V3 Domain Research Articles

Amplicon-Based Pyrosequencing Reveals High Diversity of Protistan Parasites in Ships' Ballast Water: Implications for Biogeography and Infectious Diseases.

Ships' ballast water (BW) commonly moves macroorganisms and microorganisms across the world's oceans and along coasts; however, the majority of these microbial transfers have gone undetected. We applied high-throughput sequencing methods to identify microbial eukaryotes, specifically emphasizing the protistan parasites, in ships' BW collected from vessels calling to the Chesapeake Bay (Virginia and Maryland, USA) from European and Eastern Canadian ports. We utilized tagged-amplicon 454 pyrosequencing with two general primer sets, amplifying either the V4 or V9 domain of the small subunit (SSU) of the ribosomal RNA (rRNA) gene complex, from total DNA extracted from water samples collected from the ballast tanks of bulk cargo vessels. We detected a diverse group of protistan taxa, with some known to contain important parasites in marine systems, including Apicomplexa (unidentified apicomplexans, unidentified gregarines, Cryptosporidium spp.), Dinophyta (Blastodinium spp., Euduboscquella sp., unidentified syndinids, Karlodinium spp., Syndinium spp.), Perkinsea (Parvilucifera sp.), Opisthokonta (Ichthyosporea sp., Pseudoperkinsidae, unidentified ichthyosporeans), and Stramenopiles (Labyrinthulomycetes). Further characterization of groups with parasitic taxa, consisting of phylogenetic analyses for four taxa (Cryptosporidium spp., Parvilucifera spp., Labyrinthulomycetes, and Ichthyosporea), revealed that sequences were obtained from both known and novel lineages. This study demonstrates that high-throughput sequencing is a viable and sensitive method for detecting parasitic protists when present and transported in the ballast water of ships. These data also underscore the potential importance of human-aided dispersal in the biogeography of these microbes and emerging diseases in the world's oceans.

Continuously Ingesting Fructooligosaccharide Can't Maintain Rats’ GutBifidobacteriumat a High Level

Fructooligosaccharide (FOS) has been reported to increase Lactobacillus and Bifidobacterium populations in animal and human gut. Hence, it has been utilized to regulate the balance of gut microbiota. In this study, we compared the effects of high-FOS (HFOS) diet on normal and obese rats' gut Lactobacillus and Bifidobacterium, with high-soybean-fibers (HSF) diet as control. The results showed that the level of Bifidobacterium population substantially increased at week 4 in groups of rats fed the HFOS diet (P < 0.05), but significantly reduced to a small level at week 8 (P < 0.05); the abundance of Lactobacillus was increased in normal rats (P < 0.05), but decreased in obese rats (P < 0.05). The HSF diet did not promote the growth of Lactobacillus and Bifidobacterium in rats' gut. The findings suggested that Bifidobacterium population could not be maintained at a high level when the rats continuously ingested the HFOS diet for 8 wk; additionally, Lactobacillus population could adapt to a relatively stable level with the consumption of HFOS diet. Fructooligosaccharide (FOS) is one of the most popular prebiotics, and it is widely used in infant formulas, which is aiming to increase the growth of probiotics like Lactobacillus and Bifidobacterium. This study discovered new growth rhythm of Bifidobacterium based on a high-FOS diet. The growth of Bifidobacterium was first promoted but receded in the end. This finding is highly instructive and meaningful for the application of fructooligosaccharide in probiotic or prebiotic food.

Identification of dual-tropic HIV-1 using evolved neural networks

Blocking the binding of the envelope HIV-1 protein to immune cells is a popular concept for development of anti-HIV therapeutics. R5 HIV-1 binds CCR5, X4 HIV-1 binds CXCR4, and dual-tropic HIV-1 can bind either coreceptor for cellular entry. R5 viruses are associated with early infection and over time can evolve to X4 viruses that are associated with immune failure. Dual-tropic HIV-1 is less studied; however, it represents functional antigenic intermediates during the transition of R5 to X4 viruses. Viral tropism is linked partly to the HIV-1 envelope V3 domain, where the amino acid sequence helps dictate the receptor a particular virus will target; however, using V3 sequence information to identify dual-tropic HIV-1 isolates has remained difficult. Our goal in this study was to elucidate features of dual-tropic HIV-1 isolates that assist in the biological understanding of dual-tropism and develop an approach for their detection. Over 1559 HIV-1 subtype B sequences with known tropisms were analyzed. Each sequence was represented by 73 structural, biochemical and regional features. These features were provided to an evolved neural network classifier and evaluated using balanced and unbalanced data sets. The study resolved R5X4 viruses from R5 with an accuracy of 81.8% and from X4 with an accuracy of 78.8%. The approach also identified a set of V3 features (hydrophobicity, structural and polarity) that are associated with tropism transitions. The ability to distinguish R5X4 isolates will improve computational tropism decisions for R5 vs. X4 and assist in HIV-1 research and drug development efforts.

Targeting Atp6v1c1 Prevents Inflammation and Bone Erosion Caused by Periodontitis and Reveals Its Critical Function in Osteoimmunology.

Periodontal disease (Periodontitis) is a serious disease that affects a majority of adult Americans and is associated with other systemic diseases, including diabetes, rheumatoid arthritis, and other inflammatory diseases. While great efforts have been devoted toward understanding the pathogenesis of periodontitis, there remains a pressing need for developing potent therapeutic strategies for targeting this pervasive and destructive disease. In this study, we utilized novel adeno-associated virus (AAV)-mediated Atp6v1c1 knockdown gene therapy to treat bone erosion and inflammatory caused by periodontitis in mouse model. Atp6v1c1 is a subunit of the V-ATPase complex and regulator of the assembly of the V0 and V1 domains of the V-ATPase complex. We demonstrated previously that Atp6v1c1 has an essential function in osteoclast mediated bone resorption. We hypothesized that Atp6v1c1 may be an ideal target to prevent the bone erosion and inflammation caused by periodontitis. To test the hypothesis, we employed AAV RNAi knockdown of Atp6v1c1 gene expression to prevent bone erosion and gingival inflammation simultaneously. We found that lesion-specific injection of AAV-shRNA-Atp6v1c1 into the periodontal disease lesions protected against bone erosion (>85%) and gingival inflammation caused by P. gingivalis W50 infection. AAV-mediated Atp6v1c1 knockdown dramatically reduced osteoclast numbers and inhibited the infiltration of dendritic cells and macrophages in the bacteria-induced inflammatory lesions in periodontitis. Silencing of Atp6v1c1 expression also prevented the expressions of osteoclast-related genes and pro-inflammatory cytokine genes. Our data suggests that AAV-shRNA-Atp6v1c1 treatment can significantly attenuate the bone erosion and inflammation caused by periodontitis, indicating the dual function of AAV-shRNA-Atp6v1c1 as an inhibitor of bone erosion mediated by osteoclasts, and as an inhibitor of inflammation through down-regulation of pro-inflammatory cytokine expression. This study demonstrated that Atp6v1c1 RNAi knockdown gene therapy mediated by AAV-shRNA-Atp6v1c1 is a promising novel therapeutic approach for the treatment of bone erosion and inflammatory related diseases, such as periodontitis and rheumatoid arthritis.

Identification of a duplicated V3 domain in NS5A associated with cirrhosis and hepatocellular carcinoma in HCV-1b patients

BackgroundThe NS5A protein of the hepatitis C virus has been shown to be involved in the development of hepatocellular carcinoma. ObjectivesIn a French multicenter study, we investigated the clinical and epidemiological features of a new HCV genotype 1b strain bearing a wide insertion into the V3 domain. Study DesignWe studied NS5A gene sequences in 821 French patients infected with genotype 1b HCV. ResultsWe identified an uncharacterized V3 insertion without ORF disruption in 3.05% of the HCV sequences. The insertion comprised 31 amino-acids for the majority of patients; 3 patients had 27 amino-acids insertions and 1 had a 12 amino-acids insertion. Sequence identity between the 31 amino-acids insertions and the V3 domain ranged from 48 to 96% with E-values above 4e−5, thus illustrating sequence homology and a partial gene duplication event that to our knowledge has never been reported in HCV. Moreover we showed the presence of the duplication at the time of infection and its persistence at least during 12 years in the entire quasispecies. No association was found with extrahepatic diseases. Conversely, patients with cirrhosis were two times more likely to have HCV with this genetic characteristic (p=0.04). Moreover, its prevalence increased with liver disease severity (from 3.0% in patients without cirrhosis to 9.4% in patients with both cirrhosis and HCC, p for trend=0.045). ConclusionsWe identified a duplicated V3 domain in the HCV-1b NS5A protein for the first time. The duplication may be associated with unfavorable evolution of liver disease including a possible involvement in liver carcinogenesis.

Compensatory branching morphogenesis of stalk cells in the Drosophila trachea.

Tubes are essential for nutrient transport and gas exchange in multicellular eukaryotes, but how connections between different tube types are maintained over time is unknown. In the Drosophila tracheal system, mutations in oak gall (okg) and conjoined (cnj) confer identical defects, including late onset blockage near the terminal cell-stalk cell junction and the ectopic extension of autocellular, seamed tubes into the terminal cell. We determined that okg and cnj encode the E and G subunits of the vacuolar ATPase (vATPase) and showed that both the V0 and V1 domains are required for terminal cell morphogenesis. Remarkably, the ectopic seamed tubes running along vATPase-deficient terminal cells belonged to the neighboring stalk cells. All vATPase-deficient tracheal cells had reduced apical domains and terminal cells displayed mislocalized apical proteins. Consistent with recent reports that the mTOR and vATPase pathways intersect, we found that mTOR pathway mutants phenocopied okg and cnj. Furthermore, terminal cells depleted for the apical determinants Par6 or aPKC had identical ectopic seamed tube defects. We thus identify a novel mechanism of compensatory branching in which stalk cells extend autocellular tubes into neighboring terminal cells with undersized apical domains. This compensatory branching also occurs in response to injury, with damaged terminal cells being rapidly invaded by their stalk cell neighbor.

Regulation of lipid droplet dynamics in Saccharomyces cerevisiae depends on the Rab7-like Ypt7p, HOPS complex and V1-ATPase.

ABSTRACTIt has now been clearly shown that lipid droplets (LDs) play a dynamic role in the cell. This was reinforced by LD proteomics which suggest that a significant number of trafficking proteins are associated with this organelle. Using microscopy, we showed that LDs partly co-localize with the vacuole in S. cerevisiae. Immunoblot experiments confirmed the association of the vacuolar Rab GTPase Rab7-like Ypt7p with LDs. We observed an increase in fatty acid content and LD number in ypt7Δ mutant and also changes in LD morphology and intra LD fusions, revealing a direct role for Ypt7p in LD dynamics. Using co-immunoprecipitation, we isolated potential Ypt7p partners including, Vma13p, the H subunit of the V1 part of the vacuolar (H+) ATPase (V-ATPase). Deletion of the VMA13 gene, as well as deletion of three other subunits of the V1 part of the V-ATPase, also increased the cell fatty acid content and LD number. Mutants of the Homotypic fusion and vacuole protein sorting (HOPS) complex showed similar phenotypes. Here, we demonstrated that LD dynamics and membrane trafficking between the vacuole and LDs are regulated by the Rab7-like Ypt7p and are impaired when the HOPS complex and the V1 domain of the V-ATPase are defective.

Atp6v1c1 Facilitates Breast Cancer Growth and Bone Metastasis through the mTORC1 Pathway

The mTORC1 kinase promotes growth in response to growth factors, energy levels, and amino acids, and its activity is often deregulated in cancers, including breast cancer. Recent studies have shown that mTORC1 senses lysosomal amino acids through an inside‐out mechanism that requires vacuolar H+‐ATPase activity. However, the mechanism underlying how V‐ATPases activate the mTORC1 pathway in cancer growth and metastasis remains largely unclear. Atp6v1c1, an assembly regulator of the V0 and V1 domains of the V‐ATPase complex, is highly expressed in metastatic cancer cells. Our data revealed that silencing Atp6v1c1 in metastatic 4T1 mouse breast cancer cells (4T1) severely impairs 4T1 cell growth, migration, and metastasis, as well as inhibiting mTORC1 pathway activation stimulated by amino acids. Notably, Atp6v1c1 knockdown also inhibited the proliferation of MCF‐7, MDA‐MB‐231, and MDA‐MB‐435s human breast cancer cells and blocked their mTORC1 pathway activation stimulated by amino acids. Surprisingly, Atp6v1c1 knockdown in normal cells C3H10T1/2 has no effect on its proliferation and mTORC1 pathway activation stimulated by amino acids. Our study demonstrates, for the first time, a potential mechanism by which Atp6v1c1 facilitates cancer cell growth and metastasis through its function in mTOR pathway activation and may provide a novel therapeutic approach for breast cancer metastasis.

Whole‐Wheat Supplemented Diet Changed Fecal Microbial Ecology of Obese Diabetic Mice

Diet affects bacterial diversity and abundance of the gastrointestinal microbiota. Our objective was to study the effect of whole‐wheat supplemented diet in fecal microbiota composition of obese diabetic (db/db) mice. The db/db mice were fed standard or whole‐wheat isocaloric diets for eight weeks. Fecal DNA was extracted and the V4 domain of the 16S rRNA was amplified using the universal primers F515 and R806; subsequently, amplicons were sequenced on an Illumina MiSeq instrument. Raw Illumina fastq files were demultiplexed, quality filtered, and analyzed using QIIME v1.8.0. Operational taxonomic units (OTUs) were assigned using QIIME's uclust‐based close‐reference OTU‐picking workflow, with a threshold of 97% pairwise identity against the Greengenes bacterial 16S rRNA database (13_5 release). Our results showed that the bacteroidetes/firmicutes ratio was three times higher in the whole‐wheat group compared to the db/db standard diet group. In addition, whole‐wheat diet decreased abundances of the families Enterococcaceae, Turicibacteraceae, and Ruminococcaceae. However, the alpha diversity, quantified by Shannon‐Wiener index was not significantly different between groups (p=0.0652). In conclusion, whole‐wheat supplemented diet changed the bacterial diversity and abundance of fecal microbiota of obese mice. Further studies are being conducted to analyze the relationship between microbiota changes and host metabolic responses.

A Plant-Derived Multi-HIV Antigen Induces Broad Immune Responses in Orally Immunized Mice.

Multi-HIV, a multiepitopic protein derived from both gp120 and gp41 envelope proteins of the human immunodeficiency virus (HIV), has been proposed as a vaccine prototype capable of inducing broad immune responses, as it carries various B and T cell epitopes from several HIV strains. In this study, the immunogenic properties of a Multi-HIV expressed in tobacco chloroplasts are evaluated in test mice. BALB/c mice orally immunized with tobacco-derived Multi-HIV have elicited antibody responses, including both the V3 loop of gp120 and the ELDKWA epitope of gp41. Based on splenocyte proliferation assays, stimulation with epitopes of the C4, V3 domain of gp120, and the ELDKWA domain of gp41 elicits positive cellular responses. Furthermore, specific interferon gamma production is observed in both CD4+ and CD8+ T cells stimulated with HIV peptides. These results demonstrate that plant-derived Multi-HIV induces T helper-specific responses. Altogether, these findings illustrate the immunogenic potential of plant-derived Multi-HIV in an oral immunization scheme. The potential of this low-cost immunization approach and its implications on HIV/AIDS vaccine development are discussed.

Crystal Structure of Subunits D and F in Complex Gives Insight into Energy Transmission of the Eukaryotic V-ATPase from Saccharomyces cerevisiae

Eukaryotic V1VO-ATPases hydrolyze ATP in the V1 domain coupled to ion pumping in VO. A unique mode of regulation of V-ATPases is the reversible disassembly of V1 and VO, which reduces ATPase activity and causes silencing of ion conduction. The subunits D and F are proposed to be key in these enzymatic processes. Here, we describe the structures of two conformations of the subunit DF assembly of Saccharomyces cerevisiae (ScDF) V-ATPase at 3.1 Å resolution. Subunit D (ScD) consists of a long pair of α-helices connected by a short helix ((79)IGYQVQE(85)) as well as a β-hairpin region, which is flanked by two flexible loops. The long pair of helices is composed of the N-terminal α-helix and the C-terminal helix, showing structural alterations in the two ScDF structures. The entire subunit F (ScF) consists of an N-terminal domain of four β-strands (β1-β4) connected by four α-helices (α1-α4). α1 and β2 are connected via the loop (26)GQITPETQEK(35), which is unique in eukaryotic V-ATPases. Adjacent to the N-terminal domain is a flexible loop, followed by a C-terminal α-helix (α5). A perpendicular and extended conformation of helix α5 was observed in the two crystal structures and in solution x-ray scattering experiments, respectively. Fitted into the nucleotide-bound A3B3 structure of the related A-ATP synthase from Enterococcus hirae, the arrangements of the ScDF molecules reflect their central function in ATPase-coupled ion conduction. Furthermore, the flexibility of the terminal helices of both subunits as well as the loop (26)GQITPETQEK(35) provides information about the regulatory step of reversible V1VO disassembly.

Superior orbital fissure syndrome caused by aspergillus infection from maxillary sinusitis

AbstractA 62‐year‐old woman who was prescribed prednisolone and cyclosporine for uveitis developed right blepharoptosis, diplopia and hypesthesia of superficial sensation of the trigeminal nerve V1 domain. Preserved right visual acuity led to a diagnosis of superior orbital fissure syndrome. We could not find any abnormalities on computed tomography except for right maxillary sinusitis associated with calcification. No infectious agents were detected in the blood or cerebrospinal fluid. We concluded that maxillary aspergillus, which was proven histopathologically after surgery, caused superior orbital fissure syndrome through the ophthalmic veins. Surgical excision of the lesion and voriconazole administration resulted in complete remission. To the best of our knowledge, there have been no other reports of superior orbital fissure syndrome caused by maxillary aspergillus sinusitis. The calcified sinusitis suggested fungal infections, which allowed us to avoid using corticosteroids.

HIV Tropism Prediction: Digital Signal Processing-Based Bioinformatics Approach is Non-Sequence Alignment Dependent

Phenotypic and genotypic predictors forHIV/SIV tropism are available. The genotypic predictors are more rational. However, they are sequence alignment dependent only. Regrettably, non-homologous proteins are found to display common biological functionality. This indicates that sequence-dependent predictors cannot be trusted with appropriate classification of the HIV and SIV isolates, especially if the isolates belong to same tropic group but share divergent sequence alignment. There is therefore need for genotypic predictors that will incorporate embedded intrinsic biological characteristics of the HIV and SIV isolates in the determination of HIV tropism. Secondly, more than 30 positions with at least single mutation outside the V3 domain have been found to influence HIV Tropism. Disappointingly, the available sequence alignment-based HIV genotypic predictors engage only the hyper-variable region (V3) of the HIV gp120. This has resulted in inaccurate classification of HIV and SIV strains. Finally, available HIV genotypic predictors are found to lack the ability to identify and accurately evaluate the sequences of most HIV-1 non B clades, HIV-2 and SIV. Against this background, the ability of the Digital Signal Processing (DSP) Technique called Informational Spectrum Method (ISM), which does not engage sequence similarity but the embedded bio-functionalities of the entire gp120 sequence length to predict HIV and SIV tropism is therefore investigated. 83 isolates of HIV and SIV are subjected to ISM and three other procedures. Results are generated and findings correlated. For isolates, which are analyzable by the four procedures, the results from ISM and three other procedures are found to correlate. Using 50% affinity for the host CD4 as the cut-off, the tropism of the uncategorized isolates are predicted. ISM-based technique is adjudged a better procedure. It analyzes the sequences of all HIV (HIV-1 together with non B, and HIV-2) as well as SIV isolates including those that could not be investigated by other genotypic predictors. It engages the embedded biological characteristics rather than sequence similarity and utilizes the entire HIV gp120 sequence-length instead of V3 domain. This makes ISM-based procedure a better tool for over 180,000 isolates of HIV-1, HIV-2 and SIV in the UNIPROT database. Clinical approaches are unfeasible. This study recommends ISM technique principally for viral tropism prediction as it does not discriminate against HIV/SIVcategories. It suggests that further work be done to determine a suitable cut-off (as in geno2pheno[CORECEPTOR]), and the procedure in combination with other genotypic predictors be engaged in developing an algorithm for determining viral tropism.

X4 Tropic Virus Prediction Is Associated with a Nadir CD4 T-Cell Count below 100 Cells/mm3

Objective: The aim of this study was to evaluate tropism prediction performances of three algorithms [geno2pheno false-positive rate 10% (G2P10), position-specific scoring matrix (PSSM) and a combination of the 11/25 and net charge rules] and to investigate the viral and host factors potentially involved in the X4 or R5 prediction in human immunodeficiency virus-1 (HIV-1) patients. Methods: Viral tropism was determined in 179 HIV-1-infected patients eligible for CCR5 antagonist therapy. HIV-1 RNA or DNA was extracted and amplified for env gp120 sequencing. In parallel, demographic, viral, immunological and clinical determinants were analyzed. Results: According to the G2P10 algorithm, 48 patients harbored X4 or X4R5 virus. The tropism prediction was concordant for 87.7 and 88.2% of samples when comparing G2P10 with PSSM or with a combination of the 11/25 and net charge rules, respectively. X4 prediction was significantly associated with more than 35 amino acids in the V3 domain (p < 0.0001) and loss of an N-linked glycosylation site (p < 0.0001). Of the factors studied, only the nadir CD4 T-cell count was significantly associated with X4 tropism (p = 0.01). Conclusion: We determined that the X4 virus detection is closely linked to the nadir CD4 T-cell count below 100 cells/mm³ that must be taken into account when considering a CCR5 antagonist therapy switch.

The Significance and Mechanisms of Clustering by SLC4 Cotransporters in the Plasma Membrane

The Solute Carrier 4 (SLC4) family are integral membrane cotransporters that reabsorb or secret anions across the basolateral membranes throughout the body, especially the kidney, eye, and red blood cells. Defects of SLC4A4 (NBCe1-A) lead to type-II acidosis and myriad ocular abnormalities; those of SLC4A1 (AE1) causes type-I acidosis and sperocytosis. We demonstrate that SLC4s form patches in the membrane of proximal tubule cells, corneal endothelial cells, and Xenopus oocytes. This new discovery of homophilic interactions or self-associations was initially suggested from our biophysical studies of the N-terminal domain of NBCe1-A (Nt) in solution, and by others studying the transmembrane domain (TMD) of AE1. The role and mechanisms of self-associations are further investigated, with insight into the role of the devastating R298S hereditary mutation found in type-II RTA afflicted individuals. We use confocal-microscopy imaging of fluorescently-tagged NBCe1-A and NBCe1-A-R298S molecules expressed in the aforementioned cells types and from in-depth biophysical studies of their Nts, including our Nt crystal structure at 2.4-Å resolution, melting-temperature, and homodimer dissociation constant (KD) analyses by composition-gradient multi-angle light scattering (CG-MALS). The structure reveals that the Nt dimerizes by two interlocking arms and contains two variable sequence regions, one at the extreme N terminus (V1) and another in the middle (V2). The V1 domains are flexible segments that gate substrate entry into the Nt, and that the V2 are large solvent accessible loops that extend ∼20 Å from the core Nt structure. Truncation mutants were rationally generated, and surface plasmon resonance (SPR) was used to investigate their roles in self-association. Based on these findings, we discuss the role of clustering in HCO3- sensing and transport in health and provide insight to the pathogenic processes observed in patients with point mutations.

Network of long-range concerted chemical shift displacements upon ligand binding to human angiogenin.

Molecular recognition models of both induced fit and conformational selection rely on coupled networks of flexible residues and/or structural rearrangements to promote protein function. While the atomic details of these motional events still remain elusive, members of the pancreatic ribonuclease superfamily were previously shown to depend on subtle conformational heterogeneity for optimal catalytic function. Human angiogenin, a structural homologue of bovine pancreatic RNase A, induces blood vessel formation and relies on a weak yet functionally mandatory ribonucleolytic activity to promote neovascularization. Here, we use the NMR chemical shift projection analysis (CHESPA) to clarify the mechanism of ligand binding in human angiogenin, further providing information on long-range intramolecular residue networks potentially involved in the function of this enzyme. We identify two main clusters of residue networks displaying correlated linear chemical shift trajectories upon binding of substrate fragments to the purine- and pyrimidine-specific subsites of the catalytic cleft. A large correlated residue network clusters in the region corresponding to the V1 domain, a site generally associated with the angiogenic response and structural stability of the enzyme. Another correlated network (residues 40-42) negatively affects the catalytic activity but also increases the angiogenic activity. (15) N-CPMG relaxation dispersion experiments could not reveal the existence of millisecond timescale conformational exchange in this enzyme, a lack of flexibility supported by the very low-binding affinities and catalytic activity of angiogenin. Altogether, the current report potentially highlights the existence of long-range dynamic reorganization of the structure upon distinct subsite binding events in human angiogenin.

F-108 polymer and capillary electrophoresis easily resolves complex environmental DNA mixtures and SNPs.

Ecological studies of microbial communities often use profiling methods but the true community diversity can be underestimated in methods that separate amplicons based on sequence length using performance optimized polymer 4. Taxonomically, unrelated organisms can produce the same length amplicon even though the amplicons have different sequences. F-108 polymer has previously been shown to resolve same length amplicons by sequence polymorphisms. In this study, we showed F-108 polymer, using the ABI Prism 310 Genetic Analyzer and CE, resolved four bacteria that produced the same length amplicon for the 16S rRNA domain V3 but have variable nucleotide content. Second, a microbial mat community profile was resolved and supported by NextGen sequencing where the number of peaks in the F-108 profile was in concordance with the confirmed species numbers in the mat. Third, equine DNA was analyzed for SNPs. The F-108 polymer was able to distinguish heterozygous and homozygous individuals for the melanocortin 1 receptor coat color gene. The method proved to be rapid, inexpensive, reproducible, and uses common CE instruments. The potential for F-108 to resolve DNA mixtures or SNPs can be applied to various sample types-from SNPs to forensic mixtures to ecological communities.

Assessment of psychometric properties of the Korean SF-12 v2 in the general population.

BackgroundThe psychometric properties of the Korean Short Form-12 Health Survey, version 2 (SF-12 v2) have not been assessed in the general population. Therefore, the aim of our study was to evaluate the psychometric properties of the Korean version of the SF-12 v2 in the general population and to provide SF-12 v2 domain scores according to the general characteristics of the study population.MethodsA total of 1,000 participants from the general Korean population were recruited using a multistage quota sampling method. Psychometric properties were evaluated by descriptive statistics, validity, reliability, and exploratory factor analysis.ResultsItem convergent and discriminant validity met the criteria established by the instrument developer. In the known-group comparison, male gender, age <60 years, high educational status, and absence of any comorbidity were significantly associated with high scale scores. The reliability of all SF-12 v2 items was 0.88.ConclusionsThe findings of this study generally support the idea that the Korean SF-12 v2 is a feasible, valid, and reliable instrument for assessing health-related quality of life in the general population. The SF-12 v2 seems to be a viable alternative health-related quality of life instrument for the Korean population.

A Mammalian Cell Based FACS-Panning Platform for the Selection of HIV-1 Envelopes for Vaccine Development

An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates.

Yeast Phosphofructokinase-1 Subunit Pfk2p Is Necessary for pH Homeostasis and Glucose-dependent Vacuolar ATPase Reassembly

V-ATPases are conserved ATP-driven proton pumps that acidify organelles. Yeast V-ATPase assembly and activity are glucose-dependent. Glucose depletion causes V-ATPase disassembly and its inactivation. Glucose readdition triggers reassembly and resumes proton transport and organelle acidification. We investigated the roles of the yeast phosphofructokinase-1 subunits Pfk1p and Pfk2p for V-ATPase function. The pfk1Δ and pfk2Δ mutants grew on glucose and assembled wild-type levels of V-ATPase pumps at the membrane. Both phosphofructokinase-1 subunits co-immunoprecipitated with V-ATPase in wild-type cells; upon deletion of one subunit, the other subunit retained binding to V-ATPase. The pfk2Δ cells exhibited a partial vma growth phenotype. In vitro ATP hydrolysis and proton transport were reduced by 35% in pfk2Δ membrane fractions; they were normal in pfk1Δ. In vivo, the pfk1Δ and pfk2Δ vacuoles were alkalinized and the cytosol acidified, suggestive of impaired V-ATPase proton transport. Overall the pH alterations were more dramatic in pfk2Δ than pfk1Δ at steady state and after readdition of glucose to glucose-deprived cells. Glucose-dependent reassembly was 50% reduced in pfk2Δ, and the vacuolar lumen was not acidified after reassembly. RAVE-assisted glucose-dependent reassembly and/or glucose signals were disturbed in pfk2Δ. Binding of disassembled V-ATPase (V1 domain) to its assembly factor RAVE (subunit Rav1p) was 5-fold enhanced, indicating that Pfk2p is necessary for V-ATPase regulation by glucose. Because Pfk1p and Pfk2p are necessary for V-ATPase proton transport at the vacuole in vivo, a role for glycolysis at regulating V-ATPase proton transport is discussed.