The inference of phased haplotypes for the immunoglobulin H chain V region gene loci by analysis of VDJ gene rearrangements

The existence of many highly similar genes in the lymphocyte receptor gene loci makes them difficult to investigate, and the determination of phased "haplotypes" has been particularly problematic. However, V(D)J gene rearrangements provide an opportunity to infer the association of Ig genes along the chromosomes. The chromosomal distribution of H chain genes in an Ig genotype can be inferred through analysis of VDJ rearrangements in individuals who are heterozygous at points within the IGH locus. We analyzed VDJ rearrangements from 44 individuals for whom sufficient unique rearrangements were available to allow comprehensive genotyping. Nine individuals were identified who were heterozygous at the IGHJ6 locus and for whom sufficient suitable VDJ rearrangements were available to allow comprehensive haplotyping. Each of the 18 resulting IGHV│IGHD│IGHJ haplotypes was unique. Apparent deletion polymorphisms were seen that involved as many as four contiguous, functional IGHV genes. Two deletion polymorphisms involving multiple contiguous IGHD genes were also inferred. Three previously unidentified gene duplications were detected, where two sequences recognized as allelic variants of a single gene were both inferred to be on a single chromosome. Phased genomic data brings clarity to the study of the contribution of each gene to the available repertoire of rearranged VDJ genes. Analysis of rearrangement frequencies suggests that particular genes may have substantially different yet predictable propensities for rearrangement within different haplotypes. Together with data highlighting the extent of haplotypic variation within the population, this suggests that there may be substantial variability in the available Ab repertoires of different individuals.     

The complete genome sequence of a single-stranded RNA virus from the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois)

The complete genome sequence of a single-stranded RNA virus infecting the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), was identified by sequencing cDNA prepared from insects collected from the Mississippi Delta. The 9655 nucleotide positive-sense single-stranded RNA genome of the L. lineolaris single-stranded RNA virus (LyLV-1) contained a single open reading frame of 8958 nucleotides encoding a 2986 amino acid genome polypeptide. The open reading frame was flanked by untranslated regions of 603 and 69 nucleotides at the 5'- and 3'- ends of the genome, respectively. Database searches and homology based modeling was used to identify four capsid proteins (VP1-VP4), helicase/AAA-ATPase, cysteine protease (C3P), protease 2A, and the RNA-directed RNA polymerase (RdRp). In addition, a region with weak similarity to the eukaryotic structural maintenance of chromosome (SMC) domain was identified near the amino-terminal of the polyprotein and adjacent to the VP1 domain. The amino acid sequence of LyLV-1 was approximately 44.4% similar to that of sacbrood virus (SBV) of the honey bee. The genomic organization of both viruses showed remarkable similarity with the exception of highly divergent amino acid regions flanking fairly conserved structural and non-structural polypeptide regions. High similarity to the SBV genome and similarities in the genome organization and amino acid sequence with the viruses of the family Iflaviridae suggested that LyLV-1 was a novel member of this family. Virus particles were 39 nm in diameter and appeared to transmit vertically via eggs. Although this virus may only cause covert infections under normal conditions, the potential for using this virus in biological control of L. lineolaris is discussed.     

Herpes simplex virus antigens directly activate NK cells via TLR2, thus facilitating their presentation to CD4 T lymphocytes

NK cells infiltrate human herpetic lesions, but their role has been underexplored. HSV can stimulate innate immune responses via surface TLR2, which is expressed on monocyte-derived dendritic cells (DCs) and NK cells. In this study, UV-inactivated HSV1/2 and immunodominant HSV2 glycoprotein D peptides conjugated to the TLR2 agonist dipalmitoyl-S-glyceryl cysteine stimulated CD4 T lymphocyte IFN-γ responses within PBMCs or in coculture with monocyte-derived DCs. NK cells contributed markedly to the PBMC responses. Furthermore, NK cells alone were activated directly by both Ags, also upregulating HLA-DR and HLA-DQ and then they activated autologous CD4 T lymphocytes. Using Transwells, Ag-stimulated NK cells and CD4 T lymphocytes were shown to interact through both cell-to-cell contact and cytokines, differing in relative importance in different donors. A distinct immunological synapse between Ag-stimulated NK cells and CD4 T lymphocytes was observed, indicating the significance of their cell-to-cell contact. A large proportion (57%) of NK cells was also in contact with CD4 T lymphocytes in the dermal infiltrate of human recurrent herpetic lesions. Thus, NK cells stimulated by TLR2-activating HSV Ags can present Ag alone or augment the role of DCs in vitro and perhaps in herpetic lesions or draining lymph nodes. In addition to DCs, NK cells should be considered as targets for adjuvants during HSV vaccine development.     

A Novel Assay for Antibody-Dependent Cell-Mediated Cytotoxicity against HIV-1- or SIV-Infected Cells Reveals Incomplete Overlap with Antibodies Measured by Neutralization and Binding Assays

The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4⁺ T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies—frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays.     

Sex Ratio, Conflict Dynamics, and Wounding in Rhesus Macaques (Macaca mulatta)

Rhesus macaques, like many other primates, live in stable, multi-male multi-female groups in which adult females typically outnumber adult males. The number of males in multi-male/multi-female groups is most commonly discussed in terms of mate competition, where the sex ratio is a function of an adult male's ability to monopolize a group of females. However, the relationship between sex ratio and group stability is unclear because the presence of many males may either reduce stability by increasing mate competition or may improve stability if adult males are key conflict managers. We investigated the relationship between sex ratio, male intervention behavior, and trauma in seven groups of captive rhesus macaques. Our results show that high-ranking adult males intervene twice as frequently as adult females (P<0.0001) and are about twice as successful (P<0.0001). Furthermore, the type of adult males present in the group affects the relationship between intervention behavior and rate of traumas: males must be unrelated to the highest-ranking matrilines. Groups with a lower ratio of females per male unrelated to the alpha and beta matrilines had better intervention success (P<0.04) and fewer traumas requiring veterinary care (P=0.003). We conclude that conflict management behavior by adult males, particularly those unrelated to the highest-ranking matrilines, is the mechanism by which sex ratio influences the frequency of traumas, and thus group stability. We therefore suggest that monitoring and managing the sex ratio of captive primate groups is one of many measurements to predict group stability.     

Individual differences in the effects of mobile phone exposure on human sleep: rethinking the problem

Mobile phone exposure‐related effects on the human electroencephalogram (EEG) have been shown during both waking and sleep states, albeit with slight differences in the frequency affected. This discrepancy, combined with studies that failed to find effects, has led many to conclude that no consistent effects exist. We hypothesised that these differences might partly be due to individual variability in response, and that mobile phone emissions may in fact have large but differential effects on human brain activity. Twenty volunteers from our previous study underwent an adaptation night followed by two experimental nights in which they were randomly exposed to two conditions (Active and Sham), followed by a full‐night sleep episode. The EEG spectral power was increased in the sleep spindle frequency range in the first 30 min of non‐rapid eye movement (non‐REM) sleep following Active exposure. This increase was more prominent in the participants that showed an increase in the original study. These results confirm previous findings of mobile phone‐like emissions affecting the EEG during non‐REM sleep. Importantly, this low‐level effect was also shown to be sensitive to individual variability. Furthermore, this indicates that previous negative results are not strong evidence for a lack of an effect and, given the far‐reaching implications of mobile phone research, we may need to rethink the interpretation of results and the manner in which research is conducted in this field. Bioelectromagnetics 33:86–93, 2012. © 2011 Wiley Periodicals, Inc.     

Pyrosequencing Reveals Diverse and Distinct Sponge-Specific Microbial Communities in Sponges from a Single Geographical Location in Irish Waters

Marine sponges are host to numerically vast and phylogenetically diverse bacterial communities, with 26 major phyla to date having been found in close association with sponge species worldwide. Analyses of these microbial communities have revealed many sponge-specific novel genera and species. These endosymbiotic microbes are believed to play significant roles in sponge physiology including the production of an array of bioactive secondary metabolites. Here, we report on the use of culture-based and culture-independent (pyrosequencing) techniques to elucidate the bacterial community profiles associated with the marine sponges Raspailia ramosa and Stelligera stuposa sampled from a single geographical location in Irish waters and with ambient seawater. To date, little is known about the microbial ecology of sponges of these genera. Culture isolation grossly underestimated sponge-associated bacterial diversity. Four bacterial phyla (Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria) were represented amongst ~200 isolates, compared with ten phyla found using pyrosequencing. Long average read lengths of ~430 bp (V1-V3 region of 16S rRNA gene) allowed for robust resolution of sequences to genus level. Bacterial OTUs (2,109 total), at 95% sequence similarity, from ten bacterial phyla were recovered from R. ramosa, 349 OTUs were identified in S. stuposa representing eight phyla, while 533 OTUs from six phyla were found in surrounding seawater. Bacterial communities differed significantly between sponge species and the seawater. Analysis of the data for sponge-specific taxa revealed that 2.8% of classified reads from the sponge R. ramosa can be defined as sponge-specific, while 26% of S. stuposa sequences represent sponge-specific bacteria. Novel sponge-specific clusters were identified, whereas the majority of previously reported sponge-specific clusters (e.g. Poribacteria) were absent from these sponge species. This deep and robust analysis provides further evidence that the microbial communities associated with marine sponge species are highly diverse and divergent from one another and appear to be host-selected through as yet unknown processes.     

Influence of a mannan binding family 32 carbohydrate binding module on the activity of the appended mannanase

In general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocellum hypothetical protein Cthe_0821, defined here as C. thermocellum Man5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermocellum Man5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted β-1,4-mannoside linkages. The CBM32 of C. thermocellum Man5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of β-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum Man5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum Man5A is affected by the presence of CBM32.