Next-generation sequencing reveals significant bacterial diversity of botrytized wine

While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies.     

Red is not a proxy signal for female genitalia in humans

Red is a colour that induces physiological and psychological effects in humans, affecting competitive and sporting success, signalling and enhancing male social dominance. The colour is also associated with increased sexual attractiveness, such that women associated with red objects or contexts are regarded as more desirable. It has been proposed that human males have a biological predisposition towards the colour red such that it is 'sexually salient'. This hypothesis argues that women use the colour red to announce impending ovulation and sexual proceptivity, with this functioning as a proxy signal for genital colour, and that men show increased attraction in consequence. In the first test of this hypothesis, we show that contrary to the hypothesis, heterosexual men did not prefer redder female genitalia and, by extension, that red is not a proxy signal for genital colour. We found a relative preference for pinker genital images with redder genitalia rated significantly less sexually attractive. This effect was independent of raters' prior sexual experience and variation in female genital morphology. Our results refute the hypothesis that men's attraction to red is linked to an implied relationship to genital colour and women's signalling of fertility and sexual proceptivity.     

Primary human airway epithelial cell-dependent inhibition of human lung mast cell degranulation

Introduction

Chronic mast cell activation is a characteristic feature of asthma. BEAS-2B human airway epithelial cells (AEC) profoundly inhibit both constitutive and IgE-dependent human lung mast cell (HLMC) histamine release. The aim of this study was to examine the regulation of HLMC degranulation by primary AEC from healthy and asthmatic subjects, and investigate further the inhibitory mechanism.

Methods

HLMC were co-cultured with both BEAS-2B and primary AEC grown as monolayers or air-liquid interface (ALI) cultures.

Results

Both constitutive and IgE-dependent HLMC histamine release were attenuated by BEAS-2B, primary AEC monolayers and ALI cultures. This occurred in the absence of HLMC-AEC contact indicating the presence of a soluble factor. Unlike healthy ALI AEC, asthmatic ALI-AEC did not significantly reduce constitutive histamine release. AEC inhibitory activity was transferable in primary AEC monolayer supernatant, but less active than with Transwell co-culture, suggesting that the inhibitory factor was labile. The AEC inhibitory effects were attenuated by both AEC wounding and pertussis toxin, indicating the involvement of a G₀/G_i receptor coupled mechanism. Solid phase extraction of lipids (<10 kDa) removed the AEC inhibitory activity. The lipid derivatives resolvin D1 and D2 and lipoxin A₄ attenuated HLMC histamine release in a dose-dependent fashion but were not detectable in co-culture supernatants.

Conclusions

Primary AEC suppress HLMC constitutive and IgE-dependent histamine secretion through the release of a soluble, labile lipid mediator(s) that signals through the G₀/G_i receptor coupled mechanism. Manipulation of this interaction may have a significant therapeutic role in asthma.     

Response of Sphagnum papillosum and Drosera rotundifolia to Reduced and Oxidized Wet Nitrogen Deposition

We transplanted Sphagnum ??turfs?? containing abundant Drosera rotundifolia into an existing nitrogen deposition experiment at Whim Moss near Edinburgh. These mesocosms received simulated N deposition as either NH ₄ ⁺ or NO ₃ ^- , to give total N deposition rates of approximately 8, 16 or 32, or 64?kg?N?ha^-1?year^-1. Simulated N deposition was added in a realistic way (i.e., with rainfall throughout the year). The ??¹⁵N of this added N was elevated relative to background N. We measured the tissue chemistry and ??¹⁵N of Sphagnum papillosum and D. rotundifolia over two years after transplant. Our aim was to determine uptake of the deposited N and the impact on S. papillosum tissue chemistry and D. rotundifolia tissue chemistry and ecology. We found clear, significant impacts of N deposition on S. papillosum, with increased capitula N content and reduced C:N ratio. Increased ??¹⁵N indicated uptake of deposited N. The response of D. rotundifolia was less clear with impacts only at the highest rate of N deposition. There was no evidence of differential uptake of reduced or oxidized wet N deposition by either S. papillosum or D. rotundifolia. Using the natural abundance stable isotope method we estimated the minimum contribution of prey N to the total N in D. rotundifolia to be 35%. The results suggest that differences in the uptake of reduced or oxidized wet N deposition might not be ecologically significant when wet N deposition is added realistically. They also support the suggestion that a model of N dynamics in Sphagnum-dominated ecosystems that includes the role of Sphagnum as a small-scale ecosystem engineer, is required to predict vascular plant responses to N deposition accurately.     

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence.     

MicroRNAs are exported from malignant cells in customized particles

MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body.     

Tempol modulates changes in xenobiotic permeability and occludin oligomeric assemblies at the blood-brain barrier during inflammatory pain

Our laboratory has shown that λ-carrageenan-induced peripheral inflammatory pain (CIP) can alter tight junction (TJ) protein expression and/or assembly leading to changes in blood-brain barrier xenobiotic permeability. However, the role of reactive oxygen species (ROS) and subsequent oxidative stress during CIP is unknown. ROS (i.e., superoxide) are known to cause cellular damage in response to pain/inflammation. Therefore, we examined oxidative stress-associated effects at the blood-brain barrier (BBB) in CIP rats. During CIP, increased staining of nitrosylated proteins was detected in hind paw tissue and enhanced presence of protein adducts containing 3-nitrotyrosine occurred at two molecular weights (i.e., 85 and 44 kDa) in brain microvessels. Tempol, a pharmacological ROS scavenger, attenuated formation of 3-nitrotyrosine-containing proteins in both the hind paw and in brain microvessels when administered 10 min before footpad injection of λ-carrageenan. Similarly, CIP increased 4-hydroxynoneal staining in brain microvessels and this effect was reduced by tempol. Brain permeability to [(14)C]sucrose and [(3)H]codeine was increased, and oligomeric assemblies of occludin, a critical TJ protein, were altered after 3 h CIP. Tempol attenuated both [(14)C]sucrose and [(3)H]codeine brain uptake as well as protected occludin oligomers from disruption in CIP animals, suggesting that ROS production/oxidative stress is involved in modulating BBB functional integrity during pain/inflammation. Interestingly, tempol administration reduced codeine analgesia in CIP animals, indicating that oxidative stress during pain/inflammation may affect opioid delivery to the brain and subsequent efficacy. Taken together, our data show for the first time that ROS pharmacological scavenging is a viable approach for maintaining BBB integrity and controlling central nervous system drug delivery during acute inflammatory pain.     

Duplication of GTF2I results in separation anxiety in mice and humans

Duplication (dup7q11.23) and deletion (Williams syndrome) of chromosomal region 7q11.23 cause neurodevelopmental disorders with contrasting anxiety phenotypes. We found that 30% of 4- to 12-year-olds with dup7q11.23 but fewer than 5% of children with WS or in the general population met diagnostic criteria for a separation-anxiety disorder. To address the role of one commonly duplicated or deleted gene in separation anxiety, we compared mice that had varying numbers of Gtf2i copies. Relative to mouse pups with one or two Gtf2i copies, pups with additional Gtf2i copies showed significantly increased maternal separation-induced anxiety as measured by ultrasonic vocalizations. This study links the copy number of a single gene from 7q11.23 to separation anxiety in both mice and humans, highlighting the utility of mouse models in dissecting specific gene functions for genomic disorders that span many genes. This study also offers insight into molecular separation-anxiety pathways that might enable the development of targeted therapeutics.     

Fine‐scale Spatial Genetic Structure of Ten Dipterocarp Tree Species in a Bornean Rain Forest

Fine‐scale spatial genetic structure is increasingly recognized as an important factor in the studies of tropical forest trees as it influences genetic diversity of local populations. The biologic mechanisms that generate fine‐scale spatial genetic structure are not fully understood. We studied fine‐scale spatial genetic structure in ten coexisting dipterocarp tree species in a Bornean rain forest using microsatellite markers. Six of the ten species showed statistically significant fine‐scale spatial genetic structure. Fine‐scale spatial genetic structure was stronger at smaller spatial scales (≤ 100 m) than at larger spatial scales (> 100 m) for each species. Multiple regression analysis suggested that seed dispersal distance was important at the smaller spatial scale. At the larger scale (> 100 m) and over the entire sample range (0–1000 m), pollinators and spatial distribution of adult trees were more important determinants of fine‐scale spatial genetic structure. Fine‐scale spatial genetic structure was stronger in species pollinated by less mobile small beetles than in species pollinated by the more mobile giant honeybee (Apis dorsata). It was also stronger in species where adult tree distributions were more clumped. The hypothesized mechanisms underlying the negative correlation between clump size and fine‐scale spatial genetic structure were a large overlap among seed shadows and genetic drift within clumped species.