bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractBackgroundThe 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.ResultsIn 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.ConclusionsIt 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

AbstractBackgroundThe 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.ResultsA 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.ConclusionsThe 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

AbstractBackgroundHuman 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 methodsTheintegrasecoding 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.ResultsWe 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.ConclusionsHIV-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

Abstract

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

Abstract

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

Abstract

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

Abstract

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

Abstract

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

Abstract

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

Abstract
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

Abstract
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

AbstractIntroductionRegulated 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.MethodsWe 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.ResultsModified 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.ConclusionsOur 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.