The CRHR1 gene,trauma exposure,and alcoholism risk: a test of G × E effects

The corticotropin‐releasing hormone type I receptor (CRHR1) gene has been implicated in the liability for neuropsychiatric disorders, particularly under conditions of stress. On the basis of the hypothesized effects of CRHR1 variation on stress reactivity, measures of adulthood traumatic stress exposure were analyzed for their interaction with CRHR1 haplotypes and single‐nucleotide polymorphisms (SNPs) in predicting the risk for alcoholism. Phenotypic data on 2533 non‐related Caucasian individuals (1167 alcoholics and 1366 controls) were culled from the publically available Study of Addiction: Genetics and Environment genome‐wide association study. Genotypes were available for 19 tag SNPs. Logistic regression models examined the interaction between CRHR1 haplotypes/SNPs and adulthood traumatic stress exposure in predicting alcoholism risk. Two haplotype blocks spanned CRHR1. Haplotype analyses identified one haplotype in the proximal block 1 (P = 0.029) and two haplotypes in the distal block 2 (P = 0.026, 0.042) that showed nominally significant (corrected P < 0.025) genotype × traumatic stress interactive effects on the likelihood of developing alcoholism. The block 1 haplotype effect was driven by SNPs rs110402 (P = 0.019) and rs242924 (P = 0.019). In block 2, rs17689966 (P = 0.018) showed significant and rs173365 (P = 0.026) showed nominally significant, gene × environment (G × E) effects on alcoholism status. This study extends the literature on the interplay between CRHR1 variation and alcoholism, in the context of exposure to traumatic stress. These findings are consistent with the hypothesized role of the extra hypothalamic corticotropin‐releasing factor system dysregulation in the initiation and maintenance of alcoholism. Molecular and experimental studies are needed to more fully understand the mechanisms of risk and protection conferred by genetic variation at the identified loci .     

Etiology and Audiological Outcomes at 3 Years for 364 Children in Australia

Hearing loss is an etiologically heterogeneous trait with differences in the age of onset, severity and site of lesion. It is caused by a combination of genetic and/or environmental factors. A longitudinal study to examine the efficacy of early intervention for improving child outcomes is ongoing in Australia. To determine the cause of hearing loss in these children we undertook molecular testing of perinatal “Guthrie” blood spots of children whose hearing loss was either detected via newborn hearing screening or detected later in infancy. We analyzed the GJB2 and SLC26A4 genes for the presence of mutations, screened for the mitochondrial DNA (mtDNA) A1555G mutation, and screened for congenital CMV infection in DNA isolated from dried newborn blood spots. Results were obtained from 364 children. We established etiology for 60% of children. One or two known GJB2 mutations were present in 82 children. Twenty-four children had one or two known SLC26A4 mutations. GJB2 or SLC26A4 changes with unknown consequences on hearing were found in 32 children. The A1555G mutation was found in one child, and CMV infection was detected in 28 children. Auditory neuropathy spectrum disorder was confirmed in 26 children whose DNA evaluations were negative. A secondary objective was to investigate the relationship between etiology and audiological outcomes over the first 3 years of life. Regression analysis was used to investigate the relationship between hearing levels and etiology. Data analysis does not support the existence of differential effects of etiology on degree of hearing loss or on progressiveness of hearing loss.     

Resident mesenchymal cells and fibrosis

Fibrosis is a major clinical problem associated with as many as 45% of all natural deaths in developed nations.It can affect all organs and accumulating evidence indicates that fibrogenesis is not merely a bystander product of injury, but is a central pathological problem directly contributing to loss of organ function. In the majority of clinical cases, fibrogenesis is strongly associated with the recruitment of leukocytes, even in the absence of infection. Although chronic infections are a significant cause of fibrogenesis, in most cases fibrotic disease occurs in the context of sterile injury, such as microvascular disease, toxic epithelial injury or diabetes mellitus. Fibrogenesis is a direct consequence of the activation of extensive, and previously poorly appreciated, populations of mesenchymal cells in our organs which are either wrapped around capillaries and known as ‘pericytes’, or embedded in interstitial spaces between cell structures and known as resident ‘fibroblasts’. Recent fate-mapping and complementary studies in several organs indicate that these cells are the precursors of the scar-forming myofibroblasts that appear in our organs in response to injury. Here we will review the literature supporting a central role for these cells in fibrogenesis, and highlight some of the critical cell to cell interactions that are necessary for the initiation and continuation of the fibrogenic process. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

A directed nucleotide-sequencing approach for single-stranded vectors based on recloning intermediates of a progressive DNA synthesis reaction

A simple method for site-directed nucleotide sequencing is presented that uses a novel procedure for generating nested 'deletions' within inserts of single-stranded clones. In this method, single-stranded template, sequencing primer, and the Klenow fragment of Escherichia coli DNA polymerase I are used to initiate progressive DNA synthesis of the entire insert of the clone. By time-dependent sampling and pooling of intermediates from the synthesis reaction a series of nested double-stranded DNA subfragments of the insert can be created. Nested subclones are then produced by S1-endonuclease treatment and oriented subcloning methods. First, smaller quantities of template DNA can be used, equivalent to a fraction of a small DNA sequencing prep. Second, it works with single-stranded M13 phage DNA rather than requiring the preparation of double-stranded replicative form DNA as in ExoIII-based methods. Third, the 'deletions' it generates can span areas of simple nucleotide sequence or secondary structure that often halt digestion in the single-stranded exonuclease-based method. Last, the method is adaptable to a larger variety of insert cloning sites than the ExoIII-based method. The main disadvantage of the method is that, due to the lower efficiency of subcloning larger DNA fragments, subclone inserts larger than 3 kb are generated only infrequently.     

Comparative susceptibilities of diamondback moth (Lepidoptera: Plutellidae) and cabbage looper (Lepidoptera: Noctuidae) from Minnesota and south Texas to lambda-cyhalothrin and indoxacarb

The susceptibility of early instars of diamondback moth, Plutella xylostella (L.), and early (first and second) and late instars (third and fourth) of cabbage looper, Trichoplusia ni (Hübner), from Minnesota and south Texas, to indoxacarb and lambda-cyhalothrin was determined in the laboratory. Susceptibilities of the two species from the two geographical locations to indoxacarb and lambda-cyhalothrin varied greatly. P. xylostella from Minnesota was as susceptible to indoxacarb as those from south Texas, whereas both early and late instar T. ni from south Texas were significantly more tolerant to indoxacarb than those from Minnesota. The LC50 values of indoxacarb for early and later instar T. ni at 48 h from south Texas were 4.3- and 34.0-fold greater than those from Minnesota, respectively. Similarly, early instar P. xylostella and late instar T. ni from south Texas were significantly less susceptible to lambda-cyhalothrin than those from Minnesota. Percentage mortality of the two insect species caused by the two insecticides varied with time of exposure and generally exhibited similar patterns of responses to different concentrations for each insecticide.     

A high-throughput Arabidopsis reverse genetics system

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.