyl-agarose adsorbents shows, for the first time, the intrinsic hydrophobicity of human leukocyte interferon. Additionally, the separation of two interferon components is achieved by use of the alkyl-agarose as well as by the omega-aminoalkyl-agarose adsorbents. Clear-cut separation of the two components was also achieved by chromatography on BSA-CH-Sepharose and on DEAE-Bio Gel A. An important feature of these separations is that they do not require the use of denaturing conditions. The molecular weights of the leukocyte interferon components, as determined on Sephadex G-75, are quite similar or identical, approximately 26,000. Thus, the molecular heterogeneity of human leukocyte interferon can be attributed, at least in part, to differences in the hydrophobicity and ionic properties of its two components.     

TGFbeta down-regulation of the CFTR: a means to limit epithelial chloride secretion

Transforming growth factor beta (TGFbeta) is a multifunctional cytokine with effects on many cell types. We recently showed that in addition to epithelial barrier enhancing properties, TGFbeta causes diminished cAMP-driven chloride secretion in colonic epithelia, in a manner that is p38 MAPK-dependent. In this study, we sought to further delineate the mechanism behind TGFbeta diminution of chloride secretion. Using colonic and kidney epithelial cell lines, we found that exposure to TGFbeta causes dramatic changes in the expression and localization of the apical membrane chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR). In TGFbeta-treated colonic epithelia (T84 and HT-29), CFTR mRNA was significantly reduced 2-24 h post-cytokine exposure. At a time consistent with decreased colonic epithelial secretory responses (16 h), TGFbeta treatment caused diminished intracellular CFTR protein expression (confocal microscopy) and reduced channel expression in the apical membrane during stimulated chloride secretion (biotinylation assay). In comparison, polarized kidney epithelia (MDCK) treated with TGFbeta displayed similarly reduced secretory responses to cAMP stimulating agents; however, a perinuclear accumulation of CFTR was observed, contrasting the diffuse cytoplasmic CFTR expression of control cells. Our data indicate that TGFbeta has profound effects on the expression and subcellular localization of an important channel involved in cAMP-driven chloride secretion, and thus suggest TGFbeta represents a key regulator of fluid movement.     

Fluorescence studies of normal and sickle beta apohemoglobin self-association

The acrylamide quenching of the intrinsic tryptophanyl fluorescence of normal and sickle apohemoglobins has been studied in 0.05 M potassium phosphate buffer,pH 7.5, at 5°C over a protein concentration range from 1 to 50M. Analysis of quenching dynamics revealed a strong dependence on acrylamide concentration for the intrinsic fluorescence of both normal and sickle apohemoglobins, suggesting that one tryptophanyl residue [presumably that at position 37(C3)], was more accessible to collisional quencher than the other tryptophanyl residue [15(A12)]. Additional studies, which altered viscosity and subunit assembly experimental parameters, supported the assignment of residue 37 as the more dynamically accessible residue. Finally, the quenching data were also found to be dependent on protein concentration, implying that this difference in the mobility between the two residues is a sensitive probe of self-aggregation. Extrapolated dynamic quenching constants at low concentration of acrylamide were used to estimate the dimer-monomer equilibrium dissociation constants of normal and sickle apohemoglobins, and were found to be 5.6 and 2.4M, respectively, thus demonstrating distinct self-association properties of _A and _S apohemoglobins.     

Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design

An optimal HIV vaccine should induce broadly neutralizing antibodies (bnAbs) that neutralize diverse viral strains and subtypes. However, potent bnAbs develop in only a small fraction of HIV-infected individuals, all contain rare features such as extensive mutation, insertions, deletions, and/or long complementarity-determining regions, and some are polyreactive, casting doubt on whether bnAbs to HIV can be reliably induced by vaccination. We engineered two potent VRC01-class bnAbs that minimized rare features. According to a quantitative features frequency analysis, the set of features for one of these minimally mutated bnAbs compared favorably with all 68 HIV bnAbs analyzed and was similar to antibodies elicited by common vaccines. This same minimally mutated bnAb lacked polyreactivity in four different assays. We then divided the minimal mutations into spatial clusters and dissected the epitope components interacting with those clusters, by mutational and crystallographic analyses coupled with neutralization assays. Finally, by synthesizing available data, we developed a working-concept boosting strategy to select the mutation clusters in a logical order following a germline-targeting prime. We have thus developed potent HIV bnAbs that may be more tractable vaccine goals compared to existing bnAbs, and we have proposed a strategy to elicit them. This reductionist approach to vaccine design, guided by antibody and antigen structure, could be applied to design candidate vaccines for other HIV bnAbs or protective Abs against other pathogens.