bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromosome 4q31 locus in COPD is also associated with lung cancer
Authors: Young R.P. , Whittington C.F. , Hopkins R.J. , Hay B.A. , Epton M.J. , Black P.N. , Gamble G.D.
Published: December 2010

Chronic obstructive pulmonary disease (COPD) is the single greatest risk factor for lung cancer in smokers and is found in 50–90% of lung cancer cases. The link between COPD and lung cancer may stem in part from the matrix remodelling and repair processes underlying COPD, and the development of epithelial–mesenchymal transition (EMT) that underlies lung carcinogenesis. The Hedgehog-interacting protein (HHIP), which mediates the epithelial response (EMT) to smoking, has been implicated in COPD and lung cancer. Recent genome-wide and candidate gene studies of COPD implicate genetic variants on the chromosomal 4q31 (HHIP/glycophorin A (GYPA)) locus.

In a case–control study of smokers with normal lung function, COPD and lung cancer (subphenotyped for COPD), we show the GG genotype of the rs 1489759HHIPsingle-nucleotide polymorphism (SNP) and the CC genotype of the rs 2202507 GYPA SNP confers a “protective” effect on COPD (OR 0.59, p = 0.006 forHHIPand OR = 0.65, p = 0.006 forGYPA) and lung cancer (OR = 0.70 (p = 0.05) forHHIPand OR 0.70 (p = 0.02) forGYPA).

This study suggests that, in smokers, genetic variants of the 4q31 locus conferring a protective effect for COPD are also protective in lung cancer. We conclude that genetic susceptibility to lung cancer includes COPD-related gene variants.
Sinus venosus atrial septal defect in a 31-year-old female patient: a case for surgical repair
Authors: Gatzoulis M.A. , Giannakoulas G.
Published: December 2010

A 31-yr-old female patient previously diagnosed with idiopathic pulmonary arterial hypertension (PAH) was referred to our centre for further evaluation. Cardiac magnetic resonance imaging (MRI) and echocardiography revealed a superior sinus venosus atrial septal defect (ASD) with partial anomalous pulmonary venous drainage. Following re-diagnosis, surgical repair was considered. Despite a disproportionately high mean pulmonary artery pressure (P̄pa) of 47 mmHg relative to the patient's age and defect, the decision to operate was based on the absence of oxygen desaturation (either at rest or during exercise), cyanosis or abnormally elevated haemoglobin. Other operability criteria included normal sinus rhythm at rest, vasoreactivity and a pulmonary to systemic blood flow ratio of 1.9 at rest.

Surgical repair and continued advanced therapy with bosentan 125 mgb.i.d. and aspirin 75 mgo.d. proved successful, with post-operative improvements in exercise capacity and dyspnoea, and a reduction inP̄pato 25 mmHg. Keen to start a family, the risks of pregnancy were discussed. This case illustrates the importance of secondary PAH in sinus venosus ASD and the need to exclude a sinus venosus ASD in unexplained right ventricular dilatation. Access to expertise in congenital heart disease and use of cardiac MRI can improve diagnosis and, in turn, treatment decisions.
A hybrid blob-slice model for accurate and efficient detection of fluorescence labeled nuclei in 3D
Authors: Santella Anthony , Du Zhuo , Nowotschin Sonja , Hadjantonakis Anna-Katerina , Bao Zhirong
Published: November 2010

Background

To exploit the flood of data from advances in high throughput imaging of optically sectioned nuclei, image analysis methods need to correctly detect thousands of nuclei, ideally in real time. Variability in nuclear appearance and undersampled volumetric data make this a challenge.

Results

We present a novel 3D nuclear identification method, which subdivides the problem, first segmenting nuclear slices within each 2D image plane, then using a shape model to assemble these slices into 3D nuclei. This hybrid 2D/3D approach allows accurate accounting for nuclear shape but exploits the clear 2D nuclear boundaries that are present in sectional slices to avoid the computational burden of fitting a complex shape model to volume data. When tested over C. elegans, Drosophila, zebrafish and mouse data, our method yielded 0 to 3.7% error, up to six times more accurate as well as being 30 times faster than published performances. We demonstrate our method's potential by reconstructing the morphogenesis of the C. elegans pharynx. This is an important and much studied developmental process that could not previously be followed at this single cell level of detail.

Conclusions

Because our approach is specialized for the characteristics of optically sectioned nuclear images, it can achieve superior accuracy in significantly less time than other approaches. Both of these characteristics are necessary for practical analysis of overwhelmingly large data sets where processing must be scalable to hundreds of thousands of cells and where the time cost of manual error correction makes it impossible to use data with high error rates. Our approach is fast, accurate, available as open source software and its learned shape model is easy to retrain. As our pharynx development example shows, these characteristics make single cell analysis relatively easy and will enable novel experimental methods utilizing complex data sets.
Signatures of selection in loci governing major colour patterns in Heliconius butterflies and related species
Authors: Wu Grace C , Joron Mathieu , Jiggins Chris D
Published: November 2010

Background

Protein-coding change is one possible genetic mechanism underlying the evolution of adaptive wing colour pattern variation in Heliconius butterflies. Here we determine whether 38 putative genes within two major Heliconius patterning loci, HmYb and HmB, show evidence of positive selection. Ratios of nonsynonymous to synonymous nucleotide changes (ω) were used to test for selection, as a means of identifying candidate genes within each locus that control wing pattern.

Results

Preliminary analyses using 454 transcriptome and Bacterial Artificial Chromosome (BAC) sequences from three Heliconius species highlighted a cluster of genes within each region showing relatively higher rates of sequence evolution. Other genes within the region appear to be highly constrained, and no ω estimates exceeded one. Three genes from each locus with the highest average pairwise ω values were amplified from additional Heliconius species and races. Two selected genes, fizzy-like (HmYb) and DALR (HmB), were too divergent for amplification across species and were excluded from further analysis. Amongst the remaining genes, HM00021 and Kinesin possessed the highest background ω values within the HmYb and HmB loci, respectively. After accounting for recombination, these two genes both showed evidence of having codons with a signature of selection, although statistical support for this signal was not strong in any case.

Conclusions

Tests of selection reveal a cluster of candidate genes in each locus, suggesting that weak directional selection may be occurring within a small region of each locus, but coding changes alone are unlikely to explain the full range of wing pattern diversity. These analyses pinpoint many of the same genes believed to be involved in the control of colour patterning in Heliconius that have been identified through other studies implementing different research methods.
Novel intron markers to study the phylogeny of closely related mammalian species
Authors: Igea Javier , Juste Javier , Castresana Jose
Published: November 2010

Background

Multilocus phylogenies can be used to infer the species tree of a group of closely related species. In species trees, the nodes represent the actual separation between species, thus providing essential information about their evolutionary history. In addition, multilocus phylogenies can help in analyses of species delimitation, gene flow and genetic differentiation within species. However, few adequate markers are available for such studies.

Results

In order to develop nuclear markers that can be useful in multilocus studies of mammals, we analyzed the mammalian genomes of human, chimpanzee, macaque, dog and cow. Rodents were excluded due to their unusual genomic features. Introns were extracted from the mammalian genomes because of their greater genetic variability and ease of amplification from the flanking exons. To an initial set of more than 10,000 one-to-one orthologous introns we applied several filters to select introns that belong to single-copy genes, show neutral evolutionary rates and have an adequate length for their amplification. This analysis led to a final list of 224 intron markers randomly distributed along the genome. To experimentally test their validity, we amplified twelve of these introns in a panel of six mammalian species. The result was that seven of these introns gave rise to a PCR band of the expected size in all species. In addition, we sequenced these bands and analyzed the accumulation of substitutions in these introns in five pairs of closely related species. The results showed that the estimated genetic distances in the five species pairs was quite variable among introns and that this divergence cannot be directly predicted from the overall intron divergence in mammals.

Conclusions

We have designed a new set of 224 nuclear introns with optimal features for the phylogeny of closely related mammalian species. A large proportion of the introns tested experimentally showed a perfect amplification and enough variability in most species, indicating that this marker set can be very helpful in multilocus phylogenetics of mammals. Due to the lower variability and stronger stochasticity of nuclear markers with respect to mitochondrial genes, studies should be designed to make use of several markers like the ones designed here.
Bacillus cereus cytotoxins Hbl, Nhe and CytK are secreted via the Sec translocation pathway
Authors: Fagerlund Annette , Lindbäck Toril , Granum Per Einar
Published: November 2010

Background

Bacillus cereus and the closely related Bacillus thuringiensis are Gram positive opportunistic pathogens that may cause food poisoning, and the three secreted pore-forming cytotoxins Hbl, Nhe and CytK have been implicated as the causative agents of diarrhoeal disease. It has been proposed that the Hbl toxin is secreted using the flagellar export apparatus (FEA) despite the presence of Sec-type signal peptides. As protein secretion is of key importance in virulence of a microorganism, the mechanisms by which these toxins are secreted were further investigated.

Results

Sec-type signal peptides were identified in all toxin components, and secretion of Hbl component B was shown to be dependent on an intact Sec-type signal peptide sequence. Further indication that secretion of Hbl, Nhe and CytK is dependent on the Sec translocation pathway, the main pathway on which bacterial secretion relies, was suggested by the observed intracellular accumulation and reduced secretion of the toxins in cultures supplemented with the SecA inhibitor sodium azide. Although a FEA deficient strain (a flhA mutant) showed reduced toxin expression and reduced cytotoxicity, it readily secreted overexpressed Hbl B, showing that the FEA is not required for Hbl secretion. Thus, the concurrent lack of flagella and reduced toxin secretion in the FEA deficient strain may point towards the presence of a regulatory link between motility and virulence genes, rather than FEA-dependent toxin secretion.

Conclusions

The Hbl, Nhe and CytK toxins appear to be secreted using the Sec pathway, and the reduced Hbl expression of a FEA deficient strain was shown not to be due to a secretion defect.
Ampicillin/Sulbactam versus Cefuroxime as antimicrobial prophylaxis for cesarean delivery: a randomized study
Authors: Ziogos Eleftherios , Tsiodras Sotirios , Matalliotakis Ioannis , Giamarellou Helen , Kanellakopoulou Kyriaki
Published: November 2010

Background

The efficacy and safety of a single dose of ampicillin/sulbactam compared to a single dose of cefuroxime at cord clamp for prevention of post-cesarean infectious morbidity has not been assessed.

Methods

Women scheduled for cesarean delivery were randomized to receive a single dose of either 3 g of ampicillin-sulbactam or 1.5 g of cefuroxime intravenously, after umbilical cord clamping. An evaluation for development of postoperative infections and risk factor analysis was performed.

Results

One hundred and seventy-six patients (median age 28 yrs, IQR: 24-32) were enrolled in the study during the period July 2004 - July 2005. Eighty-five (48.3%) received cefuroxime prophylaxis and 91 (51.7%) ampicillin/sulbactam. Postoperative infection developed in 5 of 86 (5.9%) patients that received cefuroxime compared to 8 of 91 (8.8%) patients that received ampicillin/sulbactam (p = 0.6). In univariate analyses 6 or more vaginal examinations prior to the operation (p = 0.004), membrane rupture for more than 6 hours (p = 0.08) and blood loss greater than 500 ml (p = 0.018) were associated with developing a postoperative surgical site infection (SSI). In logistic regression having 6 or more vaginal examinations was the most significant risk factor for a postoperative SSI (OR 6.8, 95% CI: 1.4-33.4, p = 0.019). Regular prenatal follow-up was associated with a protective effect (OR 0.04, 95% CI: 0.005-0.36, p = 0.004).

Conclusions

Ampicillin/sulbactam was as safe and effective as cefuroxime when administered for the prevention of infections following cesarean delivery.

Trial registration

Clinicaltrials.gov identifier: NCT01138852
Genetic variants in the TIRAP gene are associated with increased risk of sepsis-associated acute lung injury
Authors: Song Zhenju , Tong Chaoyang , Sun Zhan , Shen Yao , Yao Chenling , Jiang Jinjun , Yin Jun , Gao Lei , Song Yuanlin , Bai Chunxue
Published: November 2010

Background

Toll like receptors (TLRs) signaling pathways, including the adaptor protein Mal encoded by the TIRAP gene, play a central role in the development of acute lung injury (ALI). Recently, the TIRAP variants have been described association with susceptibility to inflammatory diseases. The aim of this study was to investigate whether genetic variants in TIRAP are associated with the development of ALI.

Methods

A case-control collection from Han Chinese of 298 healthy subjects, 278 sepsis-associated ALI and 288 sepsis alone patients were included. Three tag single nucleotide polymorphisms (SNPs) of the TIRAP gene and two additional SNPs that have previously showed association with susceptibility to other inflammatory diseases were genotyped by direct sequencing. The differences of allele, genotype and haplotype frequencies were evaluated between three groups.

Results

The minor allele frequencies of both rs595209 and rs8177375 were significantly increased in ALI patients compared with both healthy subjects (odds ratio (OR) = 1.47, 95% confidence interval (CI):1.15-1.88, P = 0.0027 and OR = 1.97, 95% CI: (1.38-2.80), P = 0.0001, respectively) and sepsis alone patients (OR = 1.44, 95% CI: 1.12-1.85, P = 0.0041 and OR = 1.82, 95% CI: 1.28-2.57, P = 0.00079, respectively). Haplotype consisting of these two associated SNPs strengthened the association with ALI susceptibility. The frequency of haplotype AG (rs595209A, rs8177375G) in the ALI samples was significantly higher than that in the healthy control group (OR = 2.13, 95% CI: 1.46-3.09, P = 0.00006) and the sepsis alone group (OR = 2.24, 95% CI: 1.52-3.29, P = 0.00003). Carriers of the haplotype CA (rs595209C, rs8177375A) had a lower risk for ALI compared with healthy control group (OR = 0.69, 95% CI: 0.54-0.88, P = 0.0003) and sepsis alone group (OR = 0.71, 95% CI: 0.55-0.91, P = 0.0006). These associations remained significant after adjustment for covariates in multiple logistic regression analysis and for multiple comparisons.

Conclusions

These results indicated that genetic variants in the TIRAP gene might be associated with susceptibility to sepsis-associated ALI in Han Chinese population. However, the association needs to be replicated in independent studies.
Focused ultrasound-mediated bbb disruption is associated with an increase in activation of AKT: experimental study in rats
Authors: Jalali Shahrzad , Huang Yuexi , Dumont Daniel J , Hynynen Kullervo
Published: December 2010

Background

The Blood Brain Barrier (BBB) maintains the homeostasis of central nervous system by preventing the free passage of macromolecules from the systemic circulation into the brain. This normal physiological function of the BBB presents a challenge for delivery of therapeutic compounds into the brain. Recent studies have shown that the application of focused ultrasound together with ultrasound contrast agent (microbubbles) temporarily increases the permeability of the BBB. This effect is associated with breakdown of tight junctions, the structures that regulate the paracellular permeability of the endothelial cell layer. The influence of this ultrasound effect on the activation of intracellular signaling proteins is currently not well understood. Therefore, the aim of this study was to investigate the activation of cell survival signaling molecules in response to ultrasound-mediated BBB opening;

Methods

The BBB was disrupted in two four-spot lines (1-1.5 mm spacing) along the right hemisphere of rat brain with ultrasound beams (0.3 MPa, 120 s, 10 ms bursts, repetition frequency = 1 Hz) in the presence Definity microbubbles. Contrast-enhanced MRI images were acquired to assess the extent of BBB opening upon which the animals were sacrificed and the brains removed and processed for biochemical and immunohistochemical analyses;

Results

Immunoblotting of sonicated brain lysates resolved by SDS-PAGE demonstrated an increase in phosphorylation of Akt and its downstream signaling molecule, GSK3β, while the phosphorylation of MAPK remained unchanged. The elevated levels of pAkt and pGSK3β are still evident after 24 hours post-sonication, a time point where the integrity of the BBB is known to be re-established. Furthermore, immunofluoresence staining localized this increase in pAkt and pGSK3β levels to neuronal cells flanking the region of the disrupted BBB;

Conclusions

Our data demonstrates that ultrasound-mediated BBB disruption causes an activation of the Akt signaling pathway in neuronal cells surrounding the disrupted BBB.
BRCA1-mutated and basal-like breast cancers have similar aCGH profiles and a high incidence of protein truncating TP53 mutations
Authors: Holstege Henne , Horlings Hugo M , Velds Arno , Langerød Anita , Børresen-Dale Anne-Lise , van de Vijver Marc J , Nederlof Petra M , Jonkers Jos
Published: November 2010

Background

Basal-like breast cancers (BLBC) are aggressive breast cancers for which, so far, no targeted therapy is available because they typically lack expression of hormone receptors and HER2. Phenotypic features of BLBCs, such as clinical presentation and early age of onset, resemble those of breast tumors fromBRCA1-mutation carriers. The genomic instability ofBRCA1-mutated tumors can be effectively targeted with DNA-damaging agents and poly-(ADP-ribose) polymerase 1 (PARP1) inhibitors. Molecular similarities between BLBCs andBRCA1-mutated tumors may therefore provide predictive markers for therapeutic response of BLBCs.

Methods

There are several known molecular features characteristic forBRCA1-mutated breast tumors: 1) increased numbers of genomic aberrations, 2) a distinct pattern of genomic aberrations, 3) a high frequency ofTP53mutations and 4) a high incidence of complex, protein-truncatingTP53mutations. We compared the frequency ofTP53mutations and the pattern and amount of genomic aberrations betweenBRCA1-mutated breast tumors, BLBCs and luminal breast tumors byTP53gene sequencing and array-based comparative genomics hybridization (aCGH) analysis.

Results

We found that the high incidence of protein truncatingTP53mutations and the pattern and amount of genomic aberrations specific for BRCA1-mutated breast tumors are also characteristic for BLBCs and different from luminal breast tumors.

Conclusions

Complex, protein truncating TP53 mutations in BRCA1-mutated tumors may be a direct consequence of genomic instability caused by BRCA1 loss, therefore, the presence of these types of TP53 mutations in sporadic BLBCs might be a hallmark of BRCAness and a potential biomarker for sensitivity to PARP inhibition. Also, our data suggest that a small subset of genomic regions may be used to identify BRCA1-like BLBCs. BLBCs share molecular features that were previously found to be specific for BRCA1-mutated breast tumors. These features might be useful for the identification of tumors with increased sensitivity to (high-dose or dose-dense) alkylating agents and PARP inhibitors.
Tissue-specific human beta-defensins (HBD)1, HBD2, and HBD3 secretion from human extra-placental membranes stimulated with Escherichia coli
Authors: Garcia-Lopez Guadalupe , Flores-Espinosa Pilar , Zaga-Clavellina Veronica
Published: December 2010

Background

During an ascending infection along the reproductive tract, the extra-placental membranes must act as a selective and competent barrier against pathogens. Human beta defensins (HBD)1, HBD2, and HBD3 are key elements of innate immunity that are secreted to neutralize/control the progression of infection.

Methods

Full-thickness membranes were mounted on a Transwell device, constituted by two independent chambers, 1 × 10(6) CFU/ml ofEscherichia coliwere added to either the amnion (AMN) or the choriodecidual (CHD) face or to both. Secretion profiles of HBD1, HBD2, and HBD3 to the culture medium were quantified by ELISA.

Results

In comparison with basal conditions, the secretion profile of HBD1 remained without significant changes; HBD2 level in CHD and AMN increased 1.9- and 1.4-times, respectively, after stimulation with bacteria. HBD3 secretion level increased significantly (7.8 +/- 1.9 pg/micrograms) in the CHD but only if the stimulus was applied on the AMN side.

Conclusions

Selective stimulation of extra-placental membranes withE. coli, results in a tissue specific secretion of HBD1, HBD2, and HBD3 mainly in the CHD, which is the first infected region during an ascending infection.
Epigenetic regulation of CD133 and tumorigenicity of CD133 positive and negative endometrial cancer cells
Authors: Friel Anne M , Zhang Ling , Curley Michael D , Therrien Vanessa A , Sergent Petra A , Belden Sarah E , Borger Darrell R , Mohapatra Gayatry , Zukerberg Lawrence R , Foster Rosemary , Rueda Bo R
Published: December 2010

Background

Recent data provide significant evidence to support the hypothesis that there are sub-populations of cells within solid tumors that have an increased tumor initiating potential relative to the total tumor population. CD133, a cell surface marker expressed on primitive cells of neural, hematopoietic, endothelial and epithelial lineages has been identified as a marker for tumor initiating cells in solid tumors of the brain, colon, pancreas, ovary and endometrium. Our objectives were to assess the relative level of CD133 expressing cells in primary human endometrial tumors, confirm their tumorigenic potential, and determine whether CD133 expression was epigenetically modified.

Methods

We assessed CD133 expression in primary human endometrial tumors by flow cytometry and analyzed the relative tumorigenicity of CD133+ and CD133- cells in anin vivoNOD/SCID mouse model. We assessed potential changes in CD133 expression over the course of serial transplantation by immunofluorescence and flow cytometry. We further examined CD133 promoter methylation and expression in normal endometrium and malignant tumors.

Results

As determined by flow cytometric analysis, the percentage of CD133+ cells in primary human endometrial cancer samples ranged from 5.7% to 27.4%. In addition, we confirmed the tumor initiating potential of CD133+ and CD133-cell fractions in NOD/SCID mice. Interestingly, the percentage of CD133+ cells in human endometrial tumor xenografts, as evidenced by immunofluorescence, increased with serial transplantation although this trend was not consistently detected by flow cytometry. We also determined that the relative levels of CD133 increased in endometrial cancer cell lines following treatment with 5-aza-2'-deoxycytidine suggesting a role for methylation in the regulation of CD133. To support this finding, we demonstrated that regions of the CD133 promoter were hypomethylated in malignant endometrial tissue relative to benign control endometrial tissue. Lastly, we determined that methylation of the CD133 promoter decreases over serial transplantation of an endometrial tumor xenograft.

Conclusions

These findings support the hypotheses that CD133 expression in endometrial cancer may be epigenetically regulated and that cell fractions enriched for CD133+ cells may well contribute to endometrial cancer tumorigenicity, pathology and recurrence.
Membrane topology analysis of HIV-1 envelope glycoprotein gp41
Authors: Liu Shujun , Kondo Naoyuki , Long Yufei , Xiao Dan , Iwamoto Aikichi , Matsuda Zene
Published: November 2010

Background

The gp41 subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP) that is only active in the cytoplasm. The tag protein (HaloTag) and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system.

Results

In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells) supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells), the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability.

Conclusions

It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.
The membrane-spanning domain of gp41 plays a critical role in intracellular trafficking of the HIV envelope protein
Authors: Miyauchi Kosuke , Curran A Rachael , Long Yufei , Kondo Naoyuki , Iwamoto Aikichi , Engelman Donald M , Matsuda Zene
Published: November 2010

Background

The sequences of membrane-spanning domains (MSDs) on the gp41 subunit are highly conserved among many isolates of HIV-1. The GXXXG motif, a potential helix-helix interaction motif, and an arginine residue (rare in hydrophobic MSDs) are especially well conserved. These two conserved elements are expected to locate on the opposite sides of the MSD, if the MSD takes a α-helical secondary structure. A scanning alanine-insertion mutagenesis was performed to elucidate the structure-function relationship of gp41 MSD.

Results

A circular dichroism analysis of a synthetic gp41 MSD peptide determined that the secondary structure of the gp41 MSD was α-helical. We then performed a scanning alanine-insertion mutagenesis of the entire gp41 MSD, progressively shifting the relative positions of MSD segments around the helix axis. Altering the position of Gly694, the last residue of the GXXXG motif, relative to Arg696 (the number indicates the position of the amino acid residues in HXB2 Env) around the axis resulted in defective fusion. These mutants showed impaired processing of the gp160 precursor into gp120 and gp41. Furthermore, these Env mutants manifested inefficient intracellular transport in the endoplasmic reticulum and Golgi regions. Indeed, a transplantation of the gp41 MSD portion into the transmembrane domain of another membrane protein, Tac, altered its intracellular distribution. Our data suggest that the intact MSD α-helix is critical in the intracellular trafficking of HIV-1 Env.

Conclusions

The relative position between the highly conserved GXXXG motif and an arginine residue around the gp41 MSD α-helix is critical for intracellular trafficking of HIV-1 Env. The gp41 MSD region not only modulates membrane fusion but also controls biosynthesis of HIV-1 Env.
Polymorphisms of HIV-2 integrase and selection of resistance to raltegravir
Authors: Perez-Bercoff Danielle , Triqueneaux Perrine , Lambert Christine , Oumar Aboubacar Alassane , Ternes Anne-Marie , Dao Sounkalo , Goubau Patrick , Schmit Jean-Claude , Ruelle Jean
Published: November 2010

Background

Human Immunodeficiency Virus type 2 is naturally resistant to some antiretroviral drugs, restricting therapeutic options for patients infected with HIV-2. Regimens including integrase inhibitors (INI) seem to be effective, but little data on HIV-2 integrase (IN) polymorphisms and resistance pathways are available.

Materials and methods

Theintegrasecoding sequence from 45 HIV-2-infected, INI-naïve, patients was sequenced and aligned against the ROD (group A) or EHO (group B) reference strains and polymorphic or conserved positions were analyzed.

To select for raltegravir (RAL)-resistant variantsin vitro, the ROD strain was cultured under increasing sub-optimal RAL concentrations for successive rounds. The phenotype of the selected variants was assessed using an MTT assay.

Results

We describeintegrasegene polymorphisms in HIV-2 clinical isolates from 45 patients. Sixty-seven percent of the integrase residues were conserved. The HHCC Zinc coordination motif, the catalytic triad DDE motif, and AA involved in IN-DNA binding and correct positioning were highly conserved and unchanged with respect to HIV-1 whereas the connecting residues of the N-terminal domain, the dimer interface and C-terminal LEDGF binding domain were highly conserved but differed from HIV-1. The N155 H INI resistance-associated mutation (RAM) was detected in the virus population from one ARV-treated, INI-naïve patient, and the 72I and 201I polymorphisms were detected in samples from 36 and 38 patients respectively. No other known INI RAM was detected.

Under RAL selective pressurein vitro, a ROD variant carrying the Q91R+I175M mutations was selected. The Q91R and I175M mutations emerged simultaneously and conferred phenotypic resistance (13-fold increase in IC50). The Q91R+I175M combination was absent from all clinical isolates. Three-dimensional modeling indicated that residue 91 lies on the enzyme surface, at the entry of a pocket containing the DDE catalytic triad and that adding a positive charge (Gln to Arg) might compromise IN-RAL affinity.

Conclusions

HIV-2 polymorphisms from 45 INI-naïve patients are described. Conserved regions as well as frequencies of HIV-2 IN polymorphisms were comparable to HIV-1. Two new mutations (Q91R and I175M) that conferred high resistance to RAL were selectedin vitro, which might affect therapeutic outcome.
Characterization of Hepatitis B virus (HBV) genotypes in patients from Rondônia, Brazil
Authors: Santos Alcione O , Alvarado-Mora Mónica V , Botelho Lívia , Vieira Deusilene S , Pinho João R Rebello , Carrilho Flair J , Honda Eduardo R , Salcedo Juan M
Published: November 2010

Background

Hepatitis B virus (HBV) can be classified into nine genotypes (A-I) defined by sequence divergence of more than 8% based on the complete genome. This study aims to identify the genotypic distribution of HBV in 40 HBsAg-positive patients from Rondônia, Brazil. A fragment of 1306 bp partially comprising surface and polymerase overlapping genes was amplified by PCR. Amplified DNA was purified and sequenced. Amplified DNA was purified and sequenced on an ABI PRISM® 377 Automatic Sequencer (Applied Biosystems, Foster City, CA, USA). The obtained sequences were aligned with reference sequences obtained from the GenBank using Clustal X software and then edited with Se-Al software. Phylogenetic analyses were conducted by the Markov Chain Monte Carlo (MCMC) approach using BEAST v.1.5.3.

Results

The subgenotypes distribution was A1 (37.1%), D3 (22.8%), F2a (20.0%), D4 (17.1%) and D2 (2.8%).

Conclusions

These results for the first HBV genotypic characterization in Rondônia state are consistent with other studies in Brazil, showing the presence of several HBV genotypes that reflects the mixed origin of the population, involving descendants from Native Americans, Europeans, and Africans.
Inflammatory responses to acute pneumovirus infection in neonatal mice
Authors: Bonville Cynthia A , Ptaschinski Catherine , Percopo Caroline M , Rosenberg Helene F , Domachowske Joseph B
Published: November 2010

Background

The innate immune responses of neonates differ dramatically from those of adults. Here we examine the acute inflammatory responses of neonatal and weanling mice infected with pneumonia virus of mice (PVM), a rodent pathogen (family Paramyxoviridae, genus Pneumovirus) that replicates the sequelae of severe respiratory syncytial virus infection.

Results

We demonstrate that virus replication proceeds indistinguishably in all age groups (inoculated at 1, 2, 3 and 4 weeks of age), although inflammatory responses vary in extent and character. Some of the biochemical mediators detected varied minimally with age at inoculation. Most of the mediators evaluated demonstrated elevated expression over baseline correlating directly with age at the time of virus inoculation. Among the latter group are CCL2, CCL3, and IFN-γ, all cytokines previously associated with PVM-induced inflammatory pathology in mature mice. Likewise, we detect neutrophil recruitment to lung tissue in all age groups, but recruitment is most pronounced among the older (3 - 4 week old) mice. Interestingly, all mice exhibit failure to thrive, lagging in expected weight gain for given age, including the youngest mice that present little overt evidence of inflammation.

Conclusions

Our findings among the youngest mice may explain in part the phenomenon of atypical or minimally symptomatic respiratory infections in human neonates, which may be explored further with this infection model.
Evaluation of unilateral cage-instrumented fixation for lumbar spine
Authors: Chang Ti-Sheng , Chang Jia-Hao , Wang Chien-Shiung , Chen Hung-Yi , Cheng Ching-Wei
Published: November 2010

Background

To investigate how unilateral cage-instrumented posterior lumbar interbody fusion (PLIF) affects the three-dimensional flexibility in degenerative disc disease by comparing the biomechanical characteristics of unilateral and bilateral cage-instrumented PLIF.

Methods

Twelve motion segments in sheep lumbar spine specimens were tested for flexion, extension, axial rotation, and lateral bending by nondestructive flexibility test method using a nonconstrained testing apparatus. The specimens were divided into two equal groups. Group 1 received unilateral procedures while group 2 received bilateral procedures. Laminectomy, facectomy, discectomy, cage insertion and transpedicle screw insertion were performed sequentially after testing the intact status. Changes in range of motion (ROM) and neutral zone (NZ) were compared between unilateral and bilateral cage-instrumented PLIF.

Results

Both ROM and NZ, unilateral cage-instrumented PLIF and bilateral cage-instrumented PLIF, transpedicle screw insertion procedure did not revealed a significant difference between flexion-extension, lateral bending and axial rotation direction except the ROM in the axial rotation. The bilateral group's ROM (-1.7 ± 0. 8) of axial rotation was decreased significantly after transpedicle screw insertion procedure in comparison with the unilateral group (-0.2 ± 0.1). In the unilateral cage-instrumented PLIF group, the transpedicle screw insertion procedure did not demonstrate a significant difference between right and left side in the lateral bending and axial rotation direction.

Conclusions

Based on the results of this study, unilateral cage-instrumented PLIF and bilateral cage-instrumented PLIF have similar stability after transpedicle screw fixation in the sheep spine model. The unilateral approach can substantially reduce exposure requirements. It also offers the biomechanics advantage of construction using anterior column support combined with pedicle screws just as the bilateral cage-instrumented group. The unpleasant effect of couple motion resulting from inherent asymmetry was absent in the unilateral group.
The diagnosis of inherited metabolic diseases by microarray gene expression profiling
Authors: Arenas Hernandez Monica , Schulz Reiner , Chaplin Tracy , Young Bryan D , Perrett David , Champion Michael P , Taanman Jan-Willem , Fensom Anthony , Marinaki Anthony M
Published: December 2010

Background

Inherited metabolic diseases (IMDs) comprise a diverse group of generally progressive genetic metabolic disorders of variable clinical presentations and severity. We have undertaken a study using microarray gene expression profiling of cultured fibroblasts to investigate 68 patients with a broad range of suspected metabolic disorders, including defects of lysosomal, mitochondrial, peroxisomal, fatty acid, carbohydrate, amino acid, molybdenum cofactor, and purine and pyrimidine metabolism. We aimed to define gene expression signatures characteristic of defective metabolic pathways.

Methods

Total mRNA extracted from cultured fibroblast cell lines was hybridized to Affymetrix U133 Plus 2.0 arrays. Expression data was analyzed for the presence of a gene expression signature characteristic of an inherited metabolic disorder and for genes expressing significantly decreased levels of mRNA.

Results

No characteristic signatures were found. However, in 16% of cases, disease-associated nonsense and frameshift mutations generating premature termination codons resulted in significantly decreased mRNA expression of the defective gene. The microarray assay detected these changes with high sensitivity and specificity.

Conclusion

In patients with a suspected familial metabolic disorder where initial screening tests have proven uninformative, microarray gene expression profiling may contribute significantly to the identification of the genetic defect, shortcutting the diagnostic cascade.
First detection of African Swine Fever Virus in Ornithodoros porcinus in Madagascar and new insights into tick distribution and taxonomy
Authors: Ravaomanana Julie , Michaud Vincent , Jori Ferran , Andriatsimahavandy Abel , Roger François , Albina Emmanuel , Vial Laurence
Published: November 2010

Background

African Swine Fever Virus has devastated more than the half of the domestic pig population in Madagascar since its introduction, probably in 1997-1998. One of the hypotheses to explain its persistence on the island is its establishment in local Ornithodoros soft ticks, whose presence has been reported in the past from the north-western coast to the Central Highlands. The aim of the present study was to verify such hypothesis by conducting tick examinations in three distinct zones of pig production in Madagascar where African Swine Fever outbreaks have been regularly reported over the past decade and then to improve our knowledge on the tick distribution and taxonomy.

Results

Ornithodoros ticks were only found in one pig farm in the village of Mahitsy, north-west of Antananarivo in the Central Highlands, whereas the tick seemed to be absent from the two other study zones near Ambatondrazaka and Marovoay. Using 16SrDNA PCR amplification and sequencing, it was confirmed that the collected ticks belonged to the O. porcinus species and is closely related to the O. p. domesticus sub-species Walton, 1962. ASFV was detected in 7.14% (13/182) of the field ticks through the amplification of part of the viral VP72 gene, and their ability to maintain long-term infections was confirmed since all the ticks came from a pig building where no pigs or any other potential vertebrate hosts had been introduced for at least four years.

Conclusions

Considering these results, O. porcinus is a reservoir for ASFV and most likely acts as vector for ASFV in Madagascar, but its apparent restricted distribution may limit its role in the epidemiology of the disease in domestic pigs.
NMDAR2B tyrosine phosphorylation regulates anxiety-like behavior and CRF expression in the amygdala
Authors: Delawary Mina , Tezuka Tohru , Kiyama Yuji , Yokoyama Kazumasa , Inoue Takeshi , Hattori Satoko , Hashimoto Ryota , Umemori Hisashi , Manabe Toshiya , Yamamoto Tadashi , Nakazawa Takanobu
Published: November 2010

Background

Anxiety disorders are a highly prevalent and disabling class of psychiatric disorders. There is growing evidence implicating the glutamate system in the pathophysiology and treatment of anxiety disorders, though the molecular mechanism by which the glutamate system regulates anxiety-like behavior remains unclear.

Results

In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to anxiety-like behavior. The GluN2B subunit of the NMDA receptor is tyrosine-phosphorylated: Tyr-1472 is the major phosphorylation site. Homozygous knock-in mice that express a Tyr-1472-Phe mutant of GluN2B, which prevents phosphorylation of this site, show enhanced anxiety-like behavior in the elevated plus-maze test. Expression of corticotropin-releasing factor (CRF), which is important for the regulation of anxiety-like behavior, is increased in the amygdala of the knock-in mice. Furthermore, injection of CRF receptor antagonist attenuated the enhanced anxiety-like behavior of the knock-in mice. We also show that elevated plus-maze exposure simultaneously induced de-phosphorylation of Tyr-1472 and increased CRF expression.

Conclusions

These data suggest that Tyr-1472 phosphorylation on GluN2B is important for anxiety-like behavior by negative regulation of CRF expression in the amygdala.
Comparative genomics reveals birth and death of fragile regions in mammalian evolution
Authors: Alekseyev Max A , Pevzner Pavel A
Published: November 2010

Background

An important question in genome evolution is whether there exist fragile regions (rearrangement hotspots) where chromosomal rearrangements are happening over and over again. Although nearly all recent studies supported the existence of fragile regions in mammalian genomes, the most comprehensive phylogenomic study of mammals raised some doubts about their existence.

Results

Here we demonstrate that fragile regions are subject to a birth and death process, implying that fragility has a limited evolutionary lifespan.

Conclusions

This finding implies that fragile regions migrate to different locations in different mammals, explaining why there exist only a few chromosomal breakpoints shared between different lineages. The birth and death of fragile regions as a phenomenon reinforces the hypothesis that rearrangements are promoted by matching segmental duplications and suggests putative locations of the currently active fragile regions in the human genome.
Population sequencing of two endocannabinoid metabolic genes identifies rare and common regulatory variants associated with extreme obesity and metabolite level
Authors: Harismendy Olivier , Bansal Vikas , Bhatia Gaurav , Nakano Masakazu , Scott Michael , Wang Xiaoyun , Dib Colette , Turlotte Edouard , Sipe Jack C , Murray Sarah S , Deleuze Jean Francois , Bafna Vineet , Topol Eric J , Frazer Kelly A
Published: November 2010

Background

Targeted re-sequencing of candidate genes in individuals at the extremes of a quantitative phenotype distribution is a method of choice to gain information on the contribution of rare variants to disease susceptibility. The endocannabinoid system mediates signaling in the brain and peripheral tissues involved in the regulation of energy balance, is highly active in obese patients, and represents a strong candidate pathway to examine for genetic association with body mass index (BMI).

Results

We sequenced two intervals (covering 188 kb) encoding the endocannabinoid metabolic enzymes fatty-acid amide hydrolase (FAAH) and monoglyceride lipase (MGLL) in 147 normal controls and 142 extremely obese cases. After applying quality filters, we called 1,393 high quality single nucleotide variants, 55% of which are rare, and 143 indels. Using single marker tests and collapsed marker tests, we identified four intervals associated with BMI: the FAAH promoter, the MGLL promoter, MGLL intron 2, and MGLL intron 3. Two of these intervals are composed of rare variants and the majority of the associated variants are located in promoter sequences or in predicted transcriptional enhancers, suggesting a regulatory role. The set of rare variants in the FAAH promoter associated with BMI is also associated with increased level of FAAH substrate anandamide, further implicating a functional role in obesity.

Conclusions

Our study, which is one of the first reports of a sequence-based association study using next-generation sequencing of candidate genes, provides insights into study design and analysis approaches and demonstrates the importance of examining regulatory elements rather than exclusively focusing on exon sequences.
Neurotransplantation of stem cells genetically modified to express human dopamine transporter reduces alcohol consumption
Authors: Grammatopoulos Tom N , Jones Susan M , Yoshimura Masami , Hoover Brian R , Das Mita , Snyder Evan Y , Larson Gaynor A , Zahniser Nancy R , Tabakoff Boris , Zawada WMichael
Published: December 2010

Introduction

Regulated neurotransmitter actions in the mammalian central nervous system determine brain function and control peripheral organs and behavior. Although drug-seeking behaviors, including alcohol consumption, depend on central neurotransmission, modification of neurotransmitter actions in specific brain nuclei remains challenging. Herein, we report a novel approach for neurotransmission modificationin vivoby transplantation of stem cells engineered to take up the neurotransmitter dopamine (DA) efficiently through the action of the human dopamine transporter (hDAT). As a functional test in mice, we used voluntary alcohol consumption, which is known to release DA in nucleus accumbens (NAC), an event hypothesized to help maintain drug-seeking behavior. We reasoned that reducing extracellular DA levels, by engrafting into NAC DA-sequestering stem cells expressing hDAT, would alter alcohol intake.

Methods

We have generated a neural stem cell line stably expressing the hDAT. Uptake kinetics of DA were determined to select a clone for transplantation. These genetically modified stem cells (or cells transfected with a construct lacking the hDAT sequence) were transplanted bilaterally into the NAC of wild-type mice trained to consume 10% alcohol in a two-bottle free-choice test for alcohol consumption. Alcohol intake was then ascertained for 1 week after transplantation, and brain sections through the NAC were examined for surviving grafted cells.

Results

Modified stem cells expressed hDAT and uptaken DA selectively via hDAT. Mice accustomed to drinking 10% ethanol by free choice reduced their alcohol consumption after being transplanted with hDAT-expressing stem cells. By contrast, control stem cells lacked that effect. Histologic examination revealed surviving stem cells in the NAC of all engrafted brains.

Conclusions

Our findings represent proof of principle suggesting that genetically engineered stem cells can be useful for exploring the role of neurotransmitters (or other signaling molecules) in alcohol consumption and potentially in other aspects of brain function.