Whole-exome sequencing links a variant in DHDDS to retinitis pigmentosa

Increasingly, mutations in genes causing Mendelian disease will be supported by individual and small families only; however, exome sequencing studies have thus far focused on syndromic phenotypes characterized by low locus heterogeneity. In contrast, retinitis pigmentosa (RP) is caused by >50 known genes, which still explain only half of the clinical cases. In a single, one-generation, nonsyndromic RP family, we have identified a gene, dehydrodolichol diphosphate synthase (DHDDS), demonstrating the power of combining whole-exome sequencing with rapid in vivo studies. DHDDS is a highly conserved essential enzyme for dolichol synthesis, permitting global N-linked glycosylation. Zebrafish studies showed virtually identical photoreceptor defects as observed with N-linked glycosylation-interfering mutations in the light-sensing protein rhodopsin. The identified Lys42Glu variant likely arose from an ancestral founder, because eight of the nine identified alleles in 27,174 control chromosomes were of confirmed Ashkenazi Jewish ethnicity. These findings demonstrate the power of exome sequencing linked to functional studies when faced with challenging study designs and, importantly, link RP to the pathways of N-linked glycosylation, which promise new avenues for therapeutic interventions.     

Isolation and characterization of a psychropiezophilic alphaproteobacterium

Cultivated psychropiezophilic (low-temperature- and high-pressure-adapted) bacteria are currently restricted to phylogenetically narrow groupings capable of growth under nutrient-replete conditions, limiting current knowledge of the extant functional attributes and evolutionary constraints of diverse microorganisms inhabiting the cold, deep ocean. This study documents the isolation of a deep-sea bacterium following dilution-to-extinction cultivation using a natural seawater medium at high hydrostatic pressure and low temperature. To our knowledge, this isolate, designated PRT1, is the slowest-growing (minimal doubling time, 36 h) and lowest cell density-producing (maximal densities of 5.0 × 10⁶ cells ml⁻¹) piezophile yet obtained. Optimal growth was at 80 MPa, correlating with the depth of capture (8,350 m), and 10°C, with average cell sizes of 1.46 μm in length and 0.59 μm in width. Through detailed growth studies, we provide further evidence for the temperature-pressure dependence of the growth rate for deep-ocean bacteria. PRT1 was phylogenetically placed within the Roseobacter clade, a bacterial lineage known for widespread geographic distribution and assorted lifestyle strategies in the marine environment. Additionally, the gene transfer agent (GTA) g5 capsid protein gene was amplified from PRT1, indicating a potential mechanism for increased genetic diversification through horizontal gene transfer within the hadopelagic environment. This study provides a phylogenetically novel isolate for future investigations of high-pressure adaptation, expands the known physiological traits of cultivated members of the Roseobacter lineage, and demonstrates the feasibility of cultivating novel microbial members from the deep ocean using natural seawater.     

Role of proline and glycinebetaine pretreatments in improving heat tolerance of sprouting sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> sp.) buds

High temperature strongly hampers the plant growth particularly at early growth stages. In this study, changes in some physiological and anatomical characteristics and possibility of mitigating the adversities of heat stress by soaking sugarcane nodal buds in 20 mM proline and glycinebetaine (GB) solutions have been explored. Heat stress reduced the rate of bud sprouting nonetheless soaking the setts in proline followed by GB was beneficial. In addition, heat stress reduced the bud fresh and dry weights, generated H₂O₂, reduced the tissue levels of K⁺ and Ca²⁺, while increased the osmolytes synthesis in a time course manner. Heat stress also delayed the emergence and expansion of new bud leaves, by restricting the number and area of mesophyll cells. It also caused poor and aberrant development and diffused appearance of mesophyll cells and vascular bundles in the bud leaves. However, soaking of buds in proline and GB solutions substantially reduced the H₂O₂ production, improved the accumulation of soluble sugars and protected the developing tissues from heat stress effects; although proline was more effective than GB. Correlations of various attributes indicated that soaking in GB and proline restricted the H₂O₂ generation, improved K⁺ and Ca²⁺ contents, and increased the concentrations of free proline, GB and soluble sugars eventually improving the heat tolerance of buds. Cost-benefit analysis showed that, considering increase in sprouting of buds, soaking in 20 mM solution of both osmoprotectants is economical.     

Quantitative role of shrimp fecal bacteria in organic matter fluxes in a recirculating shrimp aquaculture system

Microorganisms play integral roles in the cycling of carbon (C) and nitrogen (N) in recirculating aquaculture systems (RAS) for fish and shellfish production. We quantified the pathways of shrimp fecal bacterial activities and their role in C- and N-flux partitioning relevant to culturing Pacific white shrimp, Penaeus (Litopenaeus) vannamei, in RAS. Freshly produced feces from P. vannamei contained 0.6-7 × 10(10) bacteria g(-1) dry wt belonging to Bacteroidetes (7%), Alphaproteobacteria (4%), and, within the Gammaproteobacteria, almost exclusively to the genus Vibrio (61%). Because of partial disintegration of the feces (up to 27% within 12 h), the experimental seawater became inoculated with fecal bacteria. Bacteria grew rapidly in the feces and in the seawater, and exhibited high levels of aminopeptidase, chitinase, chitobiase, alkaline phosphatase, α- and β-glucosidase, and lipase activities. Moreover, fecal bacteria enriched the protein content of the feces within 12 h, potentially enriching the feces for the coprophagous shrimp. The bacterial turnover time was much faster in feces (1-10 h) than in mature RAS water (350 h). Thus, shrimp fecal bacteria not only inoculate RAS water but also contribute to bacterial abundance and productivity, and regulate system processes important for shrimp health.     

Eye‐mediated immune tolerance to Type II collagen in arthritis‐prone strains of mice

Type II collagen (CII) is a cartilage structural protein that plays important roles in joint function, arthritis and ageing. In studying the ability of CII to induce eye‐mediated specific immune tolerance, we have recently proven that CII is capable of inducing anterior chamber‐associated immune deviation (ACAID) in Balb/c mice. Here, we study the ability of CII to induce eye‐mediated immune tolerance in strains of mice that are prone to the induction of rheumatoid arthritis. Thus, we hypothesized that CII induces ACAID in DBA/1 mice and in C57BL/6 mice through the AC route (direct injection) or the intravenous route (adoptive transfer of in vitro‐generated CII‐specific ACAID macrophages or of CII‐specific in vitro‐generated T regulatory cells). Specific immune tolerance induction was assessed using both delayed‐type hypersensitivity (DTH) and local adoptive transfer (LAT) assays. Results indicated the ability of CII to generate CII‐specific ACAID‐mediated immune tolerance in vivo and in vitro in both DBA/1 mice and C57BL/6 mice. These findings could be beneficial in studies of immune tolerance induction using CII.     

Area-Wide Ground Applications of Bacillus thuringiensis var. israelensis for the Control of Aedes albopictus in Residential Neighborhoods: From Optimization to Operation

The increasing range of Aedes albopictus, the Asian tiger mosquito, in the USA and the threat of chikungunya and dengue outbreaks vectored by this species have necessitated novel approaches to control this peridomestic mosquito. Conventional methods such as adulticiding provide temporary relief, but fail to manage this pest on a sustained basis. We explored the use of cold aerosol foggers and misting machines for area-wide applications of Bacillus thuringiensis var. israelensis (VectoBac WDG) as a larvicide targeting Aedes albopictus. During 2010–2013 we performed initially open field trials and then 19 operational area-wide applications in urban and suburban residential areas in northeastern USA to test three truck-mounted sprayers at two application rates. Area-wide applications of WDG in open field conditions at 400 and 800 g/ha killed on average 87% of tested larvae. Once techniques were optimized in residential areas, applications with a Buffalo Turbine Mist Sprayer at a rate of 800 g/ha, the best combination, consistently provided over 90% mortality. Importantly, there was no significant decrease in efficacy with distance from the spray line even in blocks of row homes with trees and bushes in the backyards. Under laboratory conditions Bti deposition in bioassay cups during the operational trials resulted in over 6 weeks of residual control. Our results demonstrate that area-wide truck mounted applications of WDG can effectively suppress Ae. albopictus larvae and should be used in integrated mosquito management approaches to control this nuisance pest and disease vector.     

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.     

Eastern equine encephalitis virus rapidly infects and disseminates in the brain and spinal cord of cynomolgus macaques following aerosol challenge

Eastern equine encephalitis virus (EEEV) is mosquito-borne virus that produces fatal encephalitis in humans. We recently conducted a first of its kind study to investigate EEEV clinical disease course following aerosol challenge in a cynomolgus macaque model utilizing the state-of-the-art telemetry to measure critical physiological parameters. Here, we report the results of a comprehensive pathology study of NHP tissues collected at euthanasia to gain insights into EEEV pathogenesis. Viral RNA and proteins as well as microscopic lesions were absent in the visceral organs. In contrast, viral RNA and proteins were readily detected throughout the brain including autonomic nervous system (ANS) control centers and spinal cord. However, despite presence of viral RNA and proteins, majority of the brain and spinal cord tissues exhibited minimal or no microscopic lesions. The virus tropism was restricted primarily to neurons, and virus particles (~61–68 nm) were present within axons of neurons and throughout the extracellular spaces. However, active virus replication was absent or minimal in majority of the brain and was limited to regions proximal to the olfactory tract. These data suggest that EEEV initially replicates in/near the olfactory bulb following aerosol challenge and is rapidly transported to distal regions of the brain by exploiting the neuronal axonal transport system to facilitate neuron-to-neuron spread. Once within the brain, the virus gains access to the ANS control centers likely leading to disruption and/or dysregulation of critical physiological parameters to produce severe disease. Moreover, the absence of microscopic lesions strongly suggests that the underlying mechanism of EEEV pathogenesis is due to neuronal dysfunction rather than neuronal death. This study is the first comprehensive investigation into EEEV pathology in a NHP model and will provide significant insights into the evaluation of countermeasure.     

Single nucleotide polymorphisms in <Emphasis Type="Italic">TaER</Emphasis> genes and their association with carbon isotope discrimination in wheat genotypes under drought

Candidate gene association studies implicate the detection of contributing single nucleotide polymorphism (SNP) for the target traits and have been recommended as a promising technique to anatomize the complex characters in plants. The ERECTA gene in plants controls different physiological functions. In this study, we identified SNPs in 1.1 kb partial sequences of TaER-1 and TaER-2 of wheat (Triticum aestivum L.). Thirty-nine SNPs were identified in the coding regions of TaER-1 gene in 33 wheat genotypes, of which 20 SNPs caused non-synonymous mutations while 19 SNPs produced synonymous mutations; 31 SNPs were located in the coding regions of TaER-2 gene in 26 genotypes, of which 18 SNPs caused non-synonymous mutations and 13 SNPs caused synonymous mutations. In addition, 32 SNPs in TaER-1 and 9 SNPs in TaER-2 were also identified in the non-coding regions. Moreover, the significant genetic associations of SNPs of TaER-1 and TaER-2 genes with carbon isotope discrimination, stomatal conductance, photosynthetic rate, transpiration rate, intrinsic water use efficiency (iWUE), leaf length, leaf width, stomatal density, epidermal cell density, and stomatal index were noted in wheat genotypes. This study confirms the importance of TaER-1 and TaER-2 genes which could improve iWUE of wheat by regulating leaf gas exchange and leaf structural traits. These identified SNPs may play a critical role in molecular breeding by means of marker-assisted selection.