Self-assembling diblock copolymers of poly[N-(2-hydroxypropyl)methacrylamide] and a beta-sheet peptide

The self-assembly of hybrid diblock copolymers composed of poly(HPMA) and beta-sheet peptide P11 (CH(3)CO-QQRFQWQFEQQ-NH(2)) blocks was investigated. Copolymers were synthesized via thiol-maleimide coupling reaction, by conjugation of semitelechelic poly(HPMA)-SH with maleimide-modified beta-sheet peptide. As expected, CD and CR binding studies showed that the peptide block imposed its beta-sheet structural arrangement on the structure of diblock copolymers. TEM and AFM proved that peptide and these copolymers had the ability to self-assemble into fibrils.     

Gene structure,transcripts and calciotropic effects of the PTH family of peptides in <Emphasis Type="Italic">Xenopus</Emphasis> and chicken

Background  

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken.     

The reduction of water-soluble tetrazolium salt reagent on the plasma membrane of epidermal keratinocytes is oxygen dependent

The water-soluble tetrazolium salt (WST-1) assay is frequently used to assess cell proliferation. However, our study showed that in normal and cancerous keratinocytes, this assay is more responsive to changes in oxygenation than to rates of cell growth. Stimulation of keratinocyte proliferation by low Ca²⁺ and suppression of proliferation by nocodazole resulted in modest changes in WST-1 readings, whereas gradually reducing the level of oxygen in the cellular environment from ambient (21%) to near anoxic (0.1%) revealed a very strong negative correlation between cell oxygenation and WST-1 reagent reduction. In contrast, the very similar MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay, which uses a different tetrazolium salt, showed no sensitivity to the level of oxygen. Unlike MTT, WST-1 reagent is reduced extracellularly through trans-plasma membrane transport (tPMET), thereby suggesting that tPMET is oxygen dependent. We propose that the WST-1 assay can be developed into a sensitive quantitative method to evaluate cell oxygenation in vitro and used to study the role of hypoxia and tPMET in homeostasis and disease (e.g., cancer). At the same time, WST-1 assay should be used cautiously to assess cell viability or proliferation because readings can be affected by certain extrinsic (low atmospheric oxygen or high density culture) or intrinsic (defects in oxygen-sensing pathways) factors.     

Extent and scale of local adaptation in salmonid fishes: review and meta-analysis

What is the extent and scale of local adaptation (LA)? How quickly does LA arise? And what is its underlying molecular basis? Our review and meta-analysis on salmonid fishes estimates the frequency of LA to be ∼55–70%, with local populations having a 1.2 times average fitness advantage relative to foreign populations or to their performance in new environments. Salmonid LA is evident at a variety of spatial scales (for example, few km to>1000 km) and can manifest itself quickly (6–30 generations). As the geographic scale between populations increases, LA is generally more frequent and stronger. Yet the extent of LA in salmonids does not appear to differ from that in other assessed taxa. Moreover, the frequency with which foreign salmonid populations outperform local populations (∼23–35%) suggests that drift, gene flow and plasticity often limit or mediate LA. The relatively few studies based on candidate gene and genomewide analyses have identified footprints of selection at both small and large geographical scales, likely reflecting the specific functional properties of loci and the associated selection regimes (for example, local niche partitioning, pathogens, parasites, photoperiodicity and seasonal timing). The molecular basis of LA in salmonids is still largely unknown, but differential expression at the same few genes is implicated in the convergent evolution of certain phenotypes. Collectively, future research will benefit from an integration of classical and molecular approaches to understand: (i) species differences and how they originate, (ii) variation in adaptation across scales, life stages, population sizes and environmental gradients, and (iii) evolutionary responses to human activities.     

Reconstituted high-density lipoprotein infusion modulates fatty acid metabolism in patients with type 2 diabetes mellitus

We recently demonstrated that reconstituted high-density lipoprotein (rHDL) modulates glucose metabolism in humans via both AMP-activated protein kinase (AMPK) in muscle and by increasing plasma insulin. Given the key roles of both AMPK and insulin in fatty acid metabolism, the current study investigated the effect of rHDL infusion on fatty acid oxidation and lipolysis. Thirteen patients with type 2 diabetes received separate infusions of rHDL and placebo in a randomized, cross-over study. Fatty acid metabolism was assessed using steady-state tracer methodology, and plasma lipids were measured by mass spectrometry (lipidomics). In vitro studies were undertaken in 3T3-L1 adipocytes. rHDL infusion inhibited fasting-induced lipolysis (P = 0.03), fatty acid oxidation (P < 0.01), and circulating glycerol (P = 0.04). In vitro, HDL inhibited adipocyte lipolysis in part via activation of AMPK, providing a possible mechanistic link for the apparent reductions in lipolysis observed in vivo. In contrast, circulating NEFA increased after rHDL infusion (P < 0.01). Lipidomic analyses implicated phospholipase hydrolysis of rHDL-associated phosphatidylcholine as the cause, rather than lipolysis of endogenous fat stores. rHDL infusion inhibits fasting-induced lipolysis and oxidation in patients with type 2 diabetes, potentially through both AMPK activation in adipose tissue and elevation of plasma insulin. The phospholipid component of rHDL also has the potentially undesirable effect of increasing circulating NEFA.     

Passive immunotherapies protect WRvFire and IHD-J-Luc vaccinia virus-infected mice from lethality by reducing viral loads in the upper respiratory tract and internal organs

Whole-body bioimaging was employed to study the effects of passive immunotherapies on lethality and viral dissemination in BALB/c mice challenged with recombinant vaccinia viruses expressing luciferase. WRvFire and IHD-J-Luc vaccinia viruses induced lethality with similar times to death following intranasal infection, but WRvFire replicated at higher levels than IHD-J-Luc in the upper and lower respiratory tracts. Three types of therapies were tested: licensed human anti-vaccinia virus immunoglobulin intravenous (VIGIV); recombinant anti-vaccinia virus immunoglobulin (rVIG; Symphogen, Denmark), an investigational product containing a mixture of 26 human monoclonal antibodies (HuMAbs) against mature virion (MV) and enveloped virion (EV); and HuMAb compositions targeting subsets of MV or EV proteins. Bioluminescence recorded daily showed that pretreatment with VIGIV (30 mg) or with rVIG (100 μg) on day -2 protected mice from death but did not prevent viral replication at the site of inoculation and dissemination to internal organs. Compositions containing HuMAbs against MV or EV proteins were protective in both infection models at 100 μg per animal, but at 30 μg, only anti-EV antibodies conferred protection. Importantly, the t statistic of the mean total fluxes revealed that viral loads in surviving mice were significantly reduced in at least 3 sites for 3 consecutive days (days 3 to 5) postchallenge, while significant reduction for 1 or 2 days in any individual site did not confer protection. Our data suggest that reduction of viral replication at multiple sites, including respiratory tract, spleen, and liver, as monitored by whole-body bioluminescence can be used to predict the effectiveness of passive immunotherapies in mouse models.     

Environmental harshness,latitude and incipient speciation

Are rates of evolution and speciation fastest where diversity is greatest – the tropics? A commonly accepted theory links the latitudinal diversity gradient to a speciation pump model whereby the tropics produce species at a faster rate than extra‐tropical regions. In this issue of Molecular Ecology, Botero et al. ( 2014 ) test the speciation pump model using subspecies richness patterns for more than 9000 species of birds and mammals as a proxy for incipient speciation opportunity. Rather than using latitudinal centroids, the authors investigate the role of various environmental correlates of latitude as drivers of subspecies richness. Their key finding points to environmental harshness as a positive predictor of subspecies richness. The authors link high subspecies richness in environmental harsh areas to increased opportunities for geographic range fragmentation and/or faster rates of trait evolution as drivers of incipient speciation. Because environmental harshness generally increases with latitude, these results suggest that opportunity for incipient speciation is lowest where species richness is highest. The authors interpret this finding as incompatible with the view of the tropics as a cradle of diversity. Their results are consistent with a growing body of evidence that reproductive isolation and speciation occur fastest at high latitudes.     

Plasma lipidome is independently associated with variability in metabolic syndrome in Mexican American families

Plasma lipidome is now increasingly recognized as a potentially important marker of chronic diseases, but the exact extent of its contribution to the interindividual phenotypic variability in family studies is unknown. Here, we used the rich data from the ongoing San Antonio Family Heart Study (SAFHS) and developed a novel statistical approach to quantify the independent and additive value of the plasma lipidome in explaining metabolic syndrome (MS) variability in Mexican American families recruited in the SAFHS. Our analytical approach included two preprocessing steps: principal components analysis of the high-resolution plasma lipidomics data and construction of a subject-subject lipidomic similarity matrix. We then used the Sequential Oligogenic Linkage Analysis Routines software to model the complex family relationships, lipidomic similarities, and other important covariates in a variance components framework. Our results suggested that even after accounting for the shared genetic influences, indicators of lipemic status (total serum cholesterol, TGs, and HDL cholesterol), and obesity, the plasma lipidome independently explained 22% of variability in the homeostatic model of assessment-insulin resistance trait and 16% to 22% variability in glucose, insulin, and waist circumference. Our results demonstrate that plasma lipidomic studies can additively contribute to an understanding of the interindividual variability in MS.     

Simultaneous measurement of O2 radicals and pulmonary vascular reactivity in rat lung

The role of endogenous radicals in the regulation of pulmonary vascular tone was evaluated by simultaneous measurement of pulmonary artery pressure and lung radical levels during exposure of isolated rat lungs to varying inspired O2 concentrations (0-95%) and angiotensin II. Lung radical levels, measured "on-line" using luminol and lucigenin-enhanced chemiluminescence, decreased in proportion to the degree of alveolar hypoxia. Radical levels fell during hypoxia before the onset of pulmonary vasoconstriction and promptly returned to basal levels with restoration of normoxic ventilation. Mild alveolar hypoxia (10% O2), which failed to decrease chemiluminescence, did not trigger pulmonary vasoconstriction. Although chemiluminescence tended to decrease more as the hypoxic response strengthened, there was not a simple correlation between the magnitude of the change in chemiluminescence induced by hypoxia and the strength of the hypoxic pressor response. Normoxic chemiluminescence was largely inhibited by superoxide dismutase but not catalase. Superoxide dismutase also increased normoxic pulmonary vascular tone and the strength of the pressor response to hypoxia and angiotensin II. Thus the predominant activated O2 species in the lung, during normoxia, was the superoxide anion or a closely related substance. Alteration of endogenous radical levels can result in changes in vascular tone. It remains uncertain whether the decrease in lung radical production during hypoxia caused pulmonary vasoconstriction or was merely associated with hypoxic ventilation.     

Population Structure With Localized Haplotype Clusters

We propose a multilocus version of F_ST and a measure of haplotype diversity using localized haplotype clusters. Specifically, we use haplotype clusters identified with BEAGLE, which is a program implementing a hidden Markov model for localized haplotype clustering and performing several functions including inference of haplotype phase. We apply this methodology to HapMap phase 3 data. With this haplotype-cluster approach, African populations have highest diversity and lowest divergence from the ancestral population, East Asian populations have lowest diversity and highest divergence, and other populations (European, Indian, and Mexican) have intermediate levels of diversity and divergence. These relationships accord with expectation based on other studies and accepted models of human history. In contrast, the population-specific F_ST estimates obtained directly from single-nucleotide polymorphisms (SNPs) do not reflect such expected relationships. We show that ascertainment bias of SNPs has less impact on the proposed haplotype-cluster-based F_ST than on the SNP-based version, which provides a potential explanation for these results. Thus, these new measures of F_ST and haplotype-cluster diversity provide an important new tool for population genetic analysis of high-density SNP data.GENOME-WIDE data sets from worldwide panels of individuals provide an outstanding opportunity to investigate the genetic structure of human populations (Conrad et al. 2006; International Hapmap Consortium 2007; Jakobsson et al. 2008; Auton et al. 2009). Populations around the globe form a continuum rather than discrete units (Serre and Paabo 2004; Weiss and Long 2009). However, notions of discrete populations can be appropriate when, for example, ancestral populations were separated by geographic distance or barriers such that little gene flow occurred.F_ST (Wright 1951; Weir and Cockerham 1984; Holsinger and Weir 2009) is a measure of population divergence. It measures variation between populations vs. within populations. One can calculate a global measure, assuming that all populations are equally diverged from an ancestral population, or one can calculate F_ST for specific populations or for pairs of populations while utilizing data from all populations (Weir and Hill 2002). One use of F_ST is to test for signatures of selection (reviewed in Oleksyk et al. 2010).F_ST may be calculated for single genetic markers. For multiallelic markers, such as microsatellites, this is useful, but single-nucleotide polymorphisms (SNPs) contain much less information when taken one at a time, and thus it is advantageous to calculate averages over windows of markers (Weir et al. 2005) or even over the whole genome. The advantage of windowed F_ST is that it can be used to find regions of the genome that show different patterns of divergence, indicative of selective forces at work during human history.Another measure of human evolutionary history is haplotype diversity. Haplotype diversity may be measured using a count of the number of observed haplotypes in a region or by the expected haplotype heterozygosity based on haplotype frequencies in a region. Application of this regional measure to chromosomal data can be achieved by a haplotype block strategy (Patil et al. 2001) or by windowing (Conrad et al. 2006; Auton et al. 2009).One problem with the analysis of population structure based on genome-wide panels of SNPs is that a large proportion of the SNPs were ascertained in Caucasians, potentially biasing the results of the analyses. Analysis based on haplotypes is less susceptible to such bias (Conrad et al. 2006). This is because haplotypes can be represented by multiple patterns of SNPs; thus lack of ascertainment of a particular SNP does not usually prevent observation of the haplotype. On a chromosome-wide scale, one cannot directly use entire haplotypes, because all the haplotypes in the sample will almost certainly be unique, thus providing no information on population structure. Instead one can use haplotypes on a local basis, either by using windows of adjacent markers or by using localized haplotype clusters, for example those obtained from fastPHASE (Scheet and Stephens 2006) or BEAGLE (Browning 2006; Browning and Browning 2007a).Localized haplotype clusters are a clustering of haplotypes on a localized basis. At the position of each genetic marker, haplotypes are clustered according to their similarity in the vicinity of the position. Both fastPHASE and BEAGLE use hidden Markov modeling to perform the clustering, although the specific models used by the two programs differ.Localized haplotype clusters derived from fastPHASE have been used to investigate haplotype diversity, to create neighbor-joining trees of populations, and to create multidimensional scaling (MDS) plots (Jakobsson et al. 2008). It was found that haplotype clusters showed different patterns of diversity to SNPs, while the neighbor-joining and MDS plots were similar between haplotype clusters and SNPs.In this work, we apply windowed F_ST methods to localized haplotype clusters derived from the BEAGLE program (Browning and Browning 2007a,b, 2009). We consider population-average, population-specific, and pairwise F_ST estimates (Weir and Hill 2002). Population-average F_ST''s either assume that all the populations are equally diverged from a common ancestor, which is not realistic, or represent the average of a set of population-specific values. This can be convenient in that the results are summarized by a single statistic; however, information is lost. A common procedure is to calculate F_ST for each pair of populations, and these values reflect the degree of divergence between the two populations. Different levels of divergence are allowed for each pair of populations but each estimate uses data from only that pair of populations. On the other hand, population-specific F_ST''s allow unequal levels of divergence in a single analysis that makes use of all the data.We compare results from the localized haplotype clusters to those using SNPs directly. The results of applying localized haplotype clusters to population-specific F_ST estimation are very striking, showing better separation of populations and a more realistic pattern of divergence than for population-specific F_ST estimation using SNPs directly. We also use BEAGLE''s haplotype clusters in a haplotype diversity measure and investigate the relationship between this measure of haplotype-cluster diversity and the recombination rate.