Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.     

Human monkeypox and smallpox viruses: genomic comparison.

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person-to-person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197-kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.     

Reproductive history and nest environment are correlated with circulating androgen and glucocorticoid concentrations in a parental care-providing teleost fish

Using a long-term study population of wild smallmouth bass Micropterus dolomieu in a connected river-lake system, we investigated whether circulating glucocorticoid (cortisol) and androgen (testosterone) concentrations are influenced by reproductive investment and nesting environment in fish providing nest-guarding paternal care. For all individuals, we collected measures of reproductive history and the value of current parental care. We assessed nest environment and monitored individuals to quantify seasonal reproductive success. Finally, we measured circulating cortisol concentrations following a standardized stressor and circulating testosterone concentrations. Using general linear models, we found that poststress circulating cortisol concentrations were positively related to water temperature and were higher in fish nesting in the river than in the lake. Circulating testosterone concentrations were negatively related to water temperature and were higher in reproductively experienced fish. When considering the factors that influence reproductive success, we found that only parental size was positively related to current nest success. In summary, the results demonstrate that nesting environment is correlated with parental stress responses during parental care, while reproductive history and nesting environment are correlated with circulating androgen concentrations. Collectively, these results offer insight into the roles of both glucocorticoid and androgen steroid hormones during parental care in teleost fish.     

Temporal requirement of the alternative-splicing factor Sfrs1 for the survival of retinal neurons

Alternative splicing is the primary mechanism by which a limited number of protein-coding genes can generate proteome diversity. We have investigated the role of the alternative-splicing factor Sfrs1, an arginine/serine-rich (SR) protein family member, during mouse retinal development. Loss of Sfrs1 function during embryonic retinal development had a profound effect, leading to a small retina at birth. In addition, the retina underwent further degeneration in the postnatal period. Loss of Sfrs1 function resulted in the death of retinal neurons that were born during early to mid-embryonic development. Ganglion cells, cone photoreceptors, horizontal cells and amacrine cells were produced and initiated differentiation. However, these neurons subsequently underwent cell death through apoptosis. By contrast, Sfrs1 was not required for the survival of the neurons generated later, including later-born amacrine cells, rod photoreceptors, bipolar cells and Müller glia. Our results highlight the requirement of Sfrs1-mediated alternative splicing for the survival of retinal neurons, with sensitivity defined by the window of time in which the neuron was generated.     

The floral biology and reproductive system of <Emphasis Type="Italic">Mauritia flexuosa</Emphasis> (Arecaceae) in a restinga environment in northeastern Brazil

Studies of the floral biology of the buriti palm, Mauritia flexuosa, have presented conflicting results with respect to the mechanism of pollination, indicating either cantharophily or anemophily. To resolve this question, the floral biology of M. flexuosa was studied in a coastal restinga environment in northeastern Brazil. The reproductive system was studied experimentally, and floral visitors were collected by bagging inflorescences. In this environment, M. flexuosa, a dioecious species, has several gender-specific floral features that function to attract pollinators, especially beetles. The male flowers produce large amounts of pollen as a reward, and male and female inflorescences produce similar odors that attract pollinators to female flowers, which offer only a nectar secretion as a reward. When feeding on the female flowers, the visitors frequently come into contact with the stigmata. To increase the chances of pollination, the female flowers persist longer than the male ones, and the viability of the pollen grain is very high. A curculionid beetle species of the genus Grasidius was found to be an effective pollinator. We suspect that wind also contributes to the pollination of M. flexuosa in the study area, but in a relatively minor way.     

Genome-Wide and Candidate Gene Association Study of Cigarette Smoking Behaviors

The contribution of common genetic variation to one or more established smoking behaviors was investigated in a joint analysis of two genome wide association studies (GWAS) performed as part of the Cancer Genetic Markers of Susceptibility (CGEMS) project in 2,329 men from the Prostate, Lung, Colon and Ovarian (PLCO) Trial, and 2,282 women from the Nurses'' Health Study (NHS). We analyzed seven measures of smoking behavior, four continuous (cigarettes per day [CPD], age at initiation of smoking, duration of smoking, and pack years), and three binary (ever versus never smoking, ≤10 versus >10 cigarettes per day [CPDBI], and current versus former smoking). Association testing for each single nucleotide polymorphism (SNP) was conducted by study and adjusted for age, cohabitation/marital status, education, site, and principal components of population substructure. None of the SNPs achieved genome-wide significance (p<10⁻⁷) in any combined analysis pooling evidence for association across the two studies; we observed between two and seven SNPs with p<10⁻⁵ for each of the seven measures. In the chr15q25.1 region spanning the nicotinic receptors CHRNA3 and CHRNA5, we identified multiple SNPs associated with CPD (p<10⁻³), including rs1051730, which has been associated with nicotine dependence, smoking intensity and lung cancer risk. In parallel, we selected 11,199 SNPs drawn from 359 a priori candidate genes and performed individual-gene and gene-group analyses. After adjusting for multiple tests conducted within each gene, we identified between two and five genes associated with each measure of smoking behavior. Besides CHRNA3 and CHRNA5, MAOA was associated with CPDBI (gene-level p<5.4×10⁻⁵), our analysis provides independent replication of the association between the chr15q25.1 region and smoking intensity and data for multiple other loci associated with smoking behavior that merit further follow-up.     

Molecular Typing of Lung Adenocarcinoma on Cytological Samples Using a Multigene Next Generation Sequencing Panel

Identification of driver mutations in lung adenocarcinoma has led to development of targeted agents that are already approved for clinical use or are in clinical trials. Therefore, the number of biomarkers that will be needed to assess is expected to rapidly increase. This calls for the implementation of methods probing the mutational status of multiple genes for inoperable cases, for which limited cytological or bioptic material is available. Cytology specimens from 38 lung adenocarcinomas were subjected to the simultaneous assessment of 504 mutational hotspots of 22 lung cancer-associated genes using 10 nanograms of DNA and Ion Torrent PGM next-generation sequencing. Thirty-six cases were successfully sequenced (95%). In 24/36 cases (67%) at least one mutated gene was observed, including EGFR, KRAS, PIK3CA, BRAF, TP53, PTEN, MET, SMAD4, FGFR3, STK11, MAP2K1. EGFR and KRAS mutations, respectively found in 6/36 (16%) and 10/36 (28%) cases, were mutually exclusive. Nine samples (25%) showed concurrent alterations in different genes. The next-generation sequencing test used is superior to current standard methodologies, as it interrogates multiple genes and requires limited amounts of DNA. Its applicability to routine cytology samples might allow a significant increase in the fraction of lung cancer patients eligible for personalized therapy.     

8-methyl-2'-deoxyguanosine incorporation into parallel DNA quadruplex structures

This paper concerns the Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) structural studies of the quadruple helix arrangements adopted by three tailored oligodeoxyribonucleotide analogues, namely d(TG^MeGGT), d(TGG^MeGT) and d(TGGG^MeT), where dG^Me represents a 8-methyl-2′-deoxyguanosine residue. The results of this study clearly demonstrate that the effects of the incorporation of dG^Me instead of a dG residue are strongly dependant upon the positioning of a single base replacement along the sequence. As such, d(TG^MeGGT), d(TGG^MeGT) have been found to form 4-fold symmetric quadruplexes with all strands parallel and equivalent to each other, each more stable than their natural counterpart. NMR experiments clearly indicate that [d(TG^MeGGT)]₄ possesses a G^Me-tetrad with all dG^Me residues in a syn-glycosidic conformation while an anti-arrangement is apparent for the four dG^Me of [d(TGG^MeGT)]₄. As the two complexes show a quite different CD behaviour, a possible relationship between the presence of residues adopting syn-glycosidic conformations and CD profiles is briefly discussed. As far as d(TGGG^MeT) is concerned, NMR data indicate that at 25°C it exists primarily as a single-strand conformation in equilibrium with minor amounts of a quadruplex structure.     

A Randomised Trial Evaluating the Safety and Immunogenicity of the Novel Single Oral Dose Typhoid Vaccine M01ZH09 in Healthy Vietnamese Children

Background

The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile.

Objectives

We conducted a randomised placebo controlled, single-blind trial to evaluate the safety and immunogenicity of M01ZH09 in healthy Vietnamese children aged 5 to 14 years.

Methods

Subjects were randomly assigned to receive either a nominal dose of 5×10⁹ CFU of M01ZH09 or placebo and were followed up for 28 days. The primary safety outcome was the proportion of subjects with any adverse event attributed to M01ZH09. The primary immunogenicity endpoint was the proportion of subjects who showed a positive immune response to M01ZH09 in the Salmonella Typhi lipopolysaccharide (LPS) specific serum IgA and IgG ELISA.

Principal Findings

One hundred and fifty-one children were enrolled, 101 subjects received M01ZH09 and 50 subjects received placebo. An intention to treat analysis was conducted. There were no serious adverse events and no bacteraemias. In the M01ZH09 group, 26 (26%; 95% CI, 18–5%) of 101 subjects experienced adverse events compared to 11 (22%; 95% CI, 12–36%) of 50 subjects in the placebo group (odds ratio (OR) [95%CI]  = 1.23 [0.550–2.747]; p = 0.691). Faecal shedding of S. Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 was detected in 51 (51%; 95% CI, 41–61%) of 100 M01ZH09 subjects. No shedding was detected beyond day 3. A positive immune response, defined as 70% increase (1.7 fold change) in LPS specific serum IgG (day 14 or 28) and/or 50% increase (1.5 fold change) in LPS specific serum IgA (day 7 or 14) from baseline was detected in 98 (97%; 95% CI, 92–99%) of 101 M01ZH09 recipients and 8 (16%; 95% CI, 7–29%) of 50 placebo recipients. Twenty-eight (100%; 95% CI, 88–100%) of 28 vaccine recipients who were evaluated in the LPS specific IgA ELISPOT assay showed a positive response compared to none of the 14 placebo recipients tested.

Conclusions

This was the first phase II trial of a novel oral candidate typhoid vaccine in children in an endemic country. M01ZH09 had an appropriate safety profile and was immunogenic in children.

Trial Registration

Controlled-trials.comISRCTN91111837     

Multi-organ characterization of mitochondrial genomic rearrangements in ad libitum and caloric restricted mice show striking somatic mitochondrial DNA rearrangements with age.

Mitochondrial DNA (mtDNA) rearrangements have been shown to accumulate with age in the post-mitotic tissues of a variety of animals and have been hypothesized to result in the age-related decline of mitochondrial bioenergetics leading to tissue and organ failure. Caloric restriction in rodents has been shown to extend life span supporting an association between bioenergetics and senescence. In the present study, we use full length mtDNA amplification by long-extension polymerase chain reaction (LX-PCR) to demonstrate that mice accumulate a wide variety of mtDNA rearrangements with age in post mitotic tissues. Similarly, using an alternative PCR strategy, we have found that 2-4 kb minicircles containing the origin of heavy-strand replication accumulate with age in heart but not brain. Analysis of mtDNA structure and conformation by Southern blots of unrestricted DNA resolved by field inversion gel electrophoresis have revealed that the brain mtDNAs of young animals contain the traditional linear, nicked, and supercoiled mtDNAs while old animals accumulate substantial levels of a slower migrating species we designate age-specific mtDNAs. In old caloric restricted animals, a wide variety of rearranged mtDNAs can be detected by LX-PCR in post mitotic tissues, but Southern blots of unrestricted DNA reveals a marked reduction in the levels of the age- specific mtDNA species. These observations confirm that mtDNA mutations accumulate with age in mice and suggest that caloric restriction impedes this progress.