Single-crystal Ih Ice Surfaces Unveil Connection between Macroscopic and Molecular Structure

Physics and chemistry of ice surfaces are not only of fundamental interest but also have important impacts on biological and environmental processes. As ice surfaces—particularly the two prism faces—come under greater scrutiny, it is increasingly important to connect the macroscopic faces with the molecular-level structure. The microscopic structure of the ubiquitous ice Ih crystal is well-known. It consists of stacked layers of chair-form hexagonal rings referred to as molecular hexagons. Crystallographic unit cells can be assembled into a regular right hexagonal prism. The bases are labeled crystallographic hexagons. The two hexagons are rotated 30° with respect to each other. The linkage between the familiar macroscopic shape of hexagonal snowflakes and either hexagon is not obvious per se. This report presents experimental data directly connecting the macroscopic shape of ice crystals and the microscopic hexagons. Large ice single crystals were used to fabricate samples with the basal, primary prism, or secondary prism faces exposed at the surface. In each case, the same sample was used to capture both a macroscopic etch pit image and an electron backscatter diffraction (EBSD) orientation density function (ODF) plot. Direct comparison of the etch pit image and the ODF plot compellingly connects the macroscopic etch pit hexagonal profile to the crystallographic hexagon. The most stable face at the ice–water interface is the smallest area face at the ice–vapor interface. A model based on the molecular structure of the prism faces accounts for this switch

Elucidating the structural basis for differing enzyme inhibitor potency by cryo-EM

Histidine biosynthesis is an essential process in plants and microorganisms, making it an attractive target for the development of herbicides and antibacterial agents. Imidazoleglycerol-phosphate dehydratase (IGPD), a key enzyme within this pathway, has been biochemically characterized in both Saccharomyces cerevisiae (Sc_IGPD) and Arabidopsis thaliana (At_IGPD). The plant enzyme, having been the focus of in-depth structural analysis as part of an inhibitor development program, has revealed details about the reaction mechanism of IGPD, whereas the yeast enzyme has proven intractable to crystallography studies. The structure–activity relationship of potent triazole-phosphonate inhibitors of IGPD has been determined in both homologs, revealing that the lead inhibitor (C348) is an order of magnitude more potent against Sc_IGPD than At_IGPD; however, the molecular basis of this difference has not been established. Here we have used single-particle electron microscopy (EM) to study structural differences between the At and Sc_IGPD homologs, which could influence the difference in inhibitor potency. The resulting EM maps at ∼3 Å are sufficient to de novo build the protein structure and identify the inhibitor binding site, which has been validated against the crystal structure of the At_IGPD/C348 complex. The structure of Sc_IGPD reveals that a 24-amino acid insertion forms an extended loop region on the enzyme surface that lies adjacent to the active site, forming interactions with the substrate/inhibitor binding loop that may influence inhibitor potency. Overall, this study provides insights into the IGPD family and demonstrates the power of using an EM approach to study inhibitor binding

Effect of Lactobacillus salivarius Bacteriocin Abp118 on the Mouse and Pig Intestinal Microbiota

Lactobacilli are Gram-positive bacteria that are a subdominant element in the human gastrointestinal microbiota, and which are commonly used in the food industry. Some lactobacilli are considered probiotic, and have been associated with health benefits. However, there is very little culture-independent information on how consumed probiotic microorganisms might affect the entire intestinal microbiota. We therefore studied the impact of the administration of Lactobacillus salivarius UCC118, a microorganism well characterized for its probiotic properties, on the composition of the intestinal microbiota in two model animals. UCC118 has anti-infective activity due to production of the bacteriocin Abp118, a broad-spectrum class IIb bacteriocin, which we hypothesized could impact the microbiota. Mice and pigs were administered wild-type (WT) L. salivarius UCC118 cells, or a mutant lacking bacteriocin production. The microbiota composition was determined by pyrosequencing of 16S rRNA gene amplicons from faeces. The data show that L. salivarius UCC118 administration had no significant effect on proportions of major phyla comprising the mouse microbiota, whether the strain was producing bacteriocin or not. However, L. salivarius UCC118 WT administration led to a significant decrease in Spirochaetes levels, the third major phylum in the untreated pig microbiota. In both pigs and mice, L. salivarius UCC118 administration had an effect on Firmicutes genus members. This effect was not observed when the mutant strain was administered, and was thus associated with bacteriocin production. Surprisingly, in both models, L. salivarius UCC118 administration and production of Abp118 had an effect on Gram-negative microorganisms, even though Abp118 is normally not active in vitro against this group of microorganisms. Thus L. salivarius UCC118 administration has a significant but subtle impact on mouse and pig microbiota, by a mechanism that seems at least partially bacteriocin-dependent

Experimental and theoretical evidence for bilayer-by-bilayer surface melting of crystalline ice

On the surface of water ice, a quasi-liquid layer (QLL) has been extensively reported at temperatures below its bulk melting point at 273 K. Approaching the bulk melting temperature from below, the thickness of the QLL is known to increase. To elucidate the precise temperature variation of the QLL, and its nature, we investigate the surface melting of hexagonal ice by combining noncontact, surface-specific vibrational sum frequency generation (SFG) spectroscopy and spectra calculated from molecular dynamics simulations. Using SFG, we probe the outermost water layers of distinct single crystalline ice faces at different temperatures. For the basal face, a stepwise, sudden weakening of the hydrogen-bonded structure of the outermost water layers occurs at 257 K. The spectral calculations from the molecular dynamics simulations reproduce the experimental findings; this allows us to interpret our experimental findings in terms of a stepwise change from one to two molten bilayers at the transition temperature

Patient Retention and Adherence to Antiretrovirals in a Large Antiretroviral Therapy Program in Nigeria: A Longitudinal Analysis for Risk Factors

Substantial resources and patient commitment are required to successfully scale-up antiretroviral therapy (ART) and provide appropriate HIV management in resource-limited settings. We used pharmacy refill records to evaluate risk factors for loss to follow-up (LTFU) and non-adherence to ART in a large treatment cohort in Nigeria.We reviewed clinic records of adult patients initiating ART between March 2005 and July 2006 at five health facilities. Patients were classified as LTFU if they did not return >60 days from their expected visit. Pharmacy refill rates were calculated and used to assess non-adherence. We identified risk factors associated with LTFU and non-adherence using Cox and Generalized Estimating Equation (GEE) regressions, respectively. Of 5,760 patients initiating ART, 26% were LTFU. Female gender (p < 0.001), post-secondary education (p = 0.03), and initiating treatment with zidovudine-containing (p = 0.004) or tenofovir-containing (p = 0.05) regimens were associated with decreased risk of LTFU, while patients with only primary education (p = 0.02) and those with baseline CD4 counts (cell/ml(3)) >350 and <100 were at a higher risk of LTFU compared to patients with baseline CD4 counts of 100-200. The adjusted GEE analysis showed that patients aged <35 years (p = 0.005), who traveled for >2 hours to the clinic (p = 0.03), had total ART duration of >6 months (p<0.001), and CD4 counts >200 at ART initiation were at a higher risk of non-adherence. Patients who disclosed their HIV status to spouse/family (p = 0.01) and were treated with tenofovir-containing regimens (p < or = 0.001) were more likely to be adherent.These findings formed the basis for implementing multiple pre-treatment visit preparation that promote disclosure and active community outreaching to support retention and adherence. Expansion of treatment access points of care to communities to diminish travel time may have a positive impact on adherence

High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 μM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis

Genomic ancestry estimation quantifies use of wild species in grape breeding

BACKGROUND: Grapes are one of the world’s most valuable crops and most are made into wine. Grapes belong to the genus Vitis, which includes over 60 inter-fertile species. The most common grape cultivars derive their entire ancestry from the species Vitis vinifera, but wild relatives have also been exploited to create hybrid cultivars, often with increased disease resistance. RESULTS: We evaluate the genetic ancestry of some of the most widely grown commercial hybrids from North America and Europe. Using genotyping-by-sequencing (GBS), we generated 2482 SNPs and 56 indels from 7 wild Vitis, 7 V. vinifera, and 64 hybrid cultivars. We used a principal component analysis (PCA) based ancestry estimation procedure and verified its accuracy with both empirical and simulated data. V. vinifera ancestry ranged from 11 % to 76 % across hybrids studied. Approximately one third (22/64) of the hybrids have ancestry estimates consistent with F1 hybridization: they derive half of their ancestry from wild Vitis and half from V. vinifera. CONCLUSIONS: Our results suggest that hybrid grape breeding is in its infancy. The distribution of V. vinifera ancestry across hybrids also suggests that backcrosses to wild Vitis species have been more frequent than backcrosses to V. vinifera during hybrid grape breeding. This pattern is unusual in crop breeding, as it is most common to repeatedly backcross to elite, or domesticated, germplasm. We anticipate our method can be extended to facilitate marker-assisted selection in order to introgress beneficial wild Vitis traits, while allowing for offspring with the highest V. vinifera content to be selected at the seedling stage. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2834-8) contains supplementary material, which is available to authorized users

Recurrent horizontal transfer identifies mitochondrial positive selection in a transmissible cancer

Abstract: Autonomous replication and segregation of mitochondrial DNA (mtDNA) creates the potential for evolutionary conflict driven by emergence of haplotypes under positive selection for ‘selfish’ traits, such as replicative advantage. However, few cases of this phenomenon arising within natural populations have been described. Here, we survey the frequency of mtDNA horizontal transfer within the canine transmissible venereal tumour (CTVT), a contagious cancer clone that occasionally acquires mtDNA from its hosts. Remarkably, one canine mtDNA haplotype, A1d1a, has repeatedly and recently colonised CTVT cells, recurrently replacing incumbent CTVT haplotypes. An A1d1a control region polymorphism predicted to influence transcription is fixed in the products of an A1d1a recombination event and occurs somatically on other CTVT mtDNA backgrounds. We present a model whereby ‘selfish’ positive selection acting on a regulatory variant drives repeated fixation of A1d1a within CTVT cells

Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility.

Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel Na1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on Na1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings