Evolution of tonoplast P-ATPase transporters involved in vacuolar acidification.

PubMed

Li, Yanbang; Provenzano, Sofia; Bliek, Mattijs; Spelt, Cornelis; Appelhagen, Ingo; Machado de Faria, Laura; Verweij, Walter; Schubert, Andrea; Sagasser, Martin; Seidel, Thorsten; Weisshaar, Bernd; Koes, Ronald; Quattrocchio, Francesca

2016-08-01

Petunia mutants (Petunia hybrida) with blue flowers defined a novel vacuolar proton pump consisting of two interacting P-ATPases, PH1 and PH5, that hyper-acidify the vacuoles of petal cells. PH5 is similar to plasma membrane H(+) P3A -ATPase, whereas PH1 is the only known eukaryoticP3B -ATPase. As there were no indications that this tonoplast pump is widespread in plants, we investigated the distribution and evolution of PH1 and PH5. We combined database mining and phylogenetic and synteny analyses of PH1- and PH5-like proteins from all kingdoms with functional analyses (mutant complementation and intracellular localization) of homologs from diverse angiosperms. We identified functional PH1 and PH5 homologs in divergent angiosperms. PH5 homologs evolved from plasma membrane P3A -ATPases, acquiring an N-terminal tonoplast-sorting sequence and new cellular function before angiosperms appeared. PH1 is widespread among seed plants and related proteins are found in some groups of bacteria and fungi and in one moss, but is absent in most algae, suggesting that its evolution involved several cases of gene loss and possibly horizontal transfer events. The distribution of PH1 and PH5 in the plant kingdom suggests that vacuolar acidification by P-ATPases appeared in gymnosperms before flowers. This implies that, next to flower color determination, vacuolar hyper-acidification is required for yet unknown processes. © 2016 European Union. New Phytologist © 2016 New Phytologist Trust.

Genes encoding the vacuolar Na+/H+ exchanger and flower coloration.

PubMed

Yamaguchi, T; Fukada-Tanaka, S; Inagaki, Y; Saito, N; Yonekura-Sakakibara, K; Tanaka, Y; Kusumi, T; Iida, S

2001-05-01

Vacuolar pH plays an important role in flower coloration: an increase in the vacuolar pH causes blueing of flower color. In the Japanese morning glory (Ipomoea nil or Pharbitis nil), a shift from reddish-purple buds to blue open flowers correlates with an increase in the vacuolar pH. We describe details of the characterization of a mutant that carries a recessive mutation in the Purple (Pr) gene encoding a vacuolar Na+/H+ exchanger termed InNHX1. The genome of I. nil carries one copy of the Pr (or InNHX1) gene and its pseudogene, and it showed functional complementation to the yeast nhx1 mutation. The mutant of I. nil, called purple (pr), showed a partial increase in the vacuolar pH during flower-opening and its reddish-purple buds change into purple open flowers. The vacuolar pH in the purple open flowers of the mutant was significantly lower than that in the blue open flowers. The InNHX1 gene is most abundantly expressed in the petals at around 12 h before flower-opening, accompanying the increase in the vacuolar pH for the blue flower coloration. No such massive expression was observed in the petunia flowers. Since the NHX1 genes that promote the transport of Na+ into the vacuoles have been regarded to be involved in salt tolerance by accumulating Na+ in the vacuoles, we can add a new biological role for blue flower coloration in the Japanese morning glory by the vacuolar alkalization.

Microfluidic droplet enrichment for targeted sequencing

PubMed Central

Eastburn, Dennis J.; Huang, Yong; Pellegrino, Maurizio; Sciambi, Adam; Ptáček, Louis J.; Abate, Adam R.

2015-01-01

Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications. PMID:25873629

Characterization of vacuolar amino acid transporter from Fusarium oxysporum in Saccharomyces cerevisiae.

PubMed

Lunprom, Siriporn; Pongcharoen, Pongsanat; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi; Akiyama, Koichi

2015-01-01

Fusarium oxysporum causes wilt disease in many plant families, and many genes are involved in its development or growth in host plants. A recent study revealed that vacuolar amino acid transporters play an important role in spore formation in Schizosaccharomyces pombe and Saccharomyces cerevisiae. To investigate the role of vacuolar amino acid transporters of this phytopathogenic fungus, the FOXG_11334 (FoAVT3) gene from F. oxysporum was isolated and its function was characterized. Transcription of FoAVT3 was upregulated after rapamycin treatment. A green fluorescent protein fusion of FoAvt3p was localized to vacuolar membranes in both S. cerevisiae and F. oxysporum. Analysis of the amino acid content of the vacuolar fraction and amino acid transport activities using vacuolar membrane vesicles from S. cerevisiae cells heterologously expressing FoAVT3 revealed that FoAvt3p functions as a vacuolar amino acid transporter, exporting neutral amino acids. We conclude that the FoAVT3 gene encodes a vacuolar neutral amino acid transporter.

Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue.

PubMed

Hsiao, Yi Yuong; Van, Ru Chuan; Hung, Hsiao Hui; Pan, Rong Long

2002-01-01

Vacuolar proton pumping pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PPi hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H+-translocation of vacuolar H+-PPase in a concentration-dependent manner. Absorbance of vacuolar H+-PPase at 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety was probably formed. On the other hand, hydroxylamine, a reagent that can deacylate N-carbethoxyhistidine, could reverse the absorption change at 240 nm and partially restore PPi hydrolysis activity as well. The pKa of modified residues of the enzyme was determined to be 6.4, a value close to that of histidine. Thus, we speculate that inhibition of vacuolar H+-PPase by DEPC possibly could be attributed to the modification of histidyl residues on the enzyme. Furthermore, inhibition of vacuolar H+-PPase by DEPC follows pseudo-first-order rate kinetics. A reaction order of 0.85 was calculated from a double logarithmic plot of the apparent reaction constant against DEPC concentration, suggesting that the modification of one single histidine residue on the enzyme suffices to inhibit vacuolar H+-PPase. Inhibition of vacuolar H+-PPase by DEPC changes Vmax but not Km values. Moreover, DEPC inhibition of vacuolar H+-PPase could be substantially protected against by its physiological substrate, Mg2+-PPi. These results indicated that DEPC specifically competes with the substrate at the active site and the DEPC-labeled histidine residue might locate in or near the catalytic domain of the enzyme. Besides, pretreatment of the enzyme with N-ethylmaleimide decreased the degree of subsequent labeling of H+-PPase by DEPC. Taken together, we suggest that vacuolar H+-PPase likely contains a substrate-protectable histidine residue contributing to the inhibition of its activity by

Environmental Factors Influencing Blooms of a Neurotoxic Stigonematalan Cyanobacterium Responsible for Avian Vacuolar Myelinopathy

DTIC Science & Technology

2013-01-01

aquatic plants and subsequent ecological consequences. The authors of this technical note have linked avian vacuolar myelinopathy (AVM), a disease...additional cyanobacteria sequences to determine designations for probe development, to advance understanding of the species’ phylogeny , and to lay...groundwork for its formal description. Phylogeny data confirm that the species is in section V, order Stigonematales. Phylogeny also infers that the

The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier

PubMed Central

Emmerlich, Vera; Linka, Nicole; Reinhold, Thomas; Hurth, Marco A.; Traub, Michaela; Martinoia, Enrico; Neuhaus, H. Ekkehard

2003-01-01

Malate plays a central role in plant metabolism. It is an intermediate in the Krebs and glyoxylate cycles, it is the store for CO2 in C4 and crassulacean acid metabolism plants, it protects plants from aluminum toxicity, it is essential for maintaining the osmotic pressure and charge balance, and it is therefore involved in regulation of stomatal aperture. To fulfil many of these roles, malate has to be accumulated within the large central vacuole. Many unsuccessful efforts have been made in the past to identify the vacuolar malate transporter; here, we describe the identification of the vacuolar malate transporter [A. thaliana tonoplast dicarboxylate transporter (AttDT)]. This transporter exhibits highest sequence similarity to the human sodium/dicarboxylate cotransporter. Independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] Arabidopsis mutants exhibit substantially reduced levels of leaf malate, but respire exogenously applied [14C]malate faster than the WT. An AttDT-GFP fusion protein was localized to vacuole. Vacuoles isolated from Arabidopsis WT leaves exhibited carbonylcyanide m-chlorophenylhydrazone and citrate inhibitable malate transport, which was not stimulated by sodium. Vacuoles isolated from mutant plants import [14C]-malate at strongly reduced rates, confirming that this protein is the vacuolar malate transporter. PMID:12947042

The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier.

PubMed

Emmerlich, Vera; Linka, Nicole; Reinhold, Thomas; Hurth, Marco A; Traub, Michaela; Martinoia, Enrico; Neuhaus, H Ekkehard

2003-09-16

Malate plays a central role in plant metabolism. It is an intermediate in the Krebs and glyoxylate cycles, it is the store for CO2 in C4 and crassulacean acid metabolism plants, it protects plants from aluminum toxicity, it is essential for maintaining the osmotic pressure and charge balance, and it is therefore involved in regulation of stomatal aperture. To fulfil many of these roles, malate has to be accumulated within the large central vacuole. Many unsuccessful efforts have been made in the past to identify the vacuolar malate transporter; here, we describe the identification of the vacuolar malate transporter [A. thaliana tonoplast dicarboxylate transporter (AttDT)]. This transporter exhibits highest sequence similarity to the human sodium/dicarboxylate cotransporter. Independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] Arabidopsis mutants exhibit substantially reduced levels of leaf malate, but respire exogenously applied [14C]malate faster than the WT. An AttDT-GFP fusion protein was localized to vacuole. Vacuoles isolated from Arabidopsis WT leaves exhibited carbonylcyanide m-chlorophenylhydrazone and citrate inhibitable malate transport, which was not stimulated by sodium. Vacuoles isolated from mutant plants import [14C]-malate at strongly reduced rates, confirming that this protein is the vacuolar malate transporter.

Anthocyanin Vacuolar Inclusions Form by a Microautophagy Mechanism

PubMed Central

Chanoca, Alexandra; Ueda, Takashi; Grotewold, Erich

2015-01-01

Anthocyanins are flavonoid pigments synthesized in the cytoplasm and stored inside vacuoles. Many plant species accumulate densely packed, 3- to 10-μm diameter anthocyanin deposits called anthocyanin vacuolar inclusions (AVIs). Despite their conspicuousness and importance in organ coloration, the origin and nature of AVIs have remained controversial for decades. We analyzed AVI formation in cotyledons of different Arabidopsis thaliana genotypes grown under anthocyanin inductive conditions and in purple petals of lisianthus (Eustoma grandiorum). We found that cytoplasmic anthocyanin aggregates in close contact with the vacuolar surface are directly engulfed by the vacuolar membrane in a process reminiscent of microautophagy. The engulfed anthocyanin aggregates are surrounded by a single membrane derived from the tonoplast and eventually become free in the vacuolar lumen like an autophagic body. Neither endosomal/prevacuolar trafficking nor the autophagy ATG5 protein is involved in the formation of AVIs. In Arabidopsis, formation of AVIs is promoted by both an increase in cyanidin 3-O-glucoside derivatives and by depletion of the glutathione S-transferase TT19. We hypothesize that this novel microautophagy mechanism also mediates the transport of other flavonoid aggregates into the vacuole. PMID:26342015

Targeted therapy according to next generation sequencing-based panel sequencing.

PubMed

Saito, Motonobu; Momma, Tomoyuki; Kono, Koji

2018-04-17

Targeted therapy against actionable gene mutations shows a significantly higher response rate as well as longer survival compared to conventional chemotherapy, and has become a standard therapy for many cancers. Recent progress in next-generation sequencing (NGS) has enabled to identify huge number of genetic aberrations. Based on sequencing results, patients recommend to undergo targeted therapy or immunotherapy. In cases where there are no available approved drugs for the genetic mutations detected in the patients, it is recommended to be facilitate the registration for the clinical trials. For that purpose, a NGS-based sequencing panel that can simultaneously target multiple genes in a single investigation has been used in daily clinical practice. To date, various types of sequencing panels have been developed to investigate genetic aberrations with tumor somatic genome variants (gain-of-function or loss-of-function mutations, high-level copy number alterations, and gene fusions) through comprehensive bioinformatics. Because sequencing panels are efficient and cost-effective, they are quickly being adopted outside the lab, in hospitals and clinics, in order to identify personal targeted therapy for individual cancer patients.

ATP-dependent export of neutral amino acids by vacuolar membrane vesicles of Saccharomyces cerevisiae.

PubMed

Ishimoto, Masaya; Sugimoto, Naoko; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2012-01-01

Amino acid analysis of Saccharomyces cerevisiae cells indicated that neutral amino acids such as glycine and alanine were probably excluded from the vacuoles, and that vacuolar H(+)-ATPase (V-ATPase) was involved in the vacuolar compartmentalization of these amino acids. We found that vacuolar membrane vesicles export neutral amino acids in an ATP-dependent manner. This is important in identifying vacuolar transporters for neutral amino acids.

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

PubMed

Manolson, M F; Proteau, D; Preston, R A; Stenbit, A; Roberts, B T; Hoyt, M A; Preuss, D; Mulholland, J; Botstein, D; Jones, E W

1992-07-15

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.

Pth1/Vam3p is the syntaxin homolog at the vacuolar membrane of Saccharomyces cerevisiae required for the delivery of vacuolar hydrolases.

PubMed Central

Srivastava, A; Jones, E W

1998-01-01

The PEP12 homolog Pth1p (Pep twelve homolog 1) is predicted to be similar in size to Pep12p, the endosomal syntaxin homolog that mediates docking of Golgi-derived transport vesicles and, like other members of the syntaxin family, is predicted to be a cytoplasmically oriented, integral membrane protein with a C-terminal transmembrane domain. Kinetic analyses indicate that deltapth1/vam3 mutants fail to process the soluble vacuolar hydrolase precursors and that PrA, PrB and most of CpY accumulate within the cell in their Golgi-modified P2 precursor forms. This is in contrast to a pep12 mutant in which P2CpY is secreted from the cell. Furthermore, pep12 is epistatic to pth1/vam3 with respect to the CpY secretion phenotype. Alkaline phosphatase, a vacuolar membrane hydrolase, accumulates in its precursor form in the deltapth1/vam3 mutant. Maturation of pro-aminopeptidase I, a hydrolase precursor delivered directly to the vacuole from the cytoplasm, is also blocked in the deltapth1/vam3 mutant. Subcellular fractionation localizes Pth1/Vam3p to vacuolar membranes. Based on these data, we propose that Pth1/Vam3p is the vacuolar syntaxin/t-SNARE homolog that participates in docking of transport vesicles at the vacuolar membrane and that the function of Pth1/Vam3p impinges on at least three routes of protein delivery to the yeast vacuole. PMID:9475723

Cloning, 3D modeling and expression analysis of three vacuolar invertase genes from cassava (Manihot Esculenta Crantz).

PubMed

Yao, Yuan; Wu, Xiao-Hui; Geng, Meng-Ting; Li, Rui-Mei; Liu, Jiao; Hu, Xin-Wen; Guo, Jian-Chun

2014-05-15

Vacuolar invertase is one of the key enzymes in sucrose metabolism that irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose in plants. In this research, three vacuolar invertase genes, named MeVINV1-3, and with 653, 660 and 639 amino acids, respectively, were cloned from cassava. The motifs of NDPNG (β-fructosidase motif), RDP and WECVD, which are conserved and essential for catalytic activity in the vacuolar invertase family, were found in MeVINV1 and MeVINV2. Meanwhile, in MeVINV3, instead of NDPNG we found the motif NGPDG, in which the three amino acids GPD are different from those in other vacuolar invertases (DPN) that might result in MeVINV3 being an inactivated protein. The N-terminal leader sequence of MeVINVs contains a signal anchor, which is associated with the sorting of vacuolar invertase to vacuole. The overall predicted 3D structure of the MeVINVs consists of a five bladed β-propeller module at N-terminus domain, and forms a β-sandwich module at the C-terminus domain. The active site of the protein is situated in the β-propeller module. MeVINVs are classified in two subfamilies, α and β groups, in which α group members of MeVINV1 and 2 are highly expressed in reproductive organs and tuber roots (considered as sink organs), while β group members of MeVINV3 are highly expressed in leaves (source organs). All MeVINVs are highly expressed in leaves, while only MeVINV1 and 2 are highly expressed in tubers at cassava tuber maturity stage. Thus, MeVINV1 and 2 play an important role in sucrose unloading and starch accumulation, as well in buffering the pools of sucrose, hexoses and sugar phosphates in leaves, specifically at later stages of plant development.

Novel families of vacuolar amino acid transporters.

PubMed

Sekito, Takayuki; Fujiki, Yuki; Ohsumi, Yoshinori; Kakinuma, Yoshimi

2008-08-01

Amino acids are compartmentalized in the vacuoles of microorganisms and plants. In Saccharomyces cerevisiae, basic amino acids accumulate preferentially into vacuoles but acidic amino acids are almost excluded from them. This indicates that selective machineries operate at the vacuolar membrane. The members of the amino acid/auxin permease family and the major facilitator superfamily involved in the vacuolar compartmentalization of amino acids have been recently identified in studies using S. cerevisiae. Homologous genes for these transporters are also found in plant and mammalian genomes. The physiological significance in response to nitrogen starvation can now be discussed. (c) 2008 IUBMB

Multi site polyadenylation and transcriptional response to stress of a vacuolar type H+-ATPase subunit A gene in Arabidopsis thaliana

PubMed Central

Magnotta, Scot M; Gogarten, Johann Peter

2002-01-01

Background Vacuolar type H+-ATPases play a critical role in the maintenance of vacuolar homeostasis in plant cells. V-ATPases are also involved in plants' defense against environmental stress. This research examined the expression and regulation of the catalytic subunit of the vacuolar type H+-ATPase in Arabidopsis thaliana and the effect of environmental stress on multiple transcripts generated by this gene. Results Evidence suggests that subunit A of the vacuolar type H+-ATPase is encoded by a single gene in Arabidopsis thaliana. Genome blot analysis showed no indication of a second subunit A gene being present. The single gene identified was shown by whole RNA blot analysis to be transcribed in all organs of the plant. Subunit A was shown by sequencing the 3' end of multiple cDNA clones to exhibit multi site polyadenylation. Four different poly (A) tail attachment sites were revealed. Experiments were performed to determine the response of transcript levels for subunit A to environmental stress. A PCR based strategy was devised to amplify the four different transcripts from the subunit A gene. Conclusions Amplification of cDNA generated from seedlings exposed to cold, salt stress, and etiolation showed that transcript levels for subunit A of the vacuolar type H+-ATPase in Arabidopsis were responsive to stress conditions. Cold and salt stress resulted in a 2–4 fold increase in all four subunit A transcripts evaluated. Etiolation resulted in a slight increase in transcript levels. All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation. PMID:11985780

The Marine Natural Product Manzamine A Targets Vacuolar ATPases and Inhibits Autophagy in Pancreatic Cancer Cells

PubMed Central

Kallifatidis, Georgios; Hoepfner, Dominic; Jaeg, Tiphaine; Guzmán, Esther A.; Wright, Amy E.

2013-01-01

Manzamine A, a member of the manzamine alkaloids, was originally isolated from marine sponges of the genus Haliclona. It was recently shown to have activity against pancreatic cancer cells, but the precise mechanism of action remained unclear. To further our understanding of the mechanism of action of manzamine A, chemogenomic profiling in the yeast S. cerevisiae was performed, suggesting that manzamine A is an uncoupler of vacuolar ATPases. Fluorescence microscopy confirmed this effect on yeast vacuoles, where manzamine A produced a phenotype very similar to that of the established v-ATPase inhibitor bafilomycin A1. In pancreatic cancer cells, 10 µM manzamine A affected vacuolar ATPase activity and significantly increased the level of autophagosome marker LC3-II and p62/SQSTM1 as observed by western blot analysis. Treatment with manzamine A in combination with bafilomycin A1 (inhibitor of autophagosome-lysosome fusion) did not change the levels of LC3-II when compared to cells treated with bafilomycin A1 alone, suggesting that manzamine A is a potential inhibitor of autophagy by preventing autophagosome turnover. As autophagy is essential for pancreatic tumor growth, blocking this pathway with manzamine A suggests a promising strategy for the treatment of pancreatic cancer. PMID:24048269

Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H(+)-ATPase.

PubMed Central

Laitala, T; Väänänen, H K

1994-01-01

The bone resorbing cells, osteoclasts, express high levels of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) during bone resorption. We have used antisense RNA and DNA molecules targeted against CA II, and against 16- and 60-kD subunits of vacuolar H(+)-ATPase (V-ATPase), to block the expression of these proteins in vitro. Osteoclastic bone resorption was studied in two in vitro culture systems: release of 45Calcium from prelabeled newborn mouse calvaria cultures, and resorption pit assays performed with rat osteoclasts cultured on bovine bone slices. Both antisense RNA and DNA against CA II and the V-ATPase were used to compare their specificities as regards inhibiting bone resorption in vitro. The antisense molecules inhibited the synthesis of these proteins by decreasing the amounts of mRNA in the cells in a highly specific manner. In osteoclast cultures treated with the 16-kD V-ATPase antisense RNA, acidification of an unknown population of intracellular vesicles was highly stimulated. The acidification of these vesicles was not sensitive to amiloride or bafilomycin A1. This suggests the existence of a back-up system for acidification of intracellular vesicles, when the expression of the V-ATPase is blocked. Our results further indicate that blocking the expression of CA II and V-ATPase with antisense RNA or DNA leads to decreased bone resorption. Images PMID:8200964

A programmable method for massively parallel targeted sequencing

PubMed Central

Hopmans, Erik S.; Natsoulis, Georges; Bell, John M.; Grimes, Susan M.; Sieh, Weiva; Ji, Hanlee P.

2014-01-01

We have developed a targeted resequencing approach referred to as Oligonucleotide-Selective Sequencing. In this study, we report a series of significant improvements and novel applications of this method whereby the surface of a sequencing flow cell is modified in situ to capture specific genomic regions of interest from a sample and then sequenced. These improvements include a fully automated targeted sequencing platform through the use of a standard Illumina cBot fluidics station. Targeting optimization increased the yield of total on-target sequencing data 2-fold compared to the previous iteration, while simultaneously increasing the percentage of reads that could be mapped to the human genome. The described assays cover up to 1421 genes with a total coverage of 5.5 Megabases (Mb). We demonstrate a 10-fold abundance uniformity of greater than 90% in 1 log distance from the median and a targeting rate of up to 95%. We also sequenced continuous genomic loci up to 1.5 Mb while simultaneously genotyping SNPs and genes. Variants with low minor allele fraction were sensitively detected at levels of 5%. Finally, we determined the exact breakpoint sequence of cancer rearrangements. Overall, this approach has high performance for selective sequencing of genome targets, configuration flexibility and variant calling accuracy. PMID:24782526

Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers

PubMed Central

Pabinger, Stephan; Ernst, Karina; Pulverer, Walter; Kallmeyer, Rainer; Valdes, Ana M.; Metrustry, Sarah; Katic, Denis; Nuzzo, Angelo; Kriegner, Albert; Vierlinger, Klemens; Weinhaeusel, Andreas

2016-01-01

Targeted sequencing of PCR amplicons generated from bisulfite deaminated DNA is a flexible, cost-effective way to study methylation of a sample at single CpG resolution and perform subsequent multi-target, multi-sample comparisons. Currently, no platform specific protocol, support, or analysis solution is provided to perform targeted bisulfite sequencing on a Personal Genome Machine (PGM). Here, we present a novel tool, called TABSAT, for analyzing targeted bisulfite sequencing data generated on Ion Torrent sequencers. The workflow starts with raw sequencing data, performs quality assessment, and uses a tailored version of Bismark to map the reads to a reference genome. The pipeline visualizes results as lollipop plots and is able to deduce specific methylation-patterns present in a sample. The obtained profiles are then summarized and compared between samples. In order to assess the performance of the targeted bisulfite sequencing workflow, 48 samples were used to generate 53 different Bisulfite-Sequencing PCR amplicons from each sample, resulting in 2,544 amplicon targets. We obtained a mean coverage of 282X using 1,196,822 aligned reads. Next, we compared the sequencing results of these targets to the methylation level of the corresponding sites on an Illumina 450k methylation chip. The calculated average Pearson correlation coefficient of 0.91 confirms the sequencing results with one of the industry-leading CpG methylation platforms and shows that targeted amplicon bisulfite sequencing provides an accurate and cost-efficient method for DNA methylation studies, e.g., to provide platform-independent confirmation of Illumina Infinium 450k methylation data. TABSAT offers a novel way to analyze data generated by Ion Torrent instruments and can also be used with data from the Illumina MiSeq platform. It can be easily accessed via the Platomics platform, which offers a web-based graphical user interface along with sample and parameter storage. TABSAT is freely

A vacuolar iron transporter in tulip, TgVit1, is responsible for blue coloration in petal cells through iron accumulation.

PubMed

Momonoi, Kazumi; Yoshida, Kumi; Mano, Shoji; Takahashi, Hideyuki; Nakamori, Chihiro; Shoji, Kazuaki; Nitta, Akira; Nishimura, Mikio

2009-08-01

Blue color in flowers is due mainly to anthocyanins, and a considerable part of blue coloration can be attributed to metal-complexed anthocyanins. However, the mechanism of metal ion transport into vacuoles and subsequent flower color development has yet to be fully explored. Previously, we studied the mechanism of blue color development specifically at the bottom of the inner perianth in purple tulip petals of Tulipa gesneriana cv. Murasakizuisho. We found that differences in iron content were associated with the development of blue- and purple-colored cells. Here, we identify a vacuolar iron transporter in T. gesneriana (TgVit1), and characterize the localization and function of this transporter protein in tulip petals. The amino acid sequence of TgVit1 is 85% similar that of the Arabidopsis thaliana vacuolar iron transporter AtVIT1, and also showed similarity to the AtVIT1 homolog in yeast, Ca(2+)-sensitive cross-complementer 1 (CCC1). The gene TgVit1 was expressed exclusively in blue-colored epidermal cells, and protein levels increased with increasing mRNA expression and blue coloration. Transient expression experiments revealed that TgVit1 localizes to the vacuolar membrane, and is responsible for the development of the blue color in purple cells. Expression of TgVit1 in yeast rescued the growth defect of ccc1 mutant cells in the presence of high concentrations of FeSO(4). Our results indicate that TgVit1 plays an essential role in blue coloration as a vacuolar iron transporter in tulip petals. These results suggest a new role for involvement of a vacuolar iron transporter in blue flower color development.

Wheat Vacuolar Iron Transporter TaVIT2 Transports Fe and Mn and Is Effective for Biofortification.

PubMed

Connorton, James M; Jones, Eleanor R; Rodríguez-Ramiro, Ildefonso; Fairweather-Tait, Susan; Uauy, Cristobal; Balk, Janneke

2017-08-01

Increasing the intrinsic nutritional quality of crops, known as biofortification, is viewed as a sustainable approach to alleviate micronutrient deficiencies. In particular, iron deficiency anemia is a major global health issue, but the iron content of staple crops such as wheat ( Triticum aestivum ) is difficult to change because of genetic complexity and homeostasis mechanisms. To identify target genes for the biofortification of wheat, we functionally characterized homologs of the VACUOLAR IRON TRANSPORTER ( VIT ). The wheat genome contains two VIT paralogs, TaVIT1 and TaVIT2 , which have different expression patterns but are both low in the endosperm. TaVIT2, but not TaVIT1, was able to rescue the growth of a yeast ( Saccharomyces cerevisiae ) mutant defective in vacuolar iron transport. TaVIT2 also complemented a manganese transporter mutant but not a vacuolar zinc transporter mutant. By overexpressing TaVIT2 under the control of an endosperm-specific promoter, we achieved a greater than 2-fold increase in iron in white flour fractions, exceeding minimum legal fortification levels in countries such as the United Kingdom. The antinutrient phytate was not increased and the iron in the white flour fraction was bioavailable in vitro, suggesting that food products made from the biofortified flour could contribute to improved iron nutrition. The single-gene approach impacted minimally on plant growth and also was effective in barley ( Hordeum vulgare ). Our results show that by enhancing vacuolar iron transport in the endosperm, this essential micronutrient accumulated in this tissue, bypassing existing homeostatic mechanisms. © 2017 American Society of Plant Biologists. All Rights Reserved.

The vacuolar channel VvALMT9 mediates malate and tartrate accumulation in berries of Vitis vinifera.

PubMed

De Angeli, Alexis; Baetz, Ulrike; Francisco, Rita; Zhang, Jingbo; Chaves, Maria Manuela; Regalado, Ana

2013-08-01

Vitis vinifera L. represents an economically important fruit species. Grape and wine flavour is made from a complex set of compounds. The acidity of berries is a major parameter in determining grape berry quality for wine making and fruit consumption. Despite the importance of malic and tartaric acid (TA) storage and transport for grape berry acidity, no vacuolar transporter for malate or tartrate has been identified so far. Some members of the aluminium-activated malate transporter (ALMT) anion channel family from Arabidopsis thaliana have been shown to be involved in mediating malate fluxes across the tonoplast. Therefore, we hypothesised that a homologue of these channels could have a similar role in V. vinifera grape berries. We identified homologues of the Arabidopsis vacuolar anion channel AtALMT9 through a TBLASTX search on the V. vinifera genome database. We cloned the closest homologue of AtALMT9 from grape berry cDNA and designated it VvALMT9. The expression profile revealed that VvALMT9 is constitutively expressed in berry mesocarp tissue and that its transcription level increases during fruit maturation. Moreover, we found that VvALMT9 is targeted to the vacuolar membrane. Using patch-clamp analysis, we could show that, besides malate, VvALMT9 mediates tartrate currents which are higher than in its Arabidopsis homologue. In summary, in the present study we provide evidence that VvALMT9 is a vacuolar malate channel expressed in grape berries. Interestingly, in V. vinifera, a tartrate-producing plant, the permeability of the channel is apparently adjusted to TA.

Tributyltin sensitivity of vacuolar-type Na(+)-transporting ATPase from Enterococcus hirae.

PubMed

Chardwiriyapreecha, Soracom; Inoue, Tomohiro; Sugimoto, Naoko; Sekito, Takayuki; Yamato, Ichiro; Murata, Takeshi; Homma, Michio; Kakinuma, Yoshimi

2009-10-01

Tributyltin chloride (TBT), an environmental pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Some members of F-ATP synthase (F-ATPase)/vacuolar type ATPase (V-ATPase) superfamily have been identified as the molecular target of this compound. TBT inhibited the activities of H(+)-transporting or Na(+)-transporting F-ATPase as well as H(+)-transporting V-ATPase originated from various organisms. However, the sensitivity to TBT of Na(+)-transporting V-ATPase has not been investigated. We examined the effect of TBT on Na(+)-transporting V-ATPase from an eubacterium Enterococus hirae. The ATP hydrolytic activity of E. hirae V-ATPase in purified form as well as in membrane-bound form was little inhibited by less than 10 microM TBT; IC50 for TBT inhibition of purified enzyme was estimated to be about 35 microM. Active sodium transport by E. hirae cells, indicating the in vivo activity of this V-ATPase, was not inhibited by 20 microM TBT. By contrast, IC50 of H(+)-transporting V-ATPase of the vacuolar membrane vesicles from Saccharomyces cerevisiae was about 0.2 microM. E. hirae V-ATPase is thus extremely less sensitive to TBT.

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue

PubMed Central

Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

2008-01-01

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen–stigma interactions that regulate pollen tube growth in Nicotiana. PMID:18689443

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue.

PubMed

Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

2008-01-01

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen-stigma interactions that regulate pollen tube growth in Nicotiana.

Vacuolar protein sorting mechanisms in plants.

PubMed

Xiang, Li; Etxeberria, Ed; Van den Ende, Wim

2013-02-01

Plant vacuoles are unique, multifunctional organelles among eukaryotes. Considerable new insights in plant vacuolar protein sorting have been obtained recently. The basic machinery of protein export from the endoplasmic reticulum to the Golgi and the classical route to the lytic vacuole and the protein storage vacuole shows many similarities to vacuolar/lysosomal sorting in other eukaryotes. However, as a result of its unique functions in plant defence and as a storage compartment, some plant-specific entities and sorting determinants appear to exist. The alternative post-Golgi route, as found in animals and yeast, probably exists in plants as well. Likely, adaptor protein complex 3 fulfils a central role in this route. A Golgi-independent route involving plant-specific endoplasmic reticulum bodies appears to provide sedentary organisms such as plants with extra flexibility to cope with changing environmental conditions. © 2012 The Authors Journal compilation © 2012 FEBS.

Single molecule targeted sequencing for cancer gene mutation detection.

PubMed

Gao, Yan; Deng, Liwei; Yan, Qin; Gao, Yongqian; Wu, Zengding; Cai, Jinsen; Ji, Daorui; Li, Gailing; Wu, Ping; Jin, Huan; Zhao, Luyang; Liu, Song; Ge, Liangjin; Deem, Michael W; He, Jiankui

2016-05-19

With the rapid decline in cost of sequencing, it is now affordable to examine multiple genes in a single disease-targeted clinical test using next generation sequencing. Current targeted sequencing methods require a separate step of targeted capture enrichment during sample preparation before sequencing. Although there are fast sample preparation methods available in market, the library preparation process is still relatively complicated for physicians to use routinely. Here, we introduced an amplification-free Single Molecule Targeted Sequencing (SMTS) technology, which combined targeted capture and sequencing in one step. We demonstrated that this technology can detect low-frequency mutations using artificially synthesized DNA sample. SMTS has several potential advantages, including simple sample preparation thus no biases and errors are introduced by PCR reaction. SMTS has the potential to be an easy and quick sequencing technology for clinical diagnosis such as cancer gene mutation detection, infectious disease detection, inherited condition screening and noninvasive prenatal diagnosis.

Evidence for Avt6 as a vacuolar exporter of acidic amino acids in Saccharomyces cerevisiae cells.

PubMed

Chahomchuen, Thippayarat; Hondo, Kana; Ohsaki, Mariko; Sekito, Takayuki; Kakinuma, Yoshimi

2009-12-01

Here we examined the significance of Avt6, a vacuolar exporter of glutamate and aspartate suggested by the in vitro membrane vesicle experiment, in vacuolar compartmentalization of amino acids in Saccharomyces cerevisiae cells. Fluorescent microscopic observation of GFP-fused Avt6 revealed it to be exclusively localized to the vacuolar membrane, with the amount of Myc-tagged Avt6 significantly increased under nitrogen starvation. Glutamate uptake by cells was enhanced by deletion of the AVT6 gene, indicating indirect involvement of Avt6 in cellular glutamate accumulation. Differences in acidic amino acid content of both total and vacuolar fractions were insignificant between the parent and avt6Delta cells when cultured in nutrient-rich conditions. However, in nitrogen-starved conditions, the amount of glutamate and aspartate in the vacuolar fraction was notably increased in the avt6Delta cells. Avt6 is thus involved in vacuolar amino acid compartmentalization in S. cerevisiae cells, especially under conditions of nitrogen starvation.

Ypq3p-dependent histidine uptake by the vacuolar membrane vesicles of Saccharomyces cerevisiae.

PubMed

Manabe, Kunio; Kawano-Kawada, Miyuki; Ikeda, Koichi; Sekito, Takayuki; Kakinuma, Yoshimi

2016-06-01

The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.

The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells.

PubMed

Miao, Yansong; Li, Kwun Yee; Li, Hong-Ye; Yao, Xiaoqiang; Jiang, Liwen

2008-12-01

Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.

The amino-terminal hydrophilic region of the vacuolar transporter Avt3p is dispensable for the vacuolar amino acid compartmentalization of Schizosaccharomyces pombe.

PubMed

Kawano-Kawada, Miyuki; Chardwiriyapreecha, Soracom; Manabe, Kunio; Sekito, Takayuki; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

2016-12-01

Avt3p, a vacuolar amino acid exporter (656 amino acid residues) that is important for vacuolar amino acid compartmentalization as well as spore formation in Schizosaccharomyces pombe, has an extremely long hydrophilic region (approximately 290 amino acid residues) at its N-terminus. Because known functional domains have not been found in this region, its functional role was examined with a deletion mutant avt3 (∆1-270) expressed in S. pombe avt3∆ cells. The deletion of this region did not affect its intracellular localization or vacuolar contents of basic amino acids as well as neutral ones. The defect of avt3Δ cells in spore formation was rescued by the expression of avt3 + but was not completely rescued by the expression of avt3 (∆1-270) . The N-terminal region is thus dispensable for the function of Avt3p as an amino acid exporter, but it is likely to be involved in the role of Avt3p under nutritional starvation conditions.

Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae.

PubMed

Tone, Junichi; Yoshimura, Ayumi; Manabe, Kunio; Murao, Nami; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2015-01-01

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1∆ mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.

Vacuolar morphology of Saccharomyces cerevisiae during the process of wine making and Japanese sake brewing.

PubMed

Izawa, Shingo; Ikeda, Kayo; Miki, Takeo; Wakai, Yoshinori; Inoue, Yoshiharu

2010-09-01

Although ethanol and osmotic stress affect the vacuolar morphology of Saccharomyces cerevisiae, little information is available about changes in vacuolar morphology during the processes of wine making and Japanese sake (rice wine) brewing. Here, we elucidated changes in the morphology of yeast vacuoles using Zrc1p-GFP, a vacuolar membrane protein, so as to better understand yeast physiology during the brewing process. Wine yeast cells (OC-2 and EC1118) contained highly fragmented vacuoles in the sake mash (moromi) as well as in the grape must. Although sake yeast cells (Kyokai no. 9 and no. 10) also contained highly fragmented vacuoles during the wine-making process, they showed quite a distinct vacuolar morphology during sake brewing. Since the environment surrounding sake yeast cells in the sake mash did not differ much from that surrounding wine yeast cells, the difference in vacuolar morphology during sake brewing between wine yeast and sake yeast was likely caused by innate characters.

Wheat Vacuolar Iron Transporter TaVIT2 Transports Fe and Mn and Is Effective for Biofortification1[OPEN

PubMed Central

Jones, Eleanor R.; Rodríguez-Ramiro, Ildefonso

2017-01-01

Increasing the intrinsic nutritional quality of crops, known as biofortification, is viewed as a sustainable approach to alleviate micronutrient deficiencies. In particular, iron deficiency anemia is a major global health issue, but the iron content of staple crops such as wheat (Triticum aestivum) is difficult to change because of genetic complexity and homeostasis mechanisms. To identify target genes for the biofortification of wheat, we functionally characterized homologs of the VACUOLAR IRON TRANSPORTER (VIT). The wheat genome contains two VIT paralogs, TaVIT1 and TaVIT2, which have different expression patterns but are both low in the endosperm. TaVIT2, but not TaVIT1, was able to rescue the growth of a yeast (Saccharomyces cerevisiae) mutant defective in vacuolar iron transport. TaVIT2 also complemented a manganese transporter mutant but not a vacuolar zinc transporter mutant. By overexpressing TaVIT2 under the control of an endosperm-specific promoter, we achieved a greater than 2-fold increase in iron in white flour fractions, exceeding minimum legal fortification levels in countries such as the United Kingdom. The antinutrient phytate was not increased and the iron in the white flour fraction was bioavailable in vitro, suggesting that food products made from the biofortified flour could contribute to improved iron nutrition. The single-gene approach impacted minimally on plant growth and also was effective in barley (Hordeum vulgare). Our results show that by enhancing vacuolar iron transport in the endosperm, this essential micronutrient accumulated in this tissue, bypassing existing homeostatic mechanisms. PMID:28684433

Biolayer interferometry of lipid nanodisc-reconstituted yeast vacuolar H+ -ATPase.

PubMed

Sharma, Stuti; Wilkens, Stephan

2017-05-01

Vacuolar H + -ATPase (V-ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V-ATPase activity is regulated by reversible disassembly, resulting in cytosolic V 1 -ATPase and membrane-integral V 0 proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein-protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein-protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc-reconstituted V-ATPase (V 1 V 0 ND). We show that V 1 V 0 ND can be immobilized on streptavidin-coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation. © 2017 The Protein Society.

The V-ATPase subunit A is essential for salt tolerance through participating in vacuolar Na+ compartmentalization in Salicornia europaea.

PubMed

Lv, Sulian; Jiang, Ping; Tai, Fang; Wang, Duoliya; Feng, Juanjuan; Fan, Pengxiang; Bao, Hexigeduleng; Li, Yinxin

2017-12-01

The V-ATPase subunit A participates in vacuolar Na + compartmentalization in Salicornia europaea regulating V-ATPase and V-PPase activities. Na + sequestration into the vacuole is an efficient strategy in response to salinity in many halophytes. However, it is not yet fully understood how this process is achieved. Particularly, the role of vacuolar H + -ATPase (V-ATPase) in this process is controversial. Our previous proteomic investigation in the euhalophyte Salicornia europaea L. found a significant increase of the abundance of V-ATPase subunit A under salinity. Here, the gene encoding this subunit named SeVHA-A was characterized, and its role in salt tolerance was demonstrated by RNAi directed downregulation in suspension-cultured cells of S. europaea. The transcripts of genes encoding vacuolar H + -PPase (V-PPase) and vacuolar Na + /H + antiporter (SeNHX1) also decreased significantly in the RNAi cells. Knockdown of SeVHA-A resulted in a reduction in both V-ATPase and vacuolar H + -PPase (V-PPase) activities. Accordingly, the SeVHA-A-RNAi cells showed increased vacuolar pH and decreased cell viability under different NaCl concentrations. Further Na + staining showed the reduced vacuolar Na + sequestration in RNAi cells. Taken together, our results evidenced that SeVHA-A participates in vacuolar Na + sequestration regulating V-ATPase and V-PPase activities and thereby vacuolar pH in S. europaea. The possible mechanisms underlying the reduction of vacuolar V-PPase activity in SeVHA-A-RNAi cells were also discussed.

Avt5p is required for vacuolar uptake of amino acids in the fission yeast Schizosaccharomyces pombe.

PubMed

Chardwiriyapreecha, Soracom; Mukaiyama, Hiroyuki; Sekito, Takayuki; Iwaki, Tomoko; Takegawa, Kaoru; Kakinuma, Yoshimi

2010-06-03

We identified SPBC1685.07c of Schizosaccharomyces pombe as a novel vacuolar protein, Avt5p, with similarity to vacuolar amino acid transporters Avt5p from Saccharomyces cerevisiae. Avt5p localizes to the vacuolar membrane and upon disruption of avt5, uptake of histidine, glutamate, tyrosine, arginine, lysine or serine was impaired. During nitrogen starvation, the transient increase of vacuolar lysine transport observed for wild-type cells still occurred in the mutant cells, however, uptake of glutamate did not significantly increase in response to nitrogen starvation. Our results show that under diverse growth conditions Avt5p is involved in vacuolar transport of a selective set of amino acids. Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+ -pumping pyrophosphatase in pepper plants.

PubMed

Vigani, Gianpiero; Rolli, Eleonora; Marasco, Ramona; Dell'Orto, Marta; Michoud, Grégoire; Soussi, Asma; Raddadi, Noura; Borin, Sara; Sorlini, Claudia; Zocchi, Graziano; Daffonchio, Daniele

2018-05-22

It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H + -ATPase (V-ATPase) and H + -PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H + -ATPase (V-ATPase) and H + -PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes. This article is protected by copyright. All rights reserved. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Identification of the fnx1+ and fnx2+ genes for vacuolar amino acid transporters in Schizosaccharomyces pombe.

PubMed

Chardwiriyapreecha, Soracom; Shimazu, Masamitsu; Morita, Tomotake; Sekito, Takayuki; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

2008-06-25

We have identified the Schizosaccharomyces pombe SPBC3E7.06c gene (fnx2(+)) from a homology search with the fnx1(+) gene involving in G(0) arrest upon nitrogen starvation. Green fluorescent protein-fused Fnx1p and Fnx2p localized exclusively to the vacuolar membrane. Uptake of histidine or isoleucine by S. pombe cells was inhibited by concanamycin A, a specific inhibitor of the vacuolar H(+)-ATPase. Amino acid uptake was also defective in the vacuolar ATPase mutant, suggesting that vacuolar compartmentalization is critical for amino acid uptake by whole cells. In both Deltafnx1 and Deltafnx2 mutant cells, uptake of lysine, isoleucine or asparagine was impaired. These results suggest that fnx1(+) and fnx2(+) are involved in vacuolar amino acid uptake in S. pombe.

Vacuolar transporter Avt4 is involved in excretion of basic amino acids from the vacuoles of Saccharomyces cerevisiae.

PubMed

Sekito, Takayuki; Chardwiriyapreecha, Soracom; Sugimoto, Naoko; Ishimoto, Masaya; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2014-01-01

Basic amino acids (lysine, histidine and arginine) accumulated in Saccharomyces cerevisiae vacuoles should be mobilized to cytosolic nitrogen metabolism under starvation. We found that the decrease of vacuolar basic amino acids in response to nitrogen starvation was impaired by the deletion of AVT4 gene encoding a vacuolar transporter. In addition, overexpression of AVT4 reduced the accumulation of basic amino acids in vacuoles under nutrient-rich condition. In contrast to AVT4, the deletion and overexpression of AVT3, which encodes the closest homologue of Avt4p, did not affect the contents of vacuolar basic amino acids. Consistent with these, arginine uptake into vacuolar membrane vesicles was decreased by Avt4p-, but not by Avt3p-overproduction, whereas various neutral amino acids were excreted from vacuolar membrane vesicles in a manner dependent on either Avt4p or Avt3p. These results suggest that Avt4p is a vacuolar amino acid exporter involving in the recycling of basic amino acids.

Biolayer interferometry of lipid nanodisc‐reconstituted yeast vacuolar H+‐ATPase

PubMed Central

Sharma, Stuti

2017-01-01

Abstract Vacuolar H+‐ATPase (V‐ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V‐ATPase activity is regulated by reversible disassembly, resulting in cytosolic V 1‐ATPase and membrane‐integral V 0 proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein‐protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein–protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc‐reconstituted V‐ATPase (V 1 V 0ND). We show that V 1 V 0ND can be immobilized on streptavidin‐coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation. PMID:28241399

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

PubMed Central

Blomquist, Thomas M.; Crawford, Erin L.; Lovett, Jennie L.; Yeo, Jiyoun; Stanoszek, Lauren M.; Levin, Albert; Li, Jia; Lu, Mei; Shi, Leming; Muldrew, Kenneth; Willey, James C.

2013-01-01

Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1

Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

PubMed

Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

2017-01-01

Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

Vacuolar Chloride Fluxes Impact Ion Content and Distribution during Early Salinity Stress1

PubMed Central

Baetz, Ulrike; Tohge, Takayuki; Martinoia, Enrico; De Angeli, Alexis

2016-01-01

The ability to control the cytoplasmic environment is a prerequisite for plants to cope with changing environmental conditions. During salt stress, for instance, Na+ and Cl− are sequestered into the vacuole to help maintain cytosolic ion homeostasis and avoid cellular damage. It has been observed that vacuolar ion uptake is tied to fluxes across the plasma membrane. The coordination of both transport processes and relative contribution to plant adaptation, however, is still poorly understood. To investigate the link between vacuolar anion uptake and whole-plant ion distribution during salinity, we used mutants of the only vacuolar Cl− channel described to date: the Arabidopsis (Arabidopsis thaliana) ALMT9. After 24-h NaCl treatment, almt9 knock-out mutants had reduced shoot accumulation of both Cl− and Na+. In contrast, almt9 plants complemented with a mutant variant of ALMT9 that exhibits enhanced channel activity showed higher Cl− and Na+ accumulation. The altered shoot ion contents were not based on differences in transpiration, pointing to a vacuolar function in regulating xylem loading during salinity. In line with this finding, GUS staining demonstrated that ALMT9 is highly expressed in the vasculature of shoots and roots. RNA-seq analysis of almt9 mutants under salinity revealed specific expression profiles of transporters involved in long-distance ion translocation. Taken together, our study uncovers that the capacity of vacuolar Cl− loading in vascular cells plays a crucial role in controlling whole-plant ion movement rapidly after onset of salinity. PMID:27503602

Accurate and exact CNV identification from targeted high-throughput sequence data.

PubMed

Nord, Alex S; Lee, Ming; King, Mary-Claire; Walsh, Tom

2011-04-12

Massively parallel sequencing of barcoded DNA samples significantly increases screening efficiency for clinically important genes. Short read aligners are well suited to single nucleotide and indel detection. However, methods for CNV detection from targeted enrichment are lacking. We present a method combining coverage with map information for the identification of deletions and duplications in targeted sequence data. Sequencing data is first scanned for gains and losses using a comparison of normalized coverage data between samples. CNV calls are confirmed by testing for a signature of sequences that span the CNV breakpoint. With our method, CNVs can be identified regardless of whether breakpoints are within regions targeted for sequencing. For CNVs where at least one breakpoint is within targeted sequence, exact CNV breakpoints can be identified. In a test data set of 96 subjects sequenced across ~1 Mb genomic sequence using multiplexing technology, our method detected mutations as small as 31 bp, predicted quantitative copy count, and had a low false-positive rate. Application of this method allows for identification of gains and losses in targeted sequence data, providing comprehensive mutation screening when combined with a short read aligner.

The siRNA Non-seed Region and Its Target Sequences Are Auxiliary Determinants of Off-Target Effects.

PubMed

Kamola, Piotr J; Nakano, Yuko; Takahashi, Tomoko; Wilson, Paul A; Ui-Tei, Kumiko

2015-12-01

RNA interference (RNAi) is a powerful tool for post-transcriptional gene silencing. However, the siRNA guide strand may bind unintended off-target transcripts via partial sequence complementarity by a mechanism closely mirroring micro RNA (miRNA) silencing. To better understand these off-target effects, we investigated the correlation between sequence features within various subsections of siRNA guide strands, and its corresponding target sequences, with off-target activities. Our results confirm previous reports that strength of base-pairing in the siRNA seed region is the primary factor determining the efficiency of off-target silencing. However, the degree of downregulation of off-target transcripts with shared seed sequence is not necessarily similar, suggesting that there are additional auxiliary factors that influence the silencing potential. Here, we demonstrate that both the melting temperature (Tm) in a subsection of siRNA non-seed region, and the GC contents of its corresponding target sequences, are negatively correlated with the efficiency of off-target effect. Analysis of experimentally validated miRNA targets demonstrated a similar trend, indicating a putative conserved mechanistic feature of seed region-dependent targeting mechanism. These observations may prove useful as parameters for off-target prediction algorithms and improve siRNA 'specificity' design rules.

Highly multiplexed targeted DNA sequencing from single nuclei.

PubMed

Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

2016-02-01

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae.

PubMed

Chardwiriyapreecha, Soracom; Manabe, Kunio; Iwaki, Tomoko; Kawano-Kawada, Miyuki; Sekito, Takayuki; Lunprom, Siriporn; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

2015-01-01

In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3+-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3+ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3+-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles.

Regulation of Vacuolar pH in Citrus limon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lincoln Taiz

The primary objective of this grant was to characterize the vacuolar V-ATPase of lemon fruits. Lemon fruit vacuoles have an internal pH of about 2.5. Since a typical plant vacuole has a luminal pH of around 5.5, the lemon fruit V-APTase must have special properties which allow it to acidify the lumen to such a low pH: (1) it might have a different structure; (2) it might have a different H{sup +}/ATP stoichiometry; and (3) it might be regulated differently. During the course of the investigations (which began in 1996) they characterized these aspects of the V-ATPases of both lemonmore » fruits and lime fruits. They examined lime fruits because of the availability of both acidic limes with a low vacuolar pH and sweet limes, which have a much higher vacuolar pH. The existence of two types of lime fruits allowed a comparison of the V-ATPases of the two varieties. In this report they are including two publications from 1996 and 1997 as background for the later publications. A review article with Heven Sze on V-ATPase nomenclature was also generated during the funding period. In addition to the studies on citrus fruit vacuoles, they also initiated studies in two new areas: polar auxin transport and the regulation of stomatal opening by UV-B irradiation. These studies were intended to serve as a basis of future separate grants, but the proposals they submitted on these topics were not funded.« less

Targeted Re-Sequencing Emulsion PCR Panel for Myopathies: Results in 94 Cases.

PubMed

Punetha, Jaya; Kesari, Akanchha; Uapinyoying, Prech; Giri, Mamta; Clarke, Nigel F; Waddell, Leigh B; North, Kathryn N; Ghaoui, Roula; O'Grady, Gina L; Oates, Emily C; Sandaradura, Sarah A; Bönnemann, Carsten G; Donkervoort, Sandra; Plotz, Paul H; Smith, Edward C; Tesi-Rocha, Carolina; Bertorini, Tulio E; Tarnopolsky, Mark A; Reitter, Bernd; Hausmanowa-Petrusewicz, Irena; Hoffman, Eric P

2016-05-27

Molecular diagnostics in the genetic myopathies often requires testing of the largest and most complex transcript units in the human genome (DMD, TTN, NEB). Iteratively targeting single genes for sequencing has traditionally entailed high costs and long turnaround times. Exome sequencing has begun to supplant single targeted genes, but there are concerns regarding coverage and needed depth of the very large and complex genes that frequently cause myopathies. To evaluate efficiency of next-generation sequencing technologies to provide molecular diagnostics for patients with previously undiagnosed myopathies. We tested a targeted re-sequencing approach, using a 45 gene emulsion PCR myopathy panel, with subsequent sequencing on the Illumina platform in 94 undiagnosed patients. We compared the targeted re-sequencing approach to exome sequencing for 10 of these patients studied. We detected likely pathogenic mutations in 33 out of 94 patients with a molecular diagnostic rate of approximately 35%. The remaining patients showed variants of unknown significance (35/94 patients) or no mutations detected in the 45 genes tested (26/94 patients). Mutation detection rates for targeted re-sequencing vs. whole exome were similar in both methods; however exome sequencing showed better distribution of reads and fewer exon dropouts. Given that costs of highly parallel re-sequencing and whole exome sequencing are similar, and that exome sequencing now takes considerably less laboratory processing time than targeted re-sequencing, we recommend exome sequencing as the standard approach for molecular diagnostics of myopathies.

The eukaryotic signal sequence, YGRL, targets the chlamydial inclusion

PubMed Central

Kabeiseman, Emily J.; Cichos, Kyle H.; Moore, Elizabeth R.

2014-01-01

Understanding how host proteins are targeted to pathogen-specified organelles, like the chlamydial inclusion, is fundamentally important to understanding the biogenesis of these unique subcellular compartments and how they maintain autonomy within the cell. Syntaxin 6, which localizes to the chlamydial inclusion, contains an YGRL signal sequence. The YGRL functions to return syntaxin 6 to the trans-Golgi from the plasma membrane, and deletion of the YGRL signal sequence from syntaxin 6 also prevents the protein from localizing to the chlamydial inclusion. YGRL is one of three YXXL (YGRL, YQRL, and YKGL) signal sequences which target proteins to the trans-Golgi. We designed various constructs of eukaryotic proteins to test the specificity and propensity of YXXL sequences to target the inclusion. The YGRL signal sequence redirects proteins (e.g., Tgn38, furin, syntaxin 4) that normally do not localize to the chlamydial inclusion. Further, the requirement of the YGRL signal sequence for syntaxin 6 localization to inclusions formed by different species of Chlamydia is conserved. These data indicate that there is an inherent property of the chlamydial inclusion, which allows it to recognize the YGRL signal sequence. To examine whether this “inherent property” was protein or lipid in nature, we asked if deletion of the YGRL signal sequence from syntaxin 6 altered the ability of the protein to interact with proteins or lipids. Deletion or alteration of the YGRL from syntaxin 6 does not appreciably impact syntaxin 6-protein interactions, but does decrease syntaxin 6-lipid interactions. Intriguingly, data also demonstrate that YKGL or YQRL can successfully substitute for YGRL in localization of syntaxin 6 to the chlamydial inclusion. Importantly and for the first time, we are establishing that a eukaryotic signal sequence targets the chlamydial inclusion. PMID:25309881

A tale of two sequences: microRNA-target chimeric reads.

PubMed

Broughton, James P; Pasquinelli, Amy E

2016-04-04

In animals, a functional interaction between a microRNA (miRNA) and its target RNA requires only partial base pairing. The limited number of base pair interactions required for miRNA targeting provides miRNAs with broad regulatory potential and also makes target prediction challenging. Computational approaches to target prediction have focused on identifying miRNA target sites based on known sequence features that are important for canonical targeting and may miss non-canonical targets. Current state-of-the-art experimental approaches, such as CLIP-seq (cross-linking immunoprecipitation with sequencing), PAR-CLIP (photoactivatable-ribonucleoside-enhanced CLIP), and iCLIP (individual-nucleotide resolution CLIP), require inference of which miRNA is bound at each site. Recently, the development of methods to ligate miRNAs to their target RNAs during the preparation of sequencing libraries has provided a new tool for the identification of miRNA target sites. The chimeric, or hybrid, miRNA-target reads that are produced by these methods unambiguously identify the miRNA bound at a specific target site. The information provided by these chimeric reads has revealed extensive non-canonical interactions between miRNAs and their target mRNAs, and identified many novel interactions between miRNAs and noncoding RNAs.

A vacuolar membrane protein Avt7p is involved in transport of amino acid and spore formation in Saccharomyces cerevisiae.

PubMed

Tone, Junichi; Yamanaka, Atsushi; Manabe, Kunio; Murao, Nami; Kawano-Kawada, Miyuki; Sekito, Takayuki; Kakinuma, Yoshimi

2015-01-01

Active transport systems for various amino acids operate in the vacuolar membrane of Saccharomyces cerevisiae. The gene families for vacuolar amino acid transporters were identified by reverse genetics experiments. In the AVT transporter family, Avt1p works for active uptake of amino acid into vacuole, and Avt3p, Avt4p, and Avt6p for active extrusion of amino acid from vacuole to cytosol. Here, we found green fluorescent protein-tagged Avt7p, an unidentified member of the AVT family, localized to the vacuolar membrane of S. cerevisiae. Disruption of the AVT7 gene enhanced both vacuolar contents of several amino acids and uptake activities of glutamine and proline by vacuolar membrane vesicles. Efficiency of spore formation was impaired by the disruption of the AVT7 gene, suggesting the physiological importance of Avt7p-dependent efflux of amino acid from vacuoles under nutrient-poor condition.

Defects of Vps15 in skeletal muscles lead to autophagic vacuolar myopathy and lysosomal disease

PubMed Central

Nemazanyy, Ivan; Blaauw, Bert; Paolini, Cecilia; Caillaud, Catherine; Protasi, Feliciano; Mueller, Amelie; Proikas-Cezanne, Tassula; Russell, Ryan C; Guan, Kun-Liang; Nishino, Ichizo; Sandri, Marco; Pende, Mario; Panasyuk, Ganna

2013-01-01

The complex of Vacuolar Protein Sorting 34 and 15 (Vps34 and Vps15) has Class III phosphatidylinositol 3-kinase activity and putative roles in nutrient sensing, mammalian Target Of Rapamycin (mTOR) activation by amino acids, cell growth, vesicular trafficking and autophagy. Contrary to expectations, here we show that Vps15-deficient mouse tissues are competent for LC3-positive autophagosome formation and maintain mTOR activation. However, an impaired lysosomal function in mutant cells is traced by accumulation of adaptor protein p62, LC3 and Lamp2 positive vesicles, which can be reverted to normal levels after ectopic overexpression of Vps15. Mice lacking Vps15 in skeletal muscles, develop a severe myopathy. Distinct from the autophagy deficient Atg7−/− mutants, pathognomonic morphological hallmarks of autophagic vacuolar myopathy (AVM) are observed in Vps15−/− mutants, including elevated creatine kinase plasma levels, accumulation of autophagosomes, glycogen and sarcolemmal features within the fibres. Importantly, Vps34/Vps15 overexpression in myoblasts of Danon AVM disease patients alleviates the glycogen accumulation. Thus, the activity of the Vps34/Vps15 complex is critical in disease conditions such as AVMs, and possibly a variety of other lysosomal storage diseases. PMID:23630012

Calmodulin-stimulated Ca(2+)-ATPases in the vacuolar and plasma membranes in cauliflower.

PubMed

Askerlund, P

1997-07-01

The subcellular locations of Ca(2+)-ATPases in the membranes of cauliflower (Brassica oleracea L.) inflorescences were investigated. After continuous sucrose gradient centrifugation a 111-kD calmodulin (CaM)-stimulated and caM-binding Ca(2+)-ATPase (BCA1; P. Askerlund [1996] Plant Physiol 110: 913-922; S. Malmström, P. Askerlund, M.G. Plamgren [1997] FEBS Lett 400: 324-328) comigrated with vacuolar membrane markers, whereas a 116-kD caM-binding Ca(2+)-ATPase co-migrated with a marker for the plasma membrane. The 116 kD Ca(2+)-ATPase was enriched in plasma membranes obtained by aqueous two-phase partitioning, which is in agreement with a plasma membrane location of this Ca(2+)-ATPase. Countercurrent distribution of a low-density intracellular membrane fraction in an aqueous two-phase system resulted in the separation of the endoplasmic reticulum and vacuolar membranes. The 111-kD Ca(2+)-ATPase co-migrated with a vacuolar membrane marker after countercurrent distribution but not with markers for the endoplasmic reticulum. A vacuolar membrane location of the 111-kD Ca(2+)-AtPase was further supported by experiments with isolated vacuoles from cauliflower: (a) Immunoblotting with an antibody against the 111-kD Ca(2+)-ATPase showed that it was associated with the vacuoles, and (b) ATP-dependent Ca2+ uptake by the intact vacuoles was found to be CaM stimulated and partly protonophore insensitive.

LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar cardiomyopathy: a study of 3 cases.

PubMed

Daniels, Brianne H; McComb, Rodney D; Mobley, Bret C; Gultekin, Sakir Humayun; Lee, Han S; Margeta, Marta

2013-07-01

Autophagic vacuolar cardiomyopathy is an underrecognized, but potentially fatal, complication of treatment with chloroquine (CQ) and its derivative hydroxychloroquine (HCQ), which are used as therapy for malaria and common connective tissue disorders. Currently, the diagnosis of autophagic vacuolar cardiomyopathy is established through an endomyocardial biopsy and requires electron microscopy, which is not widely available and has a significant potential for sampling error. Recently, we have reported that immunohistochemistry for autophagic markers LC3 and p62 can replace electron microscopy in the diagnosis of HCQ-induced and colchicine-induced autophagic vacuolar skeletal myopathies. In the current study, we use 3 cases of CQ-induced or HCQ-induced cardiomyopathy and 1 HCQ-treated control case to show that the same two markers can be used to diagnose autophagic vacuolar cardiomyopathies by light microscopy. CQ-induced or HCQ-induced autophagic vacuolar cardiomyopathy is not universally fatal, but successful treatment requires early detection. By lowering the barriers to diagnosis, the application of these immunohistochemical markers will decrease the number of misdiagnosed patients, thus increasing the likelihood of favorable clinical outcomes.

[Target gene sequence capture and next generation sequencing technology to diagnose four children with Alagille syndrome].

PubMed

Gao, M L; Zhong, X M; Ma, X; Ning, H J; Zhu, D; Zou, J Z

2016-06-02

To make genetic diagnosis of Alagille syndrome (ALGS) patients using target gene sequence capture and next generation sequencing technology. Target gene sequence capture and next generation sequencing were used to detect ALGS gene of 4 patients. They were hospitalized at the Affiliated Hospital, Capital Institute of Pediatrics between January 2014 and December 2015, referred to clinical diagnosis of ALGS typical and atypical respectively in 2 cases. Blood samples were collected from patients and their parents and genomic DNA was extracted from lymphocytes. Target gene sequence capture and next generation sequencing was detected. Sanger sequencing was used to confirm the results of the patients and their parents. Cholestasis, heart defects, inverted triangular face and butterfly vertebrae were presented as main clinical features in 4 male patients. The first hospital visiting ages ranged from 3 months and 14 days to 3 years and 1 month. The age of onset ranged from 3 days to 42 days (median 23 days). According to the clinical diagnostic criteria of ALGS, patient 1 and patient 2 were considered as typical ALGS. The other 2 patients were considered as atypical ALGS. Four Jagged 1(JAG1) pathogenic mutations were detected. Three different missense mutations were detected in patient 1 to patient 3 with ALGS(c.839C>T(p.W280X), c. 703G>A(p.R235X), c. 1720C>T(p.V574M)). The JAG1 mutation of patient 3 was first reported. Patient 4 had one novel insertion mutation (c.1779_1780insA(p.Ile594AsnfsTer23)). Parental analysis verified that the JAG1 missense mutation of 3 patients were de novo. The results of sanger sequencing was consistent with the results of the next generation sequencing. Target gene sequence capture combined with next generation sequencing can detect two pathogenic genes in ALGS and test genes of other related diseases in infantile cholestatic diseases simultaneously and presents a high throughput, high efficiency and low cost. It may provide molecular

Vacuolar sequestration capacity and long-distance metal transport in plants

PubMed Central

Peng, Jia-Shi; Gong, Ji-Ming

2014-01-01

The vacuole is a pivotal organelle functioning in storage of metabolites, mineral nutrients, and toxicants in higher plants. Accumulating evidence indicates that in addition to its storage role, the vacuole contributes essentially to long-distance transport of metals, through the modulation of Vacuolar sequestration capacity (VSC) which is shown to be primarily controlled by cytosolic metal chelators and tonoplast-localized transporters, or the interaction between them. Plants adapt to their environments by dynamic regulation of VSC for specific metals and hence targeting metals to specific tissues. Study of VSC provides not only a new angle to understand the long-distance root-to-shoot transport of minerals in plants, but also an efficient way to biofortify essential mineral nutrients or to phytoremediate non-essential metal pollution. The current review will focus on the most recent proceedings on the interaction mechanisms between VSC regulation and long-distance metal transport. PMID:24550927

Vacuolar sequestration capacity and long-distance metal transport in plants.

PubMed

Peng, Jia-Shi; Gong, Ji-Ming

2014-01-01

The vacuole is a pivotal organelle functioning in storage of metabolites, mineral nutrients, and toxicants in higher plants. Accumulating evidence indicates that in addition to its storage role, the vacuole contributes essentially to long-distance transport of metals, through the modulation of Vacuolar sequestration capacity (VSC) which is shown to be primarily controlled by cytosolic metal chelators and tonoplast-localized transporters, or the interaction between them. Plants adapt to their environments by dynamic regulation of VSC for specific metals and hence targeting metals to specific tissues. Study of VSC provides not only a new angle to understand the long-distance root-to-shoot transport of minerals in plants, but also an efficient way to biofortify essential mineral nutrients or to phytoremediate non-essential metal pollution. The current review will focus on the most recent proceedings on the interaction mechanisms between VSC regulation and long-distance metal transport.

HybPiper: Extracting coding sequence and introns for phylogenetics from high-throughput sequencing reads using target enrichment1

PubMed Central

Johnson, Matthew G.; Gardner, Elliot M.; Liu, Yang; Medina, Rafael; Goffinet, Bernard; Shaw, A. Jonathan; Zerega, Nyree J. C.; Wickett, Norman J.

2016-01-01

Premise of the study: Using sequence data generated via target enrichment for phylogenetics requires reassembly of high-throughput sequence reads into loci, presenting a number of bioinformatics challenges. We developed HybPiper as a user-friendly platform for assembly of gene regions, extraction of exon and intron sequences, and identification of paralogous gene copies. We test HybPiper using baits designed to target 333 phylogenetic markers and 125 genes of functional significance in Artocarpus (Moraceae). Methods and Results: HybPiper implements parallel execution of sequence assembly in three phases: read mapping, contig assembly, and target sequence extraction. The pipeline was able to recover nearly complete gene sequences for all genes in 22 species of Artocarpus. HybPiper also recovered more than 500 bp of nontargeted intron sequence in over half of the phylogenetic markers and identified paralogous gene copies in Artocarpus. Conclusions: HybPiper was designed for Linux and Mac OS X and is freely available at https://github.com/mossmatters/HybPiper. PMID:27437175

Fe deficiency differentially affects the vacuolar proton pumps in cucumber and soybean roots

PubMed Central

Dell’Orto, Marta; Nisi, Patrizia De; Vigani, Gianpiero; Zocchi, Graziano

2013-01-01

Iron uptake in dicots depends on their ability to induce a set of responses in root cells including rhizosphere acidification through H+ extrusion and apoplastic Fe(III) reduction by Fe(III)-chelate reductase. These responses must be sustained by metabolic rearrangements aimed at providing the required NAD(P)H, ATP and H+. Previous results in Fe-deficient cucumber roots showed that high H+ extrusion is accompanied by increased phosphoenolpyruvate carboxylase (PEPC) activity, involved in the cytosol pH-stat; moreover 31P-NMR analysis revealed increased vacuolar pH and decreased vacuolar [inorganic phosphate (Pi)]. The opposite was found in soybean: low rhizosphere acidification, decreased PEPC activity, vacuole acidification, and increased vacuolar [Pi]. These findings, highlighting a different impact of the Fe deficiency responses on cytosolic pH in the two species, lead to hypothesize different roles for H+ and Pi movements across the tonoplast in pH homeostasis. The role of vacuole in cytosolic pH-stat involves the vacuolar H+-ATPase (V-ATPase) and vacuolar H+-pyrophosphatase (V-PPase) activities, which generating the ΔpH and ΔΨ, mediate the transport of solutes, among which Pi, across the tonoplast. Fluxes of Pi itself in its two ionic forms, H2PO4- predominating in the vacuole and HPO42- in the cytosol, may be involved in pH homeostasis owing to its pH-dependent protonation/deprotonation reactions. Tonoplast enriched fractions were obtained from cucumber and soybean roots grown with or without Fe. Both V-ATPase and V-PPase activities were analyzed and the enrichment and localization of the corresponding proteins in root tissues were determined by Western blot and immunolocalization. V-ATPase did not change its activity and expression level in response to Fe starvation in both species. V-PPase showed a different behavior: in cucumber roots its activity and abundance were decreased, while in Fe-deficient soybean roots they were increased. The distinct role of

Enrichment of target sequences for next-generation sequencing applications in research and diagnostics.

PubMed

Altmüller, Janine; Budde, Birgit S; Nürnberg, Peter

2014-02-01

Abstract Targeted re-sequencing such as gene panel sequencing (GPS) has become very popular in medical genetics, both for research projects and in diagnostic settings. The technical principles of the different enrichment methods have been reviewed several times before; however, new enrichment products are constantly entering the market, and researchers are often puzzled about the requirement to take decisions about long-term commitments, both for the enrichment product and the sequencing technology. This review summarizes important considerations for the experimental design and provides helpful recommendations in choosing the best sequencing strategy for various research projects and diagnostic applications.

Proton Gradient-Driven Nickel Uptake by Vacuolar Membrane Vesicles of Saccharomyces cerevisiae

PubMed Central

Nishimura, Ken; Igarashi, Kazuei; Kakinuma, Yoshimi

1998-01-01

A vacuolar H+-ATPase-negative mutant of Saccharomyces cerevisiae was highly sensitive to nickel ion. Accumulation of nickel ion in the cells of this mutant of less than 60% of the value for the parent strain arrested growth, suggesting a role for this ATPase in sequestering nickel ion into vacuoles. An artificially imposed pH gradient (interior acid) induced transient nickel ion uptake by vacuolar membrane vesicles, which was inhibited by collapse of the pH difference but not of the membrane potential. Nickel ion transport into vacuoles in a pH gradient-dependent manner is thus important for its detoxification in yeast. PMID:9537401

Intravenous phage display identifies peptide sequences that target the burn-injured intestine.

PubMed

Costantini, Todd W; Eliceiri, Brian P; Putnam, James G; Bansal, Vishal; Baird, Andrew; Coimbra, Raul

2012-11-01

The injured intestine is responsible for significant morbidity and mortality after severe trauma and burn; however, targeting the intestine with therapeutics aimed at decreasing injury has proven difficult. We hypothesized that we could use intravenous phage display technology to identify peptide sequences that target the injured intestinal mucosa in a murine model, and then confirm the cross-reactivity of this peptide sequence with ex vivo human gut. Four hours following 30% TBSA burn we performed an in vivo, intravenous systemic administration of phage library containing 10(12) phage in balb/c mice to biopan for gut-targeting peptides. In vivo assessment of the candidate peptide sequences identified after 4 rounds of internalization was performed by injecting 1×10(12) copies of each selected phage clone into sham or burned animals. Internalization into the gut was assessed using quantitative polymerase chain reaction. We then incubated this gut-targeting peptide sequence with human intestine and visualized fluorescence using confocal microscopy. We identified 3 gut-targeting peptide sequences which caused collapse of the phage library (4-1: SGHQLLLNKMP, 4-5: ILANDLTAPGPR, 4-11: SFKPSGLPAQSL). Sequence 4-5 was internalized into the intestinal mucosa of burned animals 9.3-fold higher than sham animals injected with the same sequence (2.9×10(5)vs. 3.1×10(4) particles per mg tissue). Sequences 4-1 and 4-11 were both internalized into the gut, but did not demonstrate specificity for the injured mucosa. Phage sequence 4-11 demonstrated cross-reactivity with human intestine. In the future, this gut-targeting peptide sequence could serve as a platform for the delivery of biotherapeutics. Copyright © 2012 Elsevier Inc. All rights reserved.

Targeted exome sequencing of suspected mitochondrial disorders

PubMed Central

Lieber, Daniel S.; Calvo, Sarah E.; Shanahan, Kristy; Slate, Nancy G.; Liu, Shangtao; Hershman, Steven G.; Gold, Nina B.; Chapman, Brad A.; Thorburn, David R.; Berry, Gerard T.; Schmahmann, Jeremy D.; Borowsky, Mark L.; Mueller, David M.; Sims, Katherine B.

2013-01-01

Objective: To evaluate the utility of targeted exome sequencing for the molecular diagnosis of mitochondrial disorders, which exhibit marked phenotypic and genetic heterogeneity. Methods: We considered a diverse set of 102 patients with suspected mitochondrial disorders based on clinical, biochemical, and/or molecular findings, and whose disease ranged from mild to severe, with varying age at onset. We sequenced the mitochondrial genome (mtDNA) and the exons of 1,598 nuclear-encoded genes implicated in mitochondrial biology, mitochondrial disease, or monogenic disorders with phenotypic overlap. We prioritized variants likely to underlie disease and established molecular diagnoses in accordance with current clinical genetic guidelines. Results: Targeted exome sequencing yielded molecular diagnoses in established disease loci in 22% of cases, including 17 of 18 (94%) with prior molecular diagnoses and 5 of 84 (6%) without. The 5 new diagnoses implicated 2 genes associated with canonical mitochondrial disorders (NDUFV1, POLG2), and 3 genes known to underlie other neurologic disorders (DPYD, KARS, WFS1), underscoring the phenotypic and biochemical overlap with other inborn errors. We prioritized variants in an additional 26 patients, including recessive, X-linked, and mtDNA variants that were enriched 2-fold over background and await further support of pathogenicity. In one case, we modeled patient mutations in yeast to provide evidence that recessive mutations in ATP5A1 can underlie combined respiratory chain deficiency. Conclusion: The results demonstrate that targeted exome sequencing is an effective alternative to the sequential testing of mtDNA and individual nuclear genes as part of the investigation of mitochondrial disease. Our study underscores the ongoing challenge of variant interpretation in the clinical setting. PMID:23596069

Design of the hairpin ribozyme for targeting specific RNA sequences.

PubMed

Hampel, A; DeYoung, M B; Galasinski, S; Siwkowski, A

1997-01-01

The following steps should be taken when designing the hairpin ribozyme to cleave a specific target sequence: 1. Select a target sequence containing BN*GUC where B is C, G, or U. 2. Select the target sequence in areas least likely to have extensive interfering structure. 3. Design the conventional hairpin ribozyme as shown in Fig. 1, such that it can form a 4 bp helix 2 and helix 1 lengths up to 10 bp. 4. Synthesize this ribozyme from single-stranded DNA templates with a double-stranded T7 promoter. 5. Prepare a series of short substrates capable of forming a range of helix 1 lengths of 5-10 bp. 6. Identify these by direct RNA sequencing. 7. Assay the extent of cleavage of each substrate to identify the optimal length of helix 1. 8. Prepare the hairpin tetraloop ribozyme to determine if catalytic efficiency can be improved.

A long-term target detection approach in infrared image sequence

NASA Astrophysics Data System (ADS)

Li, Hang; Zhang, Qi; Wang, Xin; Hu, Chao

2016-10-01

An automatic target detection method used in long term infrared (IR) image sequence from a moving platform is proposed. Firstly, based on POME(the principle of maximum entropy), target candidates are iteratively segmented. Then the real target is captured via two different selection approaches. At the beginning of image sequence, the genuine target with litter texture is discriminated from other candidates by using contrast-based confidence measure. On the other hand, when the target becomes larger, we apply online EM method to estimate and update the distributions of target's size and position based on the prior detection results, and then recognize the genuine one which satisfies both the constraints of size and position. Experimental results demonstrate that the presented method is accurate, robust and efficient.

Appropriate vacuolar acidification in Saccharomyces cerevisiae is associated with efficient high sugar fermentation.

PubMed

Nguyen, Trung D; Walker, Michelle E; Gardner, Jennifer M; Jiranek, Vladimir

2018-04-01

Vacuolar acidification serves as a homeostatic mechanism to regulate intracellular pH, ion and chemical balance, as well as trafficking and recycling of proteins and nutrients, critical for normal cellular function. This study reports on the importance of vacuole acidification during wine-like fermentation. Ninety-three mutants (homozygous deletions in lab yeast strain, BY4743), which result in protracted fermentation when grown in a chemically defined grape juice with 200 g L -1 sugar (pH 3.5), were examined to determine whether fermentation protraction was in part due to a dysfunction in vacuolar acidification (VA) during the early stages of fermentation, and whether VA was responsive to the initial sugar concentration in the medium. Cells after 24 h growth were dual-labelled with propidium iodide and vacuolar specific probe 6-carboxyfluorescein diacetate (6-CFDA) and examined with a FACS analyser for viability and impaired VA, respectively. Twenty mutants showed a greater than two-fold increase in fluorescence intensity; the experimental indicator for vacuolar dysfunction; 10 of which have not been previously annotated to this process. With the exception of Δhog1, Δpbs2 and Δvph1 mutants, where dysfunction was directly related to osmolality; the remainder exhibited increased CF-fluorescence, independent of sugar concentration at 20 g L -1 or 200 g L -1 . These findings offer insight to the importance of VA to cell growth in high sugar media. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cytosolic Nucleotides Block and Regulate the Arabidopsis Vacuolar Anion Channel AtALMT9*

PubMed Central

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-01-01

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al3+ to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. PMID:25028514

Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9.

PubMed

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-09-12

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al(3+) to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional expression of Schizosaccharomyces pombe Vba2p in the vacuolar membrane of Saccharomyces cerevisiae.

PubMed

Pongcharoen, Pongsanat; Kawano-Kawada, Miyuki; Iwaki, Tomoko; Sugimoto, Naoko; Sekito, Takayuki; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

2013-01-01

A vacuolar membrane protein, Vba2p of Schizosaccharomyces pombe, is involved in basic amino acid uptake by intact cells. Here we found evidence that Vba2p mediated ATP-dependent lysine uptake by vacuolar membrane vesicles of Saccharomyces cerevisiae. Vba2p was also responsible for quinidine sensitivity, and the addition of lysine improved cell growth on quinidine-containing media. These findings should be useful for further characterization of Vba2p.

Genetic mutations in human rectal cancers detected by targeted sequencing.

PubMed

Bai, Jun; Gao, Jinglong; Mao, Zhijun; Wang, Jianhua; Li, Jianhui; Li, Wensheng; Lei, Yu; Li, Shuaishuai; Wu, Zhuo; Tang, Chuanning; Jones, Lindsey; Ye, Hua; Lou, Feng; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; Huang, Xue F; Chen, Si-Yi; Zhang, Enke

2015-10-01

Colorectal cancer (CRC) is widespread with significant mortality. Both inherited and sporadic mutations in various signaling pathways influence the development and progression of the cancer. Identifying genetic mutations in CRC is important for optimal patient treatment and many approaches currently exist to uncover these mutations, including next-generation sequencing (NGS) and commercially available kits. In the present study, we used a semiconductor-based targeted DNA-sequencing approach to sequence and identify genetic mutations in 91 human rectal cancer samples. Analysis revealed frequent mutations in KRAS (58.2%), TP53 (28.6%), APC (16.5%), FBXW7 (9.9%) and PIK3CA (9.9%), and additional mutations in BRAF, CTNNB1, ERBB2 and SMAD4 were also detected at lesser frequencies. Thirty-eight samples (41.8%) also contained two or more mutations, with common combination mutations occurring between KRAS and TP53 (42.1%), and KRAS and APC (31.6%). DNA sequencing for individual cancers is of clinical importance for targeted drug therapy and the advantages of such targeted gene sequencing over other NGS platforms or commercially available kits in sensitivity, cost and time effectiveness may aid clinicians in treating CRC patients in the near future.

Effect of vacuolar ATPase subunit H (VmaH) on cellular pH, asexual cycle, stress tolerance and virulence in Beauveria bassiana.

PubMed

Zhu, Jing; Zhu, Xiao-Guan; Ying, Sheng-Hua; Feng, Ming-Guang

2017-01-01

Vacuolar ATPase (V-ATPase) is a conserved multi-subunit protein complex that mediates intracellular acidification in fungi. Here we show functional diversity of V-ATPase subunit H (BbVmaH) in Beauveria bassiana, a filamentous fungal insect pathogen. Deletion of BbvmaH resulted in elevated vacuolar pH, increased Ca 2+ level in cytosol but not in vacuoles, accelerated culture acidification and reduced accumulation of extracellular ammonia. Aerial conidiation and submerged blastospore production were largely delayed and reduced in the deletion mutant, respectively, accompanied with a significant delay in conidial germination, alterations of conidia and blastospores in morphology, size and/or density, and severe growth defects in minimal media with different carbon and nitrogen sources. Despite null responses to osmotic, oxidative and cell wall perturbing stresses, the deletion mutant showed increased sensitivity to Ca 2+ , Zn 2+ and Cu 2+ during growth while its conidia were less tolerant to a wet-heat stress at 45°C and UV-B irradiation. Intracellular glycerol and mannitol contents also decreased significantly. Its virulence to Galleria mellonella larvae was significantly attenuated when conidia were topically applied for normal cuticle infection or injected into haemocoel for cuticle-bypassing infection. All phenotypic changes were restored by targeted gene complementation. Our results indicate that BbVmaH plays an important role in sustaining not only vacuolar acidification but also cytosolic calcium accumulation, ambient pH homeostasis, in vitro asexual cycle and virulence in B. bassiana. Copyright © 2016 Elsevier Inc. All rights reserved.

A long-term target detection approach in infrared image sequence

NASA Astrophysics Data System (ADS)

Li, Hang; Zhang, Qi; Li, Yuanyuan; Wang, Liqiang

2015-12-01

An automatic target detection method used in long term infrared (IR) image sequence from a moving platform is proposed. Firstly, based on non-linear histogram equalization, target candidates are coarse-to-fine segmented by using two self-adapt thresholds generated in the intensity space. Then the real target is captured via two different selection approaches. At the beginning of image sequence, the genuine target with litter texture is discriminated from other candidates by using contrast-based confidence measure. On the other hand, when the target becomes larger, we apply online EM method to iteratively estimate and update the distributions of target's size and position based on the prior detection results, and then recognize the genuine one which satisfies both the constraints of size and position. Experimental results demonstrate that the presented method is accurate, robust and efficient.

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase.

PubMed

Kriegel, Anne; Andrés, Zaida; Medzihradszky, Anna; Krüger, Falco; Scholl, Stefan; Delang, Simon; Patir-Nebioglu, M Görkem; Gute, Gezahegn; Yang, Haibing; Murphy, Angus S; Peer, Wendy Ann; Pfeiffer, Anne; Krebs, Melanie; Lohmann, Jan U; Schumacher, Karin

2015-12-01

The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH. © 2015 American Society of Plant Biologists. All rights reserved.

Phosphatidylinositol 3-Kinase Promotes V-ATPase Activation and Vacuolar Acidification and Delays Methyl Jasmonate-Induced Leaf Senescence1

PubMed Central

Liu, Jian; Ji, Yingbin; Zhou, Jun; Xing, Da

2016-01-01

PI3K and its product PI3P are both involved in plant development and stress responses. In this study, the down-regulation of PI3K activity accelerated leaf senescence induced by methyl jasmonate (MeJA) and suppressed the activation of vacuolar H+-ATPase (V-ATPase). Yeast two-hybrid analyses indicated that PI3K bound to the V-ATPase B subunit (VHA-B). Analysis of bimolecular fluorescence complementation in tobacco guard cells showed that PI3K interacted with VHA-B2 in the tonoplasts. Through the use of pharmacological and genetic tools, we found that PI3K and V-ATPase promoted vacuolar acidification and stomatal closure during leaf senescence. Vacuolar acidification was suppressed by the PIKfyve inhibitor in 35S:AtVPS34-YFP Arabidopsis during MeJA-induced leaf senescence, but the decrease was lower than that in YFP-labeled Arabidopsis. These results suggest that PI3K promotes V-ATPase activation and consequently induces vacuolar acidification and stomatal closure, thereby delaying MeJA-induced leaf senescence. PMID:26739232

Dubinett - Targeted Sequencing 2012 — EDRN Public Portal

Cancer.gov

we propose to use targeted massively parallel DNA sequencing to identify somatic alterations within mutational hotspots in matched sets of primary lung tumors, premalignant lesions, and adjacent,histologically normal lung tissue.

RISC RNA sequencing for context-specific identification of in vivo miR targets

PubMed Central

Matkovich, Scot J; Van Booven, Derek J; Eschenbacher, William H; Dorn, Gerald W

2010-01-01

Rationale MicroRNAs (miRs) are expanding our understanding of cardiac disease and have the potential to transform cardiovascular therapeutics. One miR can target hundreds of individual mRNAs, but existing methodologies are not sufficient to accurately and comprehensively identify these mRNA targets in vivo. Objective To develop methods permitting identification of in vivo miR targets in an unbiased manner, using massively parallel sequencing of mouse cardiac transcriptomes in combination with sequencing of mRNA associated with mouse cardiac RNA-induced silencing complexes (RISCs). Methods and Results We optimized techniques for expression profiling small amounts of RNA without introducing amplification bias, and applied this to anti-Argonaute 2 immunoprecipitated RISCs (RISC-Seq) from mouse hearts. By comparing RNA-sequencing results of cardiac RISC and transcriptome from the same individual hearts, we defined 1,645 mRNAs consistently targeted to mouse cardiac RISCs. We employed this approach in hearts overexpressing miRs from Myh6 promoter-driven precursors (programmed RISC-Seq) to identify 209 in vivo targets of miR-133a and 81 in vivo targets of miR-499. Consistent with the fact that miR-133a and miR-499 have widely differing ‘seed’ sequences and belong to different miR families, only 6 targets were common to miR-133a- and miR-499-programmed hearts. Conclusions RISC-sequencing is a highly sensitive method for general RISC profiling and individual miR target identification in biological context, and is applicable to any tissue and any disease state. Summary MicroRNAs (miRs) are key regulators of mRNA translation in health and disease. While bioinformatic predictions suggest that a single miR may target hundreds of mRNAs, the number of experimentally verified targets of miRs is low. To enable comprehensive, unbiased examination of miR targets, we have performed deep RNA sequencing of cardiac transcriptomes in parallel with cardiac RNA-induced silencing complex

GWASeq: targeted re-sequencing follow up to GWAS.

PubMed

Salomon, Matthew P; Li, Wai Lok Sibon; Edlund, Christopher K; Morrison, John; Fortini, Barbara K; Win, Aung Ko; Conti, David V; Thomas, Duncan C; Duggan, David; Buchanan, Daniel D; Jenkins, Mark A; Hopper, John L; Gallinger, Steven; Le Marchand, Loïc; Newcomb, Polly A; Casey, Graham; Marjoram, Paul

2016-03-03

For the last decade the conceptual framework of the Genome-Wide Association Study (GWAS) has dominated the investigation of human disease and other complex traits. While GWAS have been successful in identifying a large number of variants associated with various phenotypes, the overall amount of heritability explained by these variants remains small. This raises the question of how best to follow up on a GWAS, localize causal variants accounting for GWAS hits, and as a consequence explain more of the so-called "missing" heritability. Advances in high throughput sequencing technologies now allow for the efficient and cost-effective collection of vast amounts of fine-scale genomic data to complement GWAS. We investigate these issues using a colon cancer dataset. After QC, our data consisted of 1993 cases, 899 controls. Using marginal tests of associations, we identify 10 variants distributed among six targeted regions that are significantly associated with colorectal cancer, with eight of the variants being novel to this study. Additionally, we perform so-called 'SNP-set' tests of association and identify two sets of variants that implicate both common and rare variants in the etiology of colorectal cancer. Here we present a large-scale targeted re-sequencing resource focusing on genomic regions implicated in colorectal cancer susceptibility previously identified in several GWAS, which aims to 1) provide fine-scale targeted sequencing data for fine-mapping and 2) provide data resources to address methodological questions regarding the design of sequencing-based follow-up studies to GWAS. Additionally, we show that this strategy successfully identifies novel variants associated with colorectal cancer susceptibility and can implicate both common and rare variants.

Targeted Analysis of Whole Genome Sequence Data to Diagnose Genetic Cardiomyopathy

DOE PAGES

Golbus, Jessica R.; Puckelwartz, Megan J.; Dellefave-Castillo, Lisa; ...

2014-09-01

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of more than 50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift towards comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused onmore » 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1-14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and/or segregation analysis. Three of three previously identified primary mutations were detected by this analysis. In six subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and/or had additional pathological correlation to provide evidence for causality. For two subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. We conclude that these pilot data demonstrate that ~30-40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.« less

AtALMT9 is a malate-activated vacuolar chloride channel required for stomatal opening in Arabidopsis

PubMed Central

De Angeli, Alexis; Zhang, Jingbo; Meyer, Stefan; Martinoia, Enrico

2013-01-01

Water deficit strongly affects crop productivity. Plants control water loss and CO2 uptake by regulating the aperture of the stomatal pores within the leaf epidermis. Stomata aperture is regulated by the two guard cells forming the pore and changing their size in response to ion uptake and release. While our knowledge about potassium and chloride fluxes across the plasma membrane of guard cells is advanced, little is known about fluxes across the vacuolar membrane. Here we present the molecular identification of the long-sought-after vacuolar chloride channel. AtALMT9 is a chloride channel activated by physiological concentrations of cytosolic malate. Single-channel measurements demonstrate that this activation is due to a malate-dependent increase in the channel open probability. Arabidopsis thaliana atalmt9 knockout mutants exhibited impaired stomatal opening and wilt more slowly than the wild type. Our findings show that AtALMT9 is a vacuolar chloride channel having a major role in controlling stomata aperture. PMID:23653216

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis

PubMed Central

Luna-Tapia, Arturo; Peters, Brian M.; Eberle, Karen E.; Kerns, Morgan E.; Foster, Timothy P.; Marrero, Luis; Noverr, Mairi C.; Fidel, Paul L.

2015-01-01

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed. PMID:26231054

Loss of ATP-dependent lysine uptake in the vacuolar membrane vesicles of Saccharomyces cerevisiae ypq1∆ mutant.

PubMed

Sekito, Takayuki; Nakamura, Kyosuke; Manabe, Kunio; Tone, Junichi; Sato, Yumika; Murao, Nami; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2014-01-01

Saccharomyces cerevisiae Ypq1p is a vacuolar membrane protein of the PQ-loop protein family. We found that ATP-dependent uptake activities of amino acids by vacuolar membrane vesicles were impaired by ypq1∆ mutation. Loss of lysine uptake was most remarkable, and the uptake was recovered by overproduction of Ypq1p. Ypq1p is thus involved in transport of amino acids into vacuoles.

MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples.

PubMed

Sivaganesan, Mano; Siefring, Shawn; Varma, Manju; Haugland, Richard A

2011-12-01

DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from different studies by this approach, either a consistent source of calibrator cells must be used or the estimates must account for any differences in target sequence recoveries from different sources of calibrator cells. In this report we describe two methods for estimating target sequence recoveries from whole cell calibrator samples based on qPCR analyses of their serially diluted DNA extracts and most probable number (MPN) calculation. The first method employed a traditional MPN calculation approach. The second method employed a Bayesian hierarchical statistical modeling approach and a Monte Carlo Markov Chain (MCMC) simulation method to account for the uncertainty in these estimates associated with different individual samples of the cell preparations, different dilutions of the DNA extracts and different qPCR analytical runs. The two methods were applied to estimate mean target sequence recoveries per cell from two different lots of a commercially available source of enumerated Enterococcus cell preparations. The mean target sequence recovery estimates (and standard errors) per cell from Lot A and B cell preparations by the Bayesian method were 22.73 (3.4) and 11.76 (2.4), respectively, when the data were adjusted for potential false positive results. Means were similar for the traditional MPN approach which cannot comparably assess uncertainty in the estimates. Cell numbers and estimates of recoverable target sequences in calibrator samples prepared from the two cell sources were also used to estimate cell equivalent and target sequence quantities recovered from surface water samples in a comparative Ct method. Our results illustrate the utility of the Bayesian method in accounting for

RISC RNA sequencing for context-specific identification of in vivo microRNA targets.

PubMed

Matkovich, Scot J; Van Booven, Derek J; Eschenbacher, William H; Dorn, Gerald W

2011-01-07

MicroRNAs (miRs) are expanding our understanding of cardiac disease and have the potential to transform cardiovascular therapeutics. One miR can target hundreds of individual mRNAs, but existing methodologies are not sufficient to accurately and comprehensively identify these mRNA targets in vivo. To develop methods permitting identification of in vivo miR targets in an unbiased manner, using massively parallel sequencing of mouse cardiac transcriptomes in combination with sequencing of mRNA associated with mouse cardiac RNA-induced silencing complexes (RISCs). We optimized techniques for expression profiling small amounts of RNA without introducing amplification bias and applied this to anti-Argonaute 2 immunoprecipitated RISCs (RISC-Seq) from mouse hearts. By comparing RNA-sequencing results of cardiac RISC and transcriptome from the same individual hearts, we defined 1645 mRNAs consistently targeted to mouse cardiac RISCs. We used this approach in hearts overexpressing miRs from Myh6 promoter-driven precursors (programmed RISC-Seq) to identify 209 in vivo targets of miR-133a and 81 in vivo targets of miR-499. Consistent with the fact that miR-133a and miR-499 have widely differing "seed" sequences and belong to different miR families, only 6 targets were common to miR-133a- and miR-499-programmed hearts. RISC-sequencing is a highly sensitive method for general RISC profiling and individual miR target identification in biological context and is applicable to any tissue and any disease state.

RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection.

PubMed

Takahashi, Hirokazu; Ohkawachi, Masahiko; Horio, Kyohei; Kobori, Toshiro; Aki, Tsunehiro; Matsumura, Yukihiko; Nakashimada, Yutaka; Okamura, Yoshiko

2018-05-17

RNA-primed rolling circle amplification (RPRCA) is a useful laboratory method for RNA detection; however, the detection of RNA is limited by the lack of information on 3'-terminal sequences. We uncovered that conventional RPRCA using pre-circularized probes could potentially detect the internal sequence of target RNA molecules in combination with RNase H. However, the specificity for mRNA detection was low, presumably due to non-specific hybridization of non-target RNA with the circular probe. To overcome this technical problem, we developed a method for detecting a sequence of interest in target RNA molecules via RNase H-assisted RPRCA using padlocked probes. When padlock probes are hybridized to the target RNA molecule, they are converted to the circular form by SplintR ligase. Subsequently, RNase H creates nick sites only in the hybridized RNA sequence, and single-stranded DNA is finally synthesized from the nick site by phi29 DNA polymerase. This method could specifically detect at least 10 fmol of the target RNA molecule without reverse transcription. Moreover, this method detected GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without background DNA amplification. Therefore, this method can potentially detect almost all types of RNA molecules without reverse transcription and reveal full-length sequence information.

Modeling the vacuolar storage of malate shed lights on pre- and post-harvest fruit acidity.

PubMed

Etienne, Audrey; Génard, Michel; Lobit, Philippe; Bugaud, Christophe

2014-11-18

Malate is one of the most important organic acids in many fruits and its concentration plays a critical role in organoleptic properties. Several studies suggest that malate accumulation in fruit cells is controlled at the level of vacuolar storage. However, the regulation of vacuolar malate storage throughout fruit development, and the origins of the phenotypic variability of the malate concentration within fruit species remain to be clarified. In the present study, we adapted the mechanistic model of vacuolar storage proposed by Lobit et al. in order to study the accumulation of malate in pre and postharvest fruits. The main adaptation concerned the variation of the free energy of ATP hydrolysis during fruit development. Banana fruit was taken as a reference because it has the particularity of having separate growth and post-harvest ripening stages, during which malate concentration undergoes substantial changes. Moreover, the concentration of malate in banana pulp varies greatly among cultivars which make possible to use the model as a tool to analyze the genotypic variability. The model was calibrated and validated using data sets from three cultivars with contrasting malate accumulation, grown under different fruit loads and potassium supplies, and harvested at different stages. The model predicted the pre and post-harvest dynamics of malate concentration with fairly good accuracy for the three cultivars (mean RRMSE = 0.25-0.42). The sensitivity of the model to parameters and input variables was analyzed. According to the model, vacuolar composition, in particular potassium and organic acid concentrations, had an important effect on malate accumulation. The model suggested that rising temperatures depressed malate accumulation. The model also helped distinguish differences in malate concentration among the three cultivars and between the pre and post-harvest stages by highlighting the probable importance of proton pump activity and particularly of the free

Bioinformatics by Example: From Sequence to Target

NASA Astrophysics Data System (ADS)

Kossida, Sophia; Tahri, Nadia; Daizadeh, Iraj

2002-12-01

With the completion of the human genome, and the imminent completion of other large-scale sequencing and structure-determination projects, computer-assisted bioscience is aimed to become the new paradigm for conducting basic and applied research. The presence of these additional bioinformatics tools stirs great anxiety for experimental researchers (as well as for pedagogues), since they are now faced with a wider and deeper knowledge of differing disciplines (biology, chemistry, physics, mathematics, and computer science). This review targets those individuals who are interested in using computational methods in their teaching or research. By analyzing a real-life, pharmaceutical, multicomponent, target-based example the reader will experience this fascinating new discipline.

Targeted next-generation sequencing in monogenic dyslipidemias.

PubMed

Hegele, Robert A; Ban, Matthew R; Cao, Henian; McIntyre, Adam D; Robinson, John F; Wang, Jian

2015-04-01

To evaluate the potential clinical translation of high-throughput next-generation sequencing (NGS) methods in diagnosis and management of dyslipidemia. Recent NGS experiments indicate that most causative genes for monogenic dyslipidemias are already known. Thus, monogenic dyslipidemias can now be diagnosed using targeted NGS. Targeting of dyslipidemia genes can be achieved by either: designing custom reagents for a dyslipidemia-specific NGS panel; or performing genome-wide NGS and focusing on genes of interest. Advantages of the former approach are lower cost and limited potential to detect incidental pathogenic variants unrelated to dyslipidemia. However, the latter approach is more flexible because masking criteria can be altered as knowledge advances, with no need for re-design of reagents or follow-up sequencing runs. Also, the cost of genome-wide analysis is decreasing and ethical concerns can likely be mitigated. DNA-based diagnosis is already part of the clinical diagnostic algorithms for familial hypercholesterolemia. Furthermore, DNA-based diagnosis is supplanting traditional biochemical methods to diagnose chylomicronemia caused by deficiency of lipoprotein lipase or its co-factors. The increasing availability and decreasing cost of clinical NGS for dyslipidemia means that its potential benefits can now be evaluated on a larger scale.

Creating Drought- and Salt-Tolerant Crops by Overexpressing a Vacuolar Pyrophosphatase Gene

USDA-ARS?s Scientific Manuscript database

Increased expression of an Arabidopsis vacuolar pyrophosphatase gene, AVP1, leads to increased drought and salt tolerance in transgenic plants, which has been demonstrated in laboratory and field conditions. The molecular mechanism of AVP1-mediated drought resistance is likely due to increased proto...

[Detection of pathogenic mutations in Marfan syndrome by targeted next-generation semiconductor sequencing].

PubMed

Lu, Chaoxia; Wu, Wei; Xiao, Jifang; Meng, Yan; Zhang, Shuyang; Zhang, Xue

2013-06-01

To detect pathogenic mutations in Marfan syndrome (MFS) using an Ion Torrent Personal Genome Machine (PGM) and to validate the result of targeted next-generation semiconductor sequencing for the diagnosis of genetic disorders. Peripheral blood samples were collected from three MFS patients and a normal control with informed consent. Genomic DNA was isolated by standard method and then subjected to targeted sequencing using an Ion Ampliseq(TM) Inherited Disease Panel. Three multiplex PCR reactions were carried out to amplify the coding exons of 328 genes including FBN1, TGFBR1 and TGFBR2. DNA fragments from different samples were ligated with barcoded sequencing adaptors. Template preparation and emulsion PCR, and Ion Sphere Particles enrichment were carried out using an Ion One Touch system. The ion sphere particles were sequenced on a 318 chip using the PGM platform. Data from the PGM runs were processed using an Ion Torrent Suite 3.2 software to generate sequence reads. After sequence alignment and extraction of SNPs and indels, all the variants were filtered against dbSNP137. DNA sequences were visualized with an Integrated Genomics Viewer. The most likely disease-causing variants were analyzed by Sanger sequencing. The PGM sequencing has yielded an output of 855.80 Mb, with a > 100 × median sequencing depth and a coverage of > 98% for the targeted regions in all the four samples. After data analysis and database filtering, one known missense mutation (p.E1811K) and two novel premature termination mutations (p.E2264X and p.L871FfsX23) in the FBN1 gene were identified in the three MFS patients. All mutations were verified by conventional Sanger sequencing. Pathogenic FBN1 mutations have been identified in all patients with MFS, indicating that the targeted next-generation sequencing on the PGM sequencers can be applied for accurate and high-throughput testing of genetic disorders.

Transient anterior subcapsular vacuolar change of the crystalline lens in patients after posterior chamber phakic intraocular lens implantation.

PubMed

Chung, Jin Kwon; Shin, Jin Hee; Lee, Sung Jin

2013-10-25

We present two cases of transient vacuolar changes in the anterior subcapsular space of the crystalline lens in patients after posterior chamber phakic intraocular lens implantation. Implantable collamer lenses (ICL) were implanted in healthy myopic patients. Vacuolar changes developed just after the irrigating procedure through the narrow space between the ICL and the crystalline lens. Slit-lamp examinations and spectral domain optical coherence tomography showed bleb-like lesions in the anterior subcapsular space of one eye in each case, though the lesions gradually improved without visual deterioration. Consequently, the lesions turned into a few anterior subcapsular small faint opacities. Direct irrigation of the narrow space confined by the ICL and the crystalline lens is at risk for the development of vacuolar changes in the crystalline lens. The observed spontaneous reversal indicates that surgeons should not rush to surgical intervention but rather opt for close follow over several weeks.

Atg22p, a vacuolar membrane protein involved in the amino acid compartmentalization of Schizosaccharomyces pombe.

PubMed

Sugimoto, Naoko; Iwaki, Tomoko; Chardwiriyapreecha, Soracom; Shimazu, Masamitsu; Kawano, Miyuki; Sekito, Takayuki; Takegawa, Kaoru; Kakinuma, Yoshimi

2011-01-01

The fission yeast Schizosaccharomyces pombe has a homolog of the budding yeast Atg22p, which is involved in spore formation (Mukaiyama H. et al., Microbiology, 155, 3816-3826 (2009)). GFP-tagged Atg22p in the fission yeast was localized to the vacuolar membrane. Upon disruption of atg22, the amino acid levels of the cellular fraction as well as the vacuolar fraction decreased. The uptake of several amino acids, such as lysine, histidine, and arginine, was impaired in atg22Δ cells. S. pombe Atg22p plays an important role in the compartmentalization of amino acids.

Next-generation sequencing for targeted discovery of rare mutations in rice

USDA-ARS?s Scientific Manuscript database

Advances in DNA sequencing (i.e., next-generation sequencing, NGS) have greatly increased the power and efficiency of detecting rare mutations in large mutant populations. Targeting Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach for identifying gene mutations resulting fro...

Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing

PubMed Central

Chandran, Anandhakumar; Syed, Junetha; Taylor, Rhys D.; Kashiwazaki, Gengo; Sato, Shinsuke; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

2016-01-01

Chemically engineered small molecules targeting specific genomic sequences play an important role in drug development research. Pyrrole-imidazole polyamides (PIPs) are a group of molecules that can bind to the DNA minor-groove and can be engineered to target specific sequences. Their biological effects rely primarily on their selective DNA binding. However, the binding mechanism of PIPs at the chromatinized genome level is poorly understood. Herein, we report a method using high-throughput sequencing to identify the DNA-alkylating sites of PIP-indole-seco-CBI conjugates. High-throughput sequencing analysis of conjugate 2 showed highly similar DNA-alkylating sites on synthetic oligos (histone-free DNA) and on human genomes (chromatinized DNA context). To our knowledge, this is the first report identifying alkylation sites across genomic DNA by alkylating PIP conjugates using high-throughput sequencing. PMID:27098039

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis.

PubMed

Luna-Tapia, Arturo; Peters, Brian M; Eberle, Karen E; Kerns, Morgan E; Foster, Timothy P; Marrero, Luis; Noverr, Mairi C; Fidel, Paul L; Palmer, Glen E

2015-10-01

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

To Be Cytosolic or Vacuolar: The Double Life of Listeria monocytogenes.

PubMed

Bierne, Hélène; Milohanic, Eliane; Kortebi, Mounia

2018-01-01

Intracellular bacterial pathogens are generally classified into two types: those that exploit host membrane trafficking to construct specific niches in vacuoles (i.e., "vacuolar pathogens"), and those that escape from vacuoles into the cytosol, where they proliferate and often spread to neighboring cells (i.e., "cytosolic pathogens"). However, the boundary between these distinct intracellular phenotypes is tenuous and may depend on the timing of infection and on the host cell type. Here, we discuss recent progress highlighting this phenotypic duality in Listeria monocytogenes , which has long been a model for cytosolic pathogens, but now emerges as a bacterium also capable of residing in vacuoles, in a slow/non-growing state. The ability of L. monocytogenes to enter a persistence stage in vacuoles might play a role during the asymptomatic incubation period of listeriosis and/or the carriage of this pathogen in asymptomatic hosts. Moreover, persistent vacuolar Listeria could be less susceptible to antibiotics and more difficult to detect by routine techniques of clinical biology. These hypotheses deserve to be explored in order to better manage the risks related to this food-borne pathogen.

Targeted amplicon sequencing (TAS): a scalable next-gen approach to multilocus, multitaxa phylogenetics.

PubMed

Bybee, Seth M; Bracken-Grissom, Heather; Haynes, Benjamin D; Hermansen, Russell A; Byers, Robert L; Clement, Mark J; Udall, Joshua A; Wilcox, Edward R; Crandall, Keith A

2011-01-01

Next-gen sequencing technologies have revolutionized data collection in genetic studies and advanced genome biology to novel frontiers. However, to date, next-gen technologies have been used principally for whole genome sequencing and transcriptome sequencing. Yet many questions in population genetics and systematics rely on sequencing specific genes of known function or diversity levels. Here, we describe a targeted amplicon sequencing (TAS) approach capitalizing on next-gen capacity to sequence large numbers of targeted gene regions from a large number of samples. Our TAS approach is easily scalable, simple in execution, neither time-nor labor-intensive, relatively inexpensive, and can be applied to a broad diversity of organisms and/or genes. Our TAS approach includes a bioinformatic application, BarcodeCrucher, to take raw next-gen sequence reads and perform quality control checks and convert the data into FASTA format organized by gene and sample, ready for phylogenetic analyses. We demonstrate our approach by sequencing targeted genes of known phylogenetic utility to estimate a phylogeny for the Pancrustacea. We generated data from 44 taxa using 68 different 10-bp multiplexing identifiers. The overall quality of data produced was robust and was informative for phylogeny estimation. The potential for this method to produce copious amounts of data from a single 454 plate (e.g., 325 taxa for 24 loci) significantly reduces sequencing expenses incurred from traditional Sanger sequencing. We further discuss the advantages and disadvantages of this method, while offering suggestions to enhance the approach.

Targeted Amplicon Sequencing (TAS): A Scalable Next-Gen Approach to Multilocus, Multitaxa Phylogenetics

PubMed Central

Bybee, Seth M.; Bracken-Grissom, Heather; Haynes, Benjamin D.; Hermansen, Russell A.; Byers, Robert L.; Clement, Mark J.; Udall, Joshua A.; Wilcox, Edward R.; Crandall, Keith A.

2011-01-01

Next-gen sequencing technologies have revolutionized data collection in genetic studies and advanced genome biology to novel frontiers. However, to date, next-gen technologies have been used principally for whole genome sequencing and transcriptome sequencing. Yet many questions in population genetics and systematics rely on sequencing specific genes of known function or diversity levels. Here, we describe a targeted amplicon sequencing (TAS) approach capitalizing on next-gen capacity to sequence large numbers of targeted gene regions from a large number of samples. Our TAS approach is easily scalable, simple in execution, neither time-nor labor-intensive, relatively inexpensive, and can be applied to a broad diversity of organisms and/or genes. Our TAS approach includes a bioinformatic application, BarcodeCrucher, to take raw next-gen sequence reads and perform quality control checks and convert the data into FASTA format organized by gene and sample, ready for phylogenetic analyses. We demonstrate our approach by sequencing targeted genes of known phylogenetic utility to estimate a phylogeny for the Pancrustacea. We generated data from 44 taxa using 68 different 10-bp multiplexing identifiers. The overall quality of data produced was robust and was informative for phylogeny estimation. The potential for this method to produce copious amounts of data from a single 454 plate (e.g., 325 taxa for 24 loci) significantly reduces sequencing expenses incurred from traditional Sanger sequencing. We further discuss the advantages and disadvantages of this method, while offering suggestions to enhance the approach. PMID:22002916

Vacuolar myelinopathy in waterfowl from a North Carolina impoundment

USGS Publications Warehouse

Augspurger, T.; Fischer, John R.; Thomas, Nancy; Sileo, L.; Brannian, Roger E.; Miller, Kimberli J.; Rocke, Tonie E.

2003-01-01

Vacuolar myelinopathy was confirmed by light and electron microscopic examination of mallards (Anas platyrhynchos), ring-necked ducks (Aythya collaris), and buffleheads (Bucephala albeola) collected during an epizootic at Lake Surf in central North Carolina (USA) between November 1998 and February 1999. Clinical signs of affected birds were consistent with central nervous system impairment of motor function (incoordination, abnormal movement and posture, weakness, paralysis). This is the first report of this disease in wild waterfowl (Anseriformes).Aug

Haloarcula hispanica CRISPR authenticates PAM of a target sequence to prime discriminative adaptation

PubMed Central

Li, Ming; Wang, Rui; Xiang, Hua

2014-01-01

The prokaryotic immune system CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) adapts to foreign invaders by acquiring their short deoxyribonucleic acid (DNA) fragments as spacers, which guide subsequent interference to foreign nucleic acids based on sequence matching. The adaptation mechanism avoiding acquiring ‘self’ DNA fragments is poorly understood. In Haloarcula hispanica, we previously showed that CRISPR adaptation requires being primed by a pre-existing spacer partially matching the invader DNA. Here, we further demonstrate that flanking a fully-matched target sequence, a functional PAM (protospacer adjacent motif) is still required to prime adaptation. Interestingly, interference utilizes only four PAM sequences, whereas adaptation-priming tolerates as many as 23 PAM sequences. This relaxed PAM selectivity explains how adaptation-priming maximizes its tolerance of PAM mutations (that escape interference) while avoiding mis-targeting the spacer DNA within CRISPR locus. We propose that the primed adaptation, which hitches and cooperates with the interference pathway, distinguishes target from non-target by CRISPR ribonucleic acid guidance and PAM recognition. PMID:24803673

Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kohio, Hinissan P.; Adamson, Amy L., E-mail: aladamso@uncg.edu

As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transportmore » activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.« less

Application of Quaternion in improving the quality of global sequence alignment scores for an ambiguous sequence target in Streptococcus pneumoniae DNA

NASA Astrophysics Data System (ADS)

Lestari, D.; Bustamam, A.; Novianti, T.; Ardaneswari, G.

2017-07-01

DNA sequence can be defined as a succession of letters, representing the order of nucleotides within DNA, using a permutation of four DNA base codes including adenine (A), guanine (G), cytosine (C), and thymine (T). The precise code of the sequences is determined using DNA sequencing methods and technologies, which have been developed since the 1970s and currently become highly developed, advanced and highly throughput sequencing technologies. So far, DNA sequencing has greatly accelerated biological and medical research and discovery. However, in some cases DNA sequencing could produce any ambiguous and not clear enough sequencing results that make them quite difficult to be determined whether these codes are A, T, G, or C. To solve these problems, in this study we can introduce other representation of DNA codes namely Quaternion Q = (PA, PT, PG, PC), where PA, PT, PG, PC are the probability of A, T, G, C bases that could appear in Q and PA + PT + PG + PC = 1. Furthermore, using Quaternion representations we are able to construct the improved scoring matrix for global sequence alignment processes, by applying a dot product method. Moreover, this scoring matrix produces better and higher quality of the match and mismatch score between two DNA base codes. In implementation, we applied the Needleman-Wunsch global sequence alignment algorithm using Octave, to analyze our target sequence which contains some ambiguous sequence data. The subject sequences are the DNA sequences of Streptococcus pneumoniae families obtained from the Genebank, meanwhile the target DNA sequence are received from our collaborator database. As the results we found the Quaternion representations improve the quality of the sequence alignment score and we can conclude that DNA sequence target has maximum similarity with Streptococcus pneumoniae.

Promoter regions of potato vacuolar invertase gene in response to sugars and hormones.

PubMed

Ou, Yongbin; Song, Botao; Liu, Xun; Xie, Conghua; Li, Meng; Lin, Yuan; Zhang, Huiling; Liu, Jun

2013-08-01

Potato vacuolar acid invertase (StvacINV1) (β-fructofuranosidase; EC 3.2.1.26) has been confirmed to play an important role in cold-induced sweetening of potato tubers. However, the transcriptional regulation mechanisms of StvacINV1 are largely unknown. In this study, the 5'-flanking sequence of StvacINV1 was cloned and the cis-acting elements were predicted. Histochemical assay showed that the StvacINV1 promoter governed β-glucuronidase (GUS) expression in potato leaves, stems, roots and tubers. Quantitative analysis of GUS expression suggested that the activity of StvacINV1 promoter was suppressed by sucrose, glucose, fructose, and cold, while enhanced by indole-3-acetic acid (IAA), and gibberellic acid (GA3). Further deletion analysis clarified that the promoter regions from -118 to -551, -551 to -1021, and -1021 to -1521 were required for responding to sucrose/glucose, GA3, and IAA, respectively. These findings provide essential information regarding transcriptional regulation mechanisms of StvacINV1. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing.

PubMed

Liu, Gary W; Livesay, Brynn R; Kacherovsky, Nataly A; Cieslewicz, Maryelise; Lutz, Emi; Waalkes, Adam; Jensen, Michael C; Salipante, Stephen J; Pun, Suzie H

2015-08-19

Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (<0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by nonspecific, preferentially amplifying "parasitic sequences" and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. In this work, we define the hallmarks of binding sequences in next-generation sequencing data, and develop a method that identifies several target-binding phage clones for murine, alternatively activated M2 macrophages with a high (100%) success rate: sequences and binding motifs were reproducibly present across biological replicates; binding motifs were identified across multiple unique sequences; and an unselected, amplified library accurately filtered out parasitic sequences. In addition, we validate the Multiple Em for Motif Elicitation tool as an efficient and principled means of discovering binding sequences.

ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miao, Y.C.; Liu, C.

2010-12-28

Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitorsmore » in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.« less

Profiling of potential driver mutations in sarcomas by targeted next generation sequencing.

PubMed

Andersson, Carola; Fagman, Henrik; Hansson, Magnus; Enlund, Fredrik

2016-04-01

Comprehensive genetic profiling by massively parallel sequencing, commonly known as next generation sequencing (NGS), is becoming the foundation of personalized oncology. For sarcomas very few targeted treatments are currently in routine use. In clinical practice the preoperative diagnostic workup of soft tissue tumours largely relies on core needle biopsies. Although mostly sufficient for histopathological diagnosis, only very limited amounts of formalin fixated paraffin embedded tissue are often available for predictive mutation analysis. Targeted NGS may thus open up new possibilities for comprehensive characterization of scarce biopsies. We therefore set out to search for driver mutations by NGS in a cohort of 55 clinically and morphologically well characterized sarcomas using low input of DNA from formalin fixated paraffin embedded tissues. The aim was to investigate if there are any recurrent or targetable aberrations in cancer driver genes in addition to known chromosome translocations in different types of sarcomas. We employed a panel covering 207 mutation hotspots in 50 cancer-associated genes to analyse DNA from nine gastrointestinal stromal tumours, 14 synovial sarcomas, seven myxoid liposarcomas, 22 Ewing sarcomas and three Ewing-like small round cell tumours at a large sequencing depth to detect also mutations that are subclonal or occur at low allele frequencies. We found nine mutations in eight different potential driver genes, some of which are potentially actionable by currently existing targeted therapies. Even though no recurrent mutations in driver genes were found in the different sarcoma groups, we show that targeted NGS-based sequencing is clearly feasible in a diagnostic setting with very limited amounts of paraffin embedded tissue and may provide novel insights into mesenchymal cell signalling and potentially druggable targets. Interestingly, we also identify five non-synonymous sequence variants in 4 established cancer driver genes in DNA

Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers.

PubMed

McKenzie, Marian J; Chen, Ronan K Y; Harris, John C; Ashworth, Matthew J; Brummell, David A

2013-01-01

Cold-induced sweetening (CIS) is a serious post-harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark-coloured and bitter-tasting product and generating the probable carcinogen acrylamide as a by-product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS-susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS-resistant line increased susceptibility to CIS. The results show that post-translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS. © 2012 Blackwell Publishing Ltd.

Expression analysis of Arabidopsis vacuolar sorting receptor 3 reveals a putative function in guard cells.

PubMed

Avila, Emily L; Brown, Michelle; Pan, Songqin; Desikan, Radhika; Neill, Steven J; Girke, Thomas; Surpin, Marci; Raikhel, Natasha V

2008-01-01

Vacuolar sorting receptors (VSRs) are responsible for the proper targeting of soluble cargo proteins to their destination compartments. The Arabidopsis genome encodes seven VSRs. In this work, the spatio-temporal expression of one of the members of this gene family, AtVSR3, was determined by RT-PCR and promoter::reporter gene fusions. AtVSR3 was expressed specifically in guard cells. Consequently, a reverse genetics approach was taken to determine the function of AtVSR3 by using RNA interference (RNAi) technology. Plants expressing little or no AtVSR3 transcript had a compressed life cycle, bolting approximately 1 week earlier and senescing up to 2 weeks earlier than the wild-type parent line. While the development and distribution of stomata in AtVSR3 RNAi plants appeared normal, stomatal function was altered. The guard cells of mutant plants did not close in response to abscisic acid treatment, and the mean leaf temperatures of the RNAi plants were on average 0.8 degrees C lower than both wild type and another vacuolar sorting receptor mutant, atvsr1-1. Furthermore, the loss of AtVSR3 protein caused the accumulation of nitric oxide and hydrogen peroxide, signalling molecules implicated in the regulation of stomatal opening and closing. Finally, proteomics and western blot analyses of cellular proteins isolated from wild-type and AtVSR3 RNAi leaves showed that phospholipase Dgamma, which may play a role in abscisic acid signalling, accumulated to higher levels in AtVSR3 RNAi guard cells. Thus, AtVSR3 may play an important role in responses to plant stress.

Identification and functional expression of the Arabidopsis thaliana vacuolar glucose transporter 1 and its role in seed germination and flowering.

PubMed

Aluri, Sirisha; Büttner, Michael

2007-02-13

Sugar compartmentation into vacuoles of higher plants is a very important physiological process, providing extra space for transient and long-term sugar storage and contributing to the osmoregulation of cell turgor and shape. Despite the long-standing knowledge of this subcellular sugar partitioning, the proteins responsible for these transport steps have remained unknown. We have identified a gene family in Arabidopsis consisting of three members homologous to known sugar transporters. One member of this family, Arabidopsis thaliana vacuolar glucose transporter 1 (AtVGT1), was localized to the vacuolar membrane. Moreover, we provide evidence for transport activity of a tonoplast sugar transporter based on its functional expression in bakers' yeast and uptake studies in isolated yeast vacuoles. Analyses of Atvgt1 mutant lines indicate an important function of this vacuolar glucose transporter during developmental processes like seed germination and flowering.

Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system.

PubMed

Chen, Xiugui; Lu, Xuke; Shu, Na; Wang, Shuai; Wang, Junjuan; Wang, Delong; Guo, Lixue; Ye, Wuwei

2017-03-13

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H + -pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6-81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome.

A Novel Arabidopsis Vacuolar Glucose Exporter Is Involved in Cellular Sugar Homeostasis and Affects the Composition of Seed Storage Compounds1[W][OA

PubMed Central

Poschet, Gernot; Hannich, Barbara; Raab, Sabine; Jungkunz, Isabel; Klemens, Patrick A.W.; Krueger, Stephan; Wic, Stefan; Neuhaus, H. Ekkehard; Büttner, Michael

2011-01-01

Subcellular sugar partitioning in plants is strongly regulated in response to developmental cues and changes in external conditions. Besides transitory starch, the vacuolar sugars represent a highly dynamic pool of instantly accessible metabolites that serve as energy source and osmoprotectant. Here, we present the molecular identification and functional characterization of the vacuolar glucose (Glc) exporter Arabidopsis (Arabidopsis thaliana) Early Responsive to Dehydration-Like6 (AtERDL6). We demonstrate tonoplast localization of AtERDL6 in plants. In Arabidopsis, AtERDL6 expression is induced in response to factors that activate vacuolar Glc pools, like darkness, heat stress, and wounding. On the other hand, AtERDL6 transcript levels drop during conditions that trigger Glc accumulation in the vacuole, like cold stress and external sugar supply. Accordingly, sugar analyses revealed that Aterdl6 mutants have elevated vacuolar Glc levels and that Glc flux across the tonoplast is impaired under stress conditions. Interestingly, overexpressor lines indicated a very similar function for the ERDL6 ortholog Integral Membrane Protein from sugar beet (Beta vulgaris). Aterdl6 mutant plants display increased sensitivity against external Glc, and mutant seeds exhibit a 10% increase in seed weight due to enhanced levels of seed sugars, proteins, and lipids. Our findings underline the importance of vacuolar Glc export during the regulation of cellular Glc homeostasis and the composition of seed reserves. PMID:21984725

An Optimized Transient Dual Luciferase Assay for Quantifying MicroRNA Directed Repression of Targeted Sequences

PubMed Central

Moyle, Richard L.; Carvalhais, Lilia C.; Pretorius, Lara-Simone; Nowak, Ekaterina; Subramaniam, Gayathery; Dalton-Morgan, Jessica; Schenk, Peer M.

2017-01-01

Studies investigating the action of small RNAs on computationally predicted target genes require some form of experimental validation. Classical molecular methods of validating microRNA action on target genes are laborious, while approaches that tag predicted target sequences to qualitative reporter genes encounter technical limitations. The aim of this study was to address the challenge of experimentally validating large numbers of computationally predicted microRNA-target transcript interactions using an optimized, quantitative, cost-effective, and scalable approach. The presented method combines transient expression via agroinfiltration of Nicotiana benthamiana leaves with a quantitative dual luciferase reporter system, where firefly luciferase is used to report the microRNA-target sequence interaction and Renilla luciferase is used as an internal standard to normalize expression between replicates. We report the appropriate concentration of N. benthamiana leaf extracts and dilution factor to apply in order to avoid inhibition of firefly LUC activity. Furthermore, the optimal ratio of microRNA precursor expression construct to reporter construct and duration of the incubation period post-agroinfiltration were determined. The optimized dual luciferase assay provides an efficient, repeatable and scalable method to validate and quantify microRNA action on predicted target sequences. The optimized assay was used to validate five predicted targets of rice microRNA miR529b, with as few as six technical replicates. The assay can be extended to assess other small RNA-target sequence interactions, including assessing the functionality of an artificial miRNA or an RNAi construct on a targeted sequence. PMID:28979287

The antiwrinkle effect of topical concentrated 2-dimethylaminoethanol involves a vacuolar cytopathology.

PubMed

Morissette, G; Germain, L; Marceau, F

2007-03-01

The 'cosmeceutical' agent 2-dimethylaminoethanol (DMAE) is a tertiary amine found in high concentration in numerous topical antiwrinkle preparations. We hypothesized that a 337 mmol L(-1) (3%) DMAE reservoir applied to the skin could reproduce the cytopathology induced by other amines by maintaining a millimolar drug concentration within a certain depth of the skin layers, and that vacuolar cell expansion could account for the very rapid effect on the apparent skin fullness. Morphological and functional assays were applied to cultured rabbit dermal fibroblasts treated with tertiary amines in vitro. A morphological verification of the vacuolization caused by topical DMAE was also attempted in vivo using the inner skin of the rabbit ear and in vitro using primary cultures of human cutaneous epithelial cells. Fibroblasts responded to DMAE (2.5-10 mmol L(-1)) by massive vacuolization (0.5-4 h; phase contrast observations). Triethanolamine, another chemical frequently used topically, was also active in this respect (10 mmol L(-1)). The vacuolar adenosine triphosphatase inhibitor bafilomycin A1 prevented DMAE- or triethanolamine-induced vacuolization; adding bafilomycin A1 or cell washout slowly reversed the established vacuolization induced by DMAE. Further effects of DMAE in cultured fibroblasts included a moderate cytotoxicity (10 mmol L(-1)) that was abated by bafilomycin A1 cotreatment, a concentration-dependent mitotic arrest (2.5 mmol L(-1)) and transient and mild effects on cell ploidy. The epidermis of the rabbit external ear was significantly thickened and exhibited clear perinuclear swelling indicative of vacuolization in response to 3% DMAE (1 h; paraffin tissue sections). Cultured human cutaneous epithelial cells responded to DMAE by vacuolization (inhibited by bafilomycin A1 cotreatment). The vacuolar cytopathology induced by concentrated organic amines may be the cellular basis of the antiwrinkle effect of DMAE.

Vacuolar invertase gene silencing in potato decreasing the frequency of sugar-end defects

USDA-ARS?s Scientific Manuscript database

Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one e...

Genome-wide evidence for local DNA methylation spreading from small RNA-targeted sequences in Arabidopsis.

PubMed

Ahmed, Ikhlak; Sarazin, Alexis; Bowler, Chris; Colot, Vincent; Quesneville, Hadi

2011-09-01

Transposable elements (TEs) and their relics play major roles in genome evolution. However, mobilization of TEs is usually deleterious and strongly repressed. In plants and mammals, this repression is typically associated with DNA methylation, but the relationship between this epigenetic mark and TE sequences has not been investigated systematically. Here, we present an improved annotation of TE sequences and use it to analyze genome-wide DNA methylation maps obtained at single-nucleotide resolution in Arabidopsis. We show that although the majority of TE sequences are methylated, ∼26% are not. Moreover, a significant fraction of TE sequences densely methylated at CG, CHG and CHH sites (where H = A, T or C) have no or few matching small interfering RNA (siRNAs) and are therefore unlikely to be targeted by the RNA-directed DNA methylation (RdDM) machinery. We provide evidence that these TE sequences acquire DNA methylation through spreading from adjacent siRNA-targeted regions. Further, we show that although both methylated and unmethylated TE sequences located in euchromatin tend to be more abundant closer to genes, this trend is least pronounced for methylated, siRNA-targeted TE sequences located 5' to genes. Based on these and other findings, we propose that spreading of DNA methylation through promoter regions explains at least in part the negative impact of siRNA-targeted TE sequences on neighboring gene expression.

Molecular cloning, functional expression and subcellular localization of two putative vacuolar voltage-gated chloride channels in rice (Oryza sativa L.).

PubMed

Nakamura, Atsuko; Fukuda, Atsunori; Sakai, Shingo; Tanaka, Yoshiyuki

2006-01-01

We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLC-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages.

A Grapevine TTG2-Like WRKY Transcription Factor Is Involved in Regulating Vacuolar Transport and Flavonoid Biosynthesis.

PubMed

Amato, Alessandra; Cavallini, Erika; Zenoni, Sara; Finezzo, Laura; Begheldo, Maura; Ruperti, Benedetto; Tornielli, Giovanni Battista

2016-01-01

A small set of TTG2-like homolog proteins from different species belonging to the WRKY family of transcription factors were shown to share a similar mechanism of action and to control partially conserved biochemical/developmental processes in their native species. In particular, by activating P-ATPases residing on the tonoplast, PH3 from Petunia hybrida promotes vacuolar acidification in petal epidermal cells whereas TTG2 from Arabidopsis thaliana enables the accumulation of proanthocyanidins in the seed coat. In this work we functionally characterized VvWRKY26 identified as the closest grapevine homolog of PhPH3 and AtTTG2 . When constitutively expressed in petunia ph3 mutant, VvWRKY26 can fulfill the PH3 function in the regulation of vacuolar pH and restores the wild type pigmentation phenotype. By a global correlation analysis of gene expression and by transient over-expression in Vitis vinifera , we showed transcriptomic relationships of VvWRKY26 with many genes related to vacuolar acidification and transport in grapevine. Moreover, our results indicate an involvement in flavonoid pathway possibly restricted to the control of proanthocyanidin biosynthesis that is consistent with its expression pattern in grape berry tissues. Overall, the results show that, in addition to regulative mechanisms and biological roles shared with TTG2-like orthologs, VvWRKY26 can play roles in fleshy fruit development that have not been previously reported in studies from dry fruit species. This study paves the way toward the comprehension of the regulatory network controlling vacuolar acidification and flavonoid accumulation mechanisms that contribute to the final berry quality traits in grapevine.

A Grapevine TTG2-Like WRKY Transcription Factor Is Involved in Regulating Vacuolar Transport and Flavonoid Biosynthesis

PubMed Central

Amato, Alessandra; Cavallini, Erika; Zenoni, Sara; Finezzo, Laura; Begheldo, Maura; Ruperti, Benedetto; Tornielli, Giovanni Battista

2017-01-01

A small set of TTG2-like homolog proteins from different species belonging to the WRKY family of transcription factors were shown to share a similar mechanism of action and to control partially conserved biochemical/developmental processes in their native species. In particular, by activating P-ATPases residing on the tonoplast, PH3 from Petunia hybrida promotes vacuolar acidification in petal epidermal cells whereas TTG2 from Arabidopsis thaliana enables the accumulation of proanthocyanidins in the seed coat. In this work we functionally characterized VvWRKY26 identified as the closest grapevine homolog of PhPH3 and AtTTG2. When constitutively expressed in petunia ph3 mutant, VvWRKY26 can fulfill the PH3 function in the regulation of vacuolar pH and restores the wild type pigmentation phenotype. By a global correlation analysis of gene expression and by transient over-expression in Vitis vinifera, we showed transcriptomic relationships of VvWRKY26 with many genes related to vacuolar acidification and transport in grapevine. Moreover, our results indicate an involvement in flavonoid pathway possibly restricted to the control of proanthocyanidin biosynthesis that is consistent with its expression pattern in grape berry tissues. Overall, the results show that, in addition to regulative mechanisms and biological roles shared with TTG2-like orthologs, VvWRKY26 can play roles in fleshy fruit development that have not been previously reported in studies from dry fruit species. This study paves the way toward the comprehension of the regulatory network controlling vacuolar acidification and flavonoid accumulation mechanisms that contribute to the final berry quality traits in grapevine. PMID:28105033

Structural and sequencing analysis of local target DNA recognition by MLV integrase.

PubMed

Aiyer, Sriram; Rossi, Paolo; Malani, Nirav; Schneider, William M; Chandar, Ashwin; Bushman, Frederic D; Montelione, Gaetano T; Roth, Monica J

2015-06-23

Target-site selection by retroviral integrase (IN) proteins profoundly affects viral pathogenesis. We describe the solution nuclear magnetic resonance structure of the Moloney murine leukemia virus IN (M-MLV) C-terminal domain (CTD) and a structural homology model of the catalytic core domain (CCD). In solution, the isolated MLV IN CTD adopts an SH3 domain fold flanked by a C-terminal unstructured tail. We generated a concordant MLV IN CCD structural model using SWISS-MODEL, MMM-tree and I-TASSER. Using the X-ray crystal structure of the prototype foamy virus IN target capture complex together with our MLV domain structures, residues within the CCD α2 helical region and the CTD β1-β2 loop were predicted to bind target DNA. The role of these residues was analyzed in vivo through point mutants and motif interchanges. Viable viruses with substitutions at the IN CCD α2 helical region and the CTD β1-β2 loop were tested for effects on integration target site selection. Next-generation sequencing and analysis of integration target sequences indicate that the CCD α2 helical region, in particular P187, interacts with the sequences distal to the scissile bonds whereas the CTD β1-β2 loop binds to residues proximal to it. These findings validate our structural model and disclose IN-DNA interactions relevant to target site selection. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Isolation and characterization of target sequences of the chicken CdxA homeobox gene.

PubMed Central

Margalit, Y; Yarus, S; Shapira, E; Gruenbaum, Y; Fainsod, A

1993-01-01

The DNA binding specificity of the chicken homeodomain protein CDXA was studied. Using a CDXA-glutathione-S-transferase fusion protein, DNA fragments containing the binding site for this protein were isolated. The sources of DNA were oligonucleotides with random sequence and chicken genomic DNA. The DNA fragments isolated were sequenced and tested in DNA binding assays. Sequencing revealed that most DNA fragments are AT rich which is a common feature of homeodomain binding sites. By electrophoretic mobility shift assays it was shown that the different target sequences isolated bind to the CDXA protein with different affinities. The specific sequences bound by the CDXA protein in the genomic fragments isolated, were determined by DNase I footprinting. From the footprinted sequences, the CDXA consensus binding site was determined. The CDXA protein binds the consensus sequence A, A/T, T, A/T, A, T, A/G. The CAUDAL binding site in the ftz promoter is also included in this consensus sequence. When tested, some of the genomic target sequences were capable of enhancing the transcriptional activity of reporter plasmids when introduced into CDXA expressing cells. This study determined the DNA sequence specificity of the CDXA protein and it also shows that this protein can further activate transcription in cells in culture. Images PMID:7909943

How proteins bind to DNA: target discrimination and dynamic sequence search by the telomeric protein TRF1

PubMed Central

2017-01-01

Abstract Target search as performed by DNA-binding proteins is a complex process, in which multiple factors contribute to both thermodynamic discrimination of the target sequence from overwhelmingly abundant off-target sites and kinetic acceleration of dynamic sequence interrogation. TRF1, the protein that binds to telomeric tandem repeats, faces an intriguing variant of the search problem where target sites are clustered within short fragments of chromosomal DNA. In this study, we use extensive (>0.5 ms in total) MD simulations to study the dynamical aspects of sequence-specific binding of TRF1 at both telomeric and non-cognate DNA. For the first time, we describe the spontaneous formation of a sequence-specific native protein–DNA complex in atomistic detail, and study the mechanism by which proteins avoid off-target binding while retaining high affinity for target sites. Our calculated free energy landscapes reproduce the thermodynamics of sequence-specific binding, while statistical approaches allow for a comprehensive description of intermediate stages of complex formation. PMID:28633355

A Cytotoxic Type III Secretion Effector of Vibrio parahaemolyticus Targets Vacuolar H+-ATPase Subunit c and Ruptures Host Cell Lysosomes

PubMed Central

Matsuda, Shigeaki; Okada, Natsumi; Kodama, Toshio; Honda, Takeshi; Iida, Tetsuya

2012-01-01

Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H+-ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death. PMID:22829766

The vacuolar protein sorting genes in insects: A comparative genome view.

PubMed

Li, Zhaofei; Blissard, Gary

2015-07-01

In eukaryotic cells, regulated vesicular trafficking is critical for directing protein transport and for recycling and degradation of membrane lipids and proteins. Through carefully regulated transport vesicles, the endomembrane system performs a large and important array of dynamic cellular functions while maintaining the integrity of the cellular membrane system. Genetic studies in yeast Saccharomyces cerevisiae have identified approximately 50 vacuolar protein sorting (VPS) genes involved in vesicle trafficking, and most of these genes are also characterized in mammals. The VPS proteins form distinct functional complexes, which include complexes known as ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III. Little is known about the orthologs of VPS proteins in insects. Here, with the newly annotated Manduca sexta genome, we carried out genomic comparative analysis of VPS proteins in yeast, humans, and 13 sequenced insect genomes representing the Orders Hymenoptera, Diptera, Hemiptera, Phthiraptera, Lepidoptera, and Coleoptera. Amino acid sequence alignments and domain/motif structure analyses reveal that most of the components of ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III are evolutionarily conserved across yeast, insects, and humans. However, in contrast to the VPS gene expansions observed in the human genome, only four VPS genes (VPS13, VPS16, VPS33, and VPS37) were expanded in the six insect Orders. Additionally, VPS2 was expanded only in species from Phthiraptera, Lepidoptera, and Coleoptera. These studies provide a baseline for understanding the evolution of vesicular trafficking across yeast, insect, and human genomes, and also provide a basis for further addressing specific functional roles of VPS proteins in insects. Copyright © 2014 Elsevier Ltd. All rights reserved.

BreaKmer: detection of structural variation in targeted massively parallel sequencing data using kmers.

PubMed

Abo, Ryan P; Ducar, Matthew; Garcia, Elizabeth P; Thorner, Aaron R; Rojas-Rudilla, Vanesa; Lin, Ling; Sholl, Lynette M; Hahn, William C; Meyerson, Matthew; Lindeman, Neal I; Van Hummelen, Paul; MacConaill, Laura E

2015-02-18

Genomic structural variation (SV), a common hallmark of cancer, has important predictive and therapeutic implications. However, accurately detecting SV using high-throughput sequencing data remains challenging, especially for 'targeted' resequencing efforts. This is critically important in the clinical setting where targeted resequencing is frequently being applied to rapidly assess clinically actionable mutations in tumor biopsies in a cost-effective manner. We present BreaKmer, a novel approach that uses a 'kmer' strategy to assemble misaligned sequence reads for predicting insertions, deletions, inversions, tandem duplications and translocations at base-pair resolution in targeted resequencing data. Variants are predicted by realigning an assembled consensus sequence created from sequence reads that were abnormally aligned to the reference genome. Using targeted resequencing data from tumor specimens with orthogonally validated SV, non-tumor samples and whole-genome sequencing data, BreaKmer had a 97.4% overall sensitivity for known events and predicted 17 positively validated, novel variants. Relative to four publically available algorithms, BreaKmer detected SV with increased sensitivity and limited calls in non-tumor samples, key features for variant analysis of tumor specimens in both the clinical and research settings. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Implementation of an Autonomous Multi-Maneuver Targeting Sequence for Lunar Trans-Earth Injection

NASA Technical Reports Server (NTRS)

Whitley, Ryan J.; Williams, Jacob

2010-01-01

Using a fully analytic initial guess estimate as a first iterate, a targeting procedure that constructs a flyable burn maneuver sequence to transfer a spacecraft from any closed Moon orbit to a desired Earth entry state is developed and implemented. The algorithm is built to support the need for an anytime abort capability for Orion. Based on project requirements, the Orion spacecraft must be able to autonomously calculate the translational maneuver targets for an entire Lunar mission. Translational maneuver target sequences for the Orion spacecraft include Lunar Orbit Insertion (LOI), Trans-Earth Injection (TEI), and Trajectory Correction Maneuvers (TCMs). This onboard capability is generally assumed to be supplemental to redundant ground computation in nominal mission operations and considered as a viable alternative primarily in loss of communications contingencies. Of these maneuvers, the ability to accurately and consistently establish a flyable 3-burn TEI target sequence is especially critical. The TEI is the sole means by which the crew can successfully return from the Moon to a narrowly banded Earth Entry Interface (EI) state. This is made even more critical by the desire for global access on the lunar surface. Currently, the designed propellant load is based on fully optimized TEI solutions for the worst case geometries associated with the accepted range of epochs and landing sites. This presents two challenges for an autonomous algorithm: in addition to being feasible, the targets must include burn sequences that do not exceed the anticipated propellant load.

Comparison of taxon-specific versus general locus sets for targeted sequence capture in plant phylogenomics.

PubMed

Chau, John H; Rahfeldt, Wolfgang A; Olmstead, Richard G

2018-03-01

Targeted sequence capture can be used to efficiently gather sequence data for large numbers of loci, such as single-copy nuclear loci. Most published studies in plants have used taxon-specific locus sets developed individually for a clade using multiple genomic and transcriptomic resources. General locus sets can also be developed from loci that have been identified as single-copy and have orthologs in large clades of plants. We identify and compare a taxon-specific locus set and three general locus sets (conserved ortholog set [COSII], shared single-copy nuclear [APVO SSC] genes, and pentatricopeptide repeat [PPR] genes) for targeted sequence capture in Buddleja (Scrophulariaceae) and outgroups. We evaluate their performance in terms of assembly success, sequence variability, and resolution and support of inferred phylogenetic trees. The taxon-specific locus set had the most target loci. Assembly success was high for all locus sets in Buddleja samples. For outgroups, general locus sets had greater assembly success. Taxon-specific and PPR loci had the highest average variability. The taxon-specific data set produced the best-supported tree, but all data sets showed improved resolution over previous non-sequence capture data sets. General locus sets can be a useful source of sequence capture targets, especially if multiple genomic resources are not available for a taxon.

Phosphoenolpyruvate carboxykinase of Trypanosoma brucei is targeted to the glycosomes by a C-terminal sequence.

PubMed

Sommer, J M; Nguyen, T T; Wang, C C

1994-08-15

Import of proteins into the glycosomes of T. brucei resembles the peroxisomal protein import in that C-terminal SKL-like tripeptide sequences can function as targeting signals. Many of the glycosomal proteins do not, however, possess such C-terminal tripeptide signals. Among these, phosphoenolpyruvate carboxykinase (PEPCK (ATP)) was thought to be targeted to the glycosomes by an N-terminal or an internal targeting signal. A limited similarity to the N-terminal targeting signal of rat peroxisomal thiolase exists at the N-terminus of T. brucei PEPCK. However, we found that this peroxisomal targeting signal does not function for glycosomal protein import in T. brucei. Further studies of the PEPCK gene revealed that the C-terminus of the predicted protein does not correspond to the previously deduced protein sequence of 472 amino acids due to a -1 frame shift error in the original DNA sequence. Readjusting the reading frame of the sequence results in a predicted protein of 525 amino acids in length ending in a tripeptide serine-arginine-leucine (SRL), which is a potential targeting signal for import into the glycosomes. A fusion protein of firefly luciferase, without its own C-terminal SKL targeting signal, and T. brucei PEPCK is efficiently imported into the glycosomes when expressed in procyclic trypanosomes. Deletion of the C-terminal SRL tripeptide or the last 29 amino acids of PEPCK reduced the import only by about 50%, while a deletion of the last 47 amino acids completely abolished the import. These results suggest that T. brucei PEPCK may contain a second, internal glycosomal targeting signal upstream of the C-terminal SRL sequence.

Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death.

PubMed

Koh, Eugene; Carmieli, Raanan; Mor, Avishai; Fluhr, Robert

2016-07-01

Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role. © 2016 American Society of Plant Biologists. All Rights Reserved.

Mapping a nucleolar targeting sequence of an RNA binding nucleolar protein, Nop25

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujiwara, Takashi; Suzuki, Shunji; Kanno, Motoko

2006-06-10

Nop25 is a putative RNA binding nucleolar protein associated with rRNA transcription. The present study was undertaken to determine the mechanism of Nop25 localization in the nucleolus. Deletion experiments of Nop25 amino acid sequence showed Nop25 to contain a nuclear targeting sequence in the N-terminal and a nucleolar targeting sequence in the C-terminal. By expressing derivative peptides from the C-terminal as GFP-fusion proteins in the cells, a lysine and arginine residue-enriched peptide (KRKHPRRAQDSTKKPPSATRTSKTQRRRR) allowed a GFP-fusion protein to be transported and fully retained in the nucleolus. When the peptide was fused with cMyc epitope and expressed in the cells, amore » cMyc epitope was then detected in the nucleolus. Nop25 did not localize in the nucleolus by deletion of the peptide from Nop25. Furthermore, deletion of a subdomain (KRKHPRRAQ) in the peptide or amino acid substitution of lysine and arginine residues in the subdomain resulted in the loss of Nop25 nucleolar localization. These results suggest that the lysine and arginine residue-enriched peptide is the most prominent nucleolar targeting sequence of Nop25 and that the long stretch of basic residues might play an important role in the nucleolar localization of Nop25. Although Nop25 contained putative SUMOylation, phosphorylation and glycosylation sites, the amino acid substitution in these sites had no effect on the nucleolar localization, thus suggesting that these post-translational modifications did not contribute to the localization of Nop25 in the nucleolus. The treatment of the cells, which expressed a GFP-fusion protein with a nucleolar targeting sequence of Nop25, with RNase A resulted in a complete dislocation of the protein from the nucleolus. These data suggested that the nucleolar targeting sequence might therefore play an important role in the binding of Nop25 to RNA molecules and that the RNA binding of Nop25 might be essential for the nucleolar localization of Nop25.« less

Expression of an arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) in cotton improves drought- and salt tolerance and increases fibre yield in the field conditions

USDA-ARS?s Scientific Manuscript database

The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase that functions as a proton pump on the vacuolar membrane. Overexpression of AVP1 in Arabidopsis, tomato and rice enhances plant performance under salt and drought stress conditions, because up-regulation of the type I H+-PPase from Arabid...

Expression of an Arabidopsis Vacuolar H+-pyrophosphatase Gene (AVP1) in Cotton Improves Drought- and Salt Tolerance and Increases Fibre Yield in the Field Conditions.

USDA-ARS?s Scientific Manuscript database

The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase that functions as a proton pump on the vacuolar membrane. Overexpression of AVP1 in Arabidopsis, tomato and rice enhances plant performance under salt and drought stress conditions, because up-regulation of the type I H+PPase from Arabido...

A Bioinformatic Pipeline for Monitoring of the Mutational Stability of Viral Drug Targets with Deep-Sequencing Technology.

PubMed

Kravatsky, Yuri; Chechetkin, Vladimir; Fedoseeva, Daria; Gorbacheva, Maria; Kravatskaya, Galina; Kretova, Olga; Tchurikov, Nickolai

2017-11-23

The efficient development of antiviral drugs, including efficient antiviral small interfering RNAs (siRNAs), requires continuous monitoring of the strict correspondence between a drug and the related highly variable viral DNA/RNA target(s). Deep sequencing is able to provide an assessment of both the general target conservation and the frequency of particular mutations in the different target sites. The aim of this study was to develop a reliable bioinformatic pipeline for the analysis of millions of short, deep sequencing reads corresponding to selected highly variable viral sequences that are drug target(s). The suggested bioinformatic pipeline combines the available programs and the ad hoc scripts based on an original algorithm of the search for the conserved targets in the deep sequencing data. We also present the statistical criteria for the threshold of reliable mutation detection and for the assessment of variations between corresponding data sets. These criteria are robust against the possible sequencing errors in the reads. As an example, the bioinformatic pipeline is applied to the study of the conservation of RNA interference (RNAi) targets in human immunodeficiency virus 1 (HIV-1) subtype A. The developed pipeline is freely available to download at the website http://virmut.eimb.ru/. Brief comments and comparisons between VirMut and other pipelines are also presented.

Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells.

PubMed

Kortebi, Mounia; Milohanic, Eliane; Mitchell, Gabriel; Péchoux, Christine; Prevost, Marie-Christine; Cossart, Pascale; Bierne, Hélène

2017-11-01

Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called "viable but non-culturable" state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy.

Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells

PubMed Central

Mitchell, Gabriel

2017-01-01

Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called “viable but non-culturable” state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy. PMID:29190284

Experience of targeted Usher exome sequencing as a clinical test

PubMed Central

Besnard, Thomas; García-García, Gema; Baux, David; Vaché, Christel; Faugère, Valérie; Larrieu, Lise; Léonard, Susana; Millan, Jose M; Malcolm, Sue; Claustres, Mireille; Roux, Anne-Françoise

2014-01-01

We show that massively parallel targeted sequencing of 19 genes provides a new and reliable strategy for molecular diagnosis of Usher syndrome (USH) and nonsyndromic deafness, particularly appropriate for these disorders characterized by a high clinical and genetic heterogeneity and a complex structure of several of the genes involved. A series of 71 patients including Usher patients previously screened by Sanger sequencing plus newly referred patients was studied. Ninety-eight percent of the variants previously identified by Sanger sequencing were found by next-generation sequencing (NGS). NGS proved to be efficient as it offers analysis of all relevant genes which is laborious to reach with Sanger sequencing. Among the 13 newly referred Usher patients, both mutations in the same gene were identified in 77% of cases (10 patients) and one candidate pathogenic variant in two additional patients. This work can be considered as pilot for implementing NGS for genetically heterogeneous diseases in clinical service. PMID:24498627

EM connectomics reveals axonal target variation in a sequence-generating network

PubMed Central

Narayanan, Rajeevan T; Svara, Fabian; Egger, Robert; Oberlaender, Marcel; Denk, Winfried; Long, Michael A

2017-01-01

The sequential activation of neurons has been observed in various areas of the brain, but in no case is the underlying network structure well understood. Here we examined the circuit anatomy of zebra finch HVC, a cortical region that generates sequences underlying the temporal progression of the song. We combined serial block-face electron microscopy with light microscopy to determine the cell types targeted by HVC(RA) neurons, which control song timing. Close to their soma, axons almost exclusively targeted inhibitory interneurons, consistent with what had been found with electrical recordings from pairs of cells. Conversely, far from the soma the targets were mostly other excitatory neurons, about half of these being other HVC(RA) cells. Both observations are consistent with the notion that the neural sequences that pace the song are generated by global synaptic chains in HVC embedded within local inhibitory networks. DOI: http://dx.doi.org/10.7554/eLife.24364.001 PMID:28346140

[Function of transport H+-ATPases in plant cell plasma and vacuolar membranes of maize under salt stress conditions and effect of adaptogenic preparations].

PubMed

Rybchenko, Zh I; Palladina, T O

2011-01-01

Participations of electrogenic H+-pumps of plasma and vacuolar membranes represented by E1-E2 and V-type H+-ATPases in plant cell adaptation to salt stress conditions has been studied by determination of their transport activities. Experiments were carried out on corn seedlings exposed during 1 or 10 days at 0.1 M NaCl. Preparations Methyure and Ivine were used by seed soaking at 10(-7) M. Plasma and vacuolar membrane fractions were isolated from corn seedling roots. In variants without NaCl a hydrolytical activity of plasma membrane H+-ATPase was increased with seedling age and its transport one was changed insignificantly, wherease the response of the weaker vacuolar H+-ATPase was opposite. NaCl exposition decreased hydrolytical activities of both H+-ATPases and increased their transport ones. These results demonstrated amplification of H+-pumps function especially represented by vacuolar H+-ATPase. Both preparations, Methyure mainly, caused a further increase of transport activity which was more expressed in NaCl variants. Obtained results showed the important role of these H+-pumps in plant adaptation under salt stress conditions realized by energetical maintenance of the secondary active Na+/H+ -antiporters which remove Na+ from cytoplasm.

A statistical approach to detection of copy number variations in PCR-enriched targeted sequencing data.

PubMed

Demidov, German; Simakova, Tamara; Vnuchkova, Julia; Bragin, Anton

2016-10-22

Multiplex polymerase chain reaction (PCR) is a common enrichment technique for targeted massive parallel sequencing (MPS) protocols. MPS is widely used in biomedical research and clinical diagnostics as the fast and accurate tool for the detection of short genetic variations. However, identification of larger variations such as structure variants and copy number variations (CNV) is still being a challenge for targeted MPS. Some approaches and tools for structural variants detection were proposed, but they have limitations and often require datasets of certain type, size and expected number of amplicons affected by CNVs. In the paper, we describe novel algorithm for high-resolution germinal CNV detection in the PCR-enriched targeted sequencing data and present accompanying tool. We have developed a machine learning algorithm for the detection of large duplications and deletions in the targeted sequencing data generated with PCR-based enrichment step. We have performed verification studies and established the algorithm's sensitivity and specificity. We have compared developed tool with other available methods applicable for the described data and revealed its higher performance. We showed that our method has high specificity and sensitivity for high-resolution copy number detection in targeted sequencing data using large cohort of samples.

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis1[OPEN

PubMed Central

Wang, Zhen-Yu; Gehring, Chris; Zhu, Jianhua; Li, Feng-Min; Zhu, Jian-Kang; Xiong, Liming

2015-01-01

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1. PMID:25416474

The formation of Anthocyanic Vacuolar Inclusions in Arabidopsis thaliana and implications for the sequestration of anthocyanin pigments.

PubMed

Pourcel, Lucille; Irani, Niloufer G; Lu, Yuhua; Riedl, Ken; Schwartz, Steve; Grotewold, Erich

2010-01-01

Anthocyanins are flavonoid pigments that accumulate in the large central vacuole of most plants. Inside the vacuole, anthocyanins can be found uniformly distributed or as part of sub-vacuolar pigment bodies, the Anthocyanic Vacuolar Inclusions (AVIs). Using Arabidopsis seedlings grown under anthocyanin-inductive conditions as a model to understand how AVIs are formed, we show here that the accumulation of AVIs strongly correlates with the formation of cyanidin 3-glucoside (C3G) and derivatives. Arabidopsis mutants that fail to glycosylate anthocyanidins at the 5-O position (5gt mutant) accumulate AVIs in almost every epidermal cell of the cotyledons, as compared to wild-type seedlings, where only a small fraction of the cells show AVIs. A similar phenomenon is observed when seedlings are treated with vanadate. Highlighting a role for autophagy in the formation of the AVIs, we show that various mutants that interfere with the autophagic process (atg mutants) display lower numbers of AVIs, in addition to a reduced accumulation of anthocyanins. Interestingly, vanadate increases the numbers of AVIs in the atg mutants, suggesting that several pathways might participate in AVI formation. Taken together, our results suggest novel mechanisms for the formation of sub-vacuolar compartments capable of accumulating anthocyanin pigments.

Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

PubMed Central

Ethier, Sylvain; Schmeing, T. Martin; Dostie, Josée; Pelletier, Jerry

2014-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5′NGG3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data. PMID:25275497

In vivo gene correction with targeted sequence substitution through microhomology-mediated end joining.

PubMed

Shin, Jeong Hong; Jung, Soobin; Ramakrishna, Suresh; Kim, Hyongbum Henry; Lee, Junwon

2018-07-07

Genome editing technology using programmable nucleases has rapidly evolved in recent years. The primary mechanism to achieve precise integration of a transgene is mainly based on homology-directed repair (HDR). However, an HDR-based genome-editing approach is less efficient than non-homologous end-joining (NHEJ). Recently, a microhomology-mediated end-joining (MMEJ)-based transgene integration approach was developed, showing feasibility both in vitro and in vivo. We expanded this method to achieve targeted sequence substitution (TSS) of mutated sequences with normal sequences using double-guide RNAs (gRNAs), and a donor template flanking the microhomologies and target sequence of the gRNAs in vitro and in vivo. Our method could realize more efficient sequence substitution than the HDR-based method in vitro using a reporter cell line, and led to the survival of a hereditary tyrosinemia mouse model in vivo. The proposed MMEJ-based TSS approach could provide a novel therapeutic strategy, in addition to HDR, to achieve gene correction from a mutated sequence to a normal sequence. Copyright © 2018 Elsevier Inc. All rights reserved.

Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system

PubMed Central

Chen, Xiugui; Lu, Xuke; Shu, Na; Wang, Shuai; Wang, Junjuan; Wang, Delong; Guo, Lixue; Ye, Wuwei

2017-01-01

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H+-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6–81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome. PMID:28287154

Noninvasive Prenatal Detection of Trisomy 21 by Targeted Semiconductor Sequencing: A Technical Feasibility Study.

PubMed

Xi, Yanwei; Arbabi, Aryan; McNaughton, Amy J M; Hamilton, Alison; Hull, Danna; Perras, Helene; Chiu, Tillie; Morrison, Shawna; Goldsmith, Claire; Creede, Emilie; Anger, Gregory J; Honeywell, Christina; Cloutier, Mireille; Macchio, Natasha; Kiss, Courtney; Liu, Xudong; Crocker, Susan; Davies, Gregory A; Brudno, Michael; Armour, Christine M

2017-01-01

To develop an alternate noninvasive prenatal testing method for the assessment of trisomy 21 (T21) using a targeted semiconductor sequencing approach. A customized AmpliSeq panel was designed with 1,067 primer pairs targeting specific regions on chromosomes 21, 18, 13, and others. A total of 235 samples, including 30 affected with T21, were sequenced with an Ion Torrent Proton sequencer, and a method was developed for assessing the probability of fetal aneuploidy via derivation of a risk score. Application of the derived risk score yields a bimodal distribution, with the affected samples clustering near 1.0 and the unaffected near 0. For a risk score cutoff of 0.345, above which all would be considered at "high risk," all 30 T21-positive pregnancies were correctly predicted to be affected, and 199 of the 205 non-T21 samples were correctly predicted. The average hands-on time spent on library preparation and sequencing was 19 h in total, and the average number of reads of sequence obtained was 3.75 million per sample. With the described targeted sequencing approach on the semiconductor platform using a custom-designed library and a probabilistic statistical approach, we have demonstrated the feasibility of an alternate method of assessment for fetal T21. © 2017 S. Karger AG, Basel.

Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

PubMed Central

Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

2012-01-01

PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25–35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein. PMID:25745458

Molecular characterization of oral squamous cell carcinoma using targeted next-generation sequencing.

PubMed

Er, Tze-Kiong; Wang, Yen-Yun; Chen, Chih-Chieh; Herreros-Villanueva, Marta; Liu, Ta-Chih; Yuan, Shyng-Shiou F

2015-10-01

Many genetic factors play an important role in the development of oral squamous cell carcinoma. The aim of this study was to assess the mutational profile in oral squamous cell carcinoma using formalin-fixed, paraffin-embedded tumors from a Taiwanese population by performing targeted sequencing of 26 cancer-associated genes that are frequently mutated in solid tumors. Next-generation sequencing was performed in 50 formalin-fixed, paraffin-embedded tumor specimens obtained from patients with oral squamous cell carcinoma. Genetic alterations in the 26 cancer-associated genes were detected using a deep sequencing (>1000X) approach. TP53, PIK3CA, MET, APC, CDH1, and FBXW7 were most frequently mutated genes. Most remarkably, TP53 mutations and PIK3CA mutations, which accounted for 68% and 18% of tumors, respectively, were more prevalent in a Taiwanese population. Other genes including MET (4%), APC (4%), CDH1 (2%), and FBXW7 (2%) were identified in our population. In summary, our study shows the feasibility of performing targeted sequencing using formalin-fixed, paraffin-embedded samples. Additionally, this study also reports the mutational landscape of oral squamous cell carcinoma in the Taiwanese population. We believe that this study will shed new light on fundamental aspects in understanding the molecular pathogenesis of oral squamous cell carcinoma and may aid in the development of new targeted therapies. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hygromycin B hypersensitive (hhy) mutants implicate an intact trans-Golgi and late endosome interface in efficient Tor1 vacuolar localization and TORC1 function.

PubMed

Ejzykowicz, Daniele E; Locken, Kristopher M; Ruiz, Fiona J; Manandhar, Surya P; Olson, Daniel K; Gharakhanian, Editte

2017-06-01

Saccharomyces cerevisiae vacuoles are functionally analogous to mammalian lysosomes. Both also serve as physical platforms for Tor Complex 1 (TORC1) signal transduction, the master regulator of cellular growth and proliferation. Hygromycin B is a eukaryotic translation inhibitor. We recently reported on hygromycin B hypersensitive (hhy) mutants that fail to grow at subtranslation inhibitory concentrations of the drug and exhibit vacuolar defects (Banuelos et al. in Curr Genet 56:121-137, 2010). Here, we show that hhy phenotype is not due to increased sensitivity to translation inhibition and establish a super HHY (s-HHY) subgroup of genes comprised of ARF1, CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52, and VPS54 that function exclusively or inclusively at trans-Golgi and late endosome interface. Live cell imaging of s-hhy mutants revealed that hygromycin B treatment disrupts vacuolar morphology and the localization of late endosome marker Pep12, but not that of late endosome-independent vacuolar SNARE Vam3. This, along with normal post-late endosome trafficking of the vital dye FM4-64, establishes that severe hypersensitivity to hygromycin B correlates specifically with compromised trans-Golgi and late endosome interface. We also show that Tor1p vacuolar localization and TORC1 anabolic functions, including growth promotion and phosphorylation of its direct substrate Sch9, are compromised in s-hhy mutants. Thus, an intact trans-Golgi and late endosome interface is a requisite for efficient Tor1 vacuolar localization and TORC1 function.

Discovery of Influenza A Virus Sequence Pairs and Their Combinations for Simultaneous Heterosubtypic Targeting that Hedge against Antiviral Resistance

PubMed Central

Lin, Jing; Pramono, Zacharias Aloysius Dwi; Maurer-Stroh, Sebastian

2016-01-01

The multiple circulating human influenza A virus subtypes coupled with the perpetual genomic mutations and segment reassortment events challenge the development of effective therapeutics. The capacity to drug most RNAs motivates the investigation on viral RNA targets. 123,060 segment sequences from 35,938 strains of the most prevalent subtypes also infecting humans–H1N1, 2009 pandemic H1N1, H3N2, H5N1 and H7N9, were used to identify 1,183 conserved RNA target sequences (≥15-mer) in the internal segments. 100% theoretical coverage in simultaneous heterosubtypic targeting is achieved by pairing specific sequences from the same segment (“Duals”) or from two segments (“Doubles”); 1,662 Duals and 28,463 Doubles identified. By combining specific Duals and/or Doubles to form a target graph wherein an edge connecting two vertices (target sequences) represents a Dual or Double, it is possible to hedge against antiviral resistance besides maintaining 100% heterosubtypic coverage. To evaluate the hedging potential, we define the hedge-factor as the minimum number of resistant target sequences that will render the graph to become resistant i.e. eliminate all the edges therein; a target sequence or a graph is considered resistant when it cannot achieve 100% heterosubtypic coverage. In an n-vertices graph (n ≥ 3), the hedge-factor is maximal (= n– 1) when it is a complete graph i.e. every distinct pair in a graph is either a Dual or Double. Computational analyses uncover an extensive number of complete graphs of different sizes. Monte Carlo simulations show that the mutation counts and time elapsed for a target graph to become resistant increase with the hedge-factor. Incidentally, target sequences which were reported to reduce virus titre in experiments are included in our target graphs. The identity of target sequence pairs for heterosubtypic targeting and their combinations for hedging antiviral resistance are useful toolkits to construct target graphs for

Robust Small Target Co-Detection from Airborne Infrared Image Sequences.

PubMed

Gao, Jingli; Wen, Chenglin; Liu, Meiqin

2017-09-29

In this paper, a novel infrared target co-detection model combining the self-correlation features of backgrounds and the commonality features of targets in the spatio-temporal domain is proposed to detect small targets in a sequence of infrared images with complex backgrounds. Firstly, a dense target extraction model based on nonlinear weights is proposed, which can better suppress background of images and enhance small targets than weights of singular values. Secondly, a sparse target extraction model based on entry-wise weighted robust principal component analysis is proposed. The entry-wise weight adaptively incorporates structural prior in terms of local weighted entropy, thus, it can extract real targets accurately and suppress background clutters efficiently. Finally, the commonality of targets in the spatio-temporal domain are used to construct target refinement model for false alarms suppression and target confirmation. Since real targets could appear in both of the dense and sparse reconstruction maps of a single frame, and form trajectories after tracklet association of consecutive frames, the location correlation of the dense and sparse reconstruction maps for a single frame and tracklet association of the location correlation maps for successive frames have strong ability to discriminate between small targets and background clutters. Experimental results demonstrate that the proposed small target co-detection method can not only suppress background clutters effectively, but also detect targets accurately even if with target-like interference.

The minimal amount of starting DNA for Agilent’s hybrid capture-based targeted massively parallel sequencing

PubMed Central

Chung, Jongsuk; Son, Dae-Soon; Jeon, Hyo-Jeong; Kim, Kyoung-Mee; Park, Gahee; Ryu, Gyu Ha; Park, Woong-Yang; Park, Donghyun

2016-01-01

Targeted capture massively parallel sequencing is increasingly being used in clinical settings, and as costs continue to decline, use of this technology may become routine in health care. However, a limited amount of tissue has often been a challenge in meeting quality requirements. To offer a practical guideline for the minimum amount of input DNA for targeted sequencing, we optimized and evaluated the performance of targeted sequencing depending on the input DNA amount. First, using various amounts of input DNA, we compared commercially available library construction kits and selected Agilent’s SureSelect-XT and KAPA Biosystems’ Hyper Prep kits as the kits most compatible with targeted deep sequencing using Agilent’s SureSelect custom capture. Then, we optimized the adapter ligation conditions of the Hyper Prep kit to improve library construction efficiency and adapted multiplexed hybrid selection to reduce the cost of sequencing. In this study, we systematically evaluated the performance of the optimized protocol depending on the amount of input DNA, ranging from 6.25 to 200 ng, suggesting the minimal input DNA amounts based on coverage depths required for specific applications. PMID:27220682

Detection of Somatic Mutations in Gastroenteropancreatic Neuroendocrine Tumors Using Targeted Deep Sequencing.

PubMed

Backman, Samuel; Norlén, Olov; Eriksson, Barbro; Skogseid, Britt; Stålberg, Peter; Crona, Joakim

2017-02-01

Mutations affecting the mechanistic target of rapamycin (MTOR) signalling pathway are frequent in human cancer and have been identified in up to 15% of pancreatic neuroendocrine tumours (NETs). Grade A evidence supports the efficacy of MTOR inhibition with everolimus in pancreatic NETs. Although a significant proportion of patients experience disease stabilization, only a minority will show objective tumour responses. It has been proposed that genomic mutations resulting in activation of MTOR signalling could be used to predict sensitivity to everolimus. Patients with NETs that underwent treatment with everolimus at our Institution were identified and those with available tumour tissue were selected for further analysis. Targeted next-generation sequencing (NGS) was used to re-sequence 22 genes that were selected on the basis of documented involvement in the MTOR signalling pathway or in the tumourigenesis of gastroenterpancreatic NETs. Radiological responses were documented using Response Evaluation Criteria in Solid Tumours. Six patients were identified, one had a partial response and four had stable disease. Sequencing of tumour tissue resulted in a median sequence depth of 667.1 (range=404-1301) with 1-fold coverage of 95.9-96.5% and 10-fold coverage of 87.6-92.2%. A total of 494 genetic variants were discovered, four of which were identified as pathogenic. All pathogenic variants were validated using Sanger sequencing and were found exclusively in menin 1 (MEN1) and death domain associated protein (DAXX) genes. No mutations in the MTOR pathway-related genes were observed. Targeted NGS is a feasible method with high diagnostic yield for genetic characterization of pancreatic NETs. A potential association between mutations in NETs and response to everolimus should be investigated by future studies. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

'2A-Like' Signal Sequences Mediating Translational Recoding: A Novel Form of Dual Protein Targeting.

PubMed

Roulston, Claire; Luke, Garry A; de Felipe, Pablo; Ruan, Lin; Cope, Jonathan; Nicholson, John; Sukhodub, Andriy; Tilsner, Jens; Ryan, Martin D

2016-08-01

We report the initial characterization of an N-terminal oligopeptide '2A-like' sequence that is able to function both as a signal sequence and as a translational recoding element. Owing to this translational recoding activity, two forms of nascent polypeptide are synthesized: (i) when 2A-mediated translational recoding has not occurred: the nascent polypeptide is fused to the 2A-like N-terminal signal sequence and the fusion translation product is targeted to the exocytic pathway, and, (ii) a translation product where 2A-mediated translational recoding has occurred: the 2A-like signal sequence is synthesized as a separate translation product and, therefore, the nascent (downstream) polypeptide lacks the 2A-like signal sequence and is localized to the cytoplasm. This type of dual-functional signal sequence results, therefore, in the partitioning of the translation products between the two sub-cellular sites and represents a newly described form of dual protein targeting. © 2016 The Authors. Traffic published by John Wiley & Sons Ltd.

Performance evaluation of Sanger sequencing for the diagnosis of primary hyperoxaluria and comparison with targeted next generation sequencing

PubMed Central

Williams, Emma L; Bagg, Eleanor A L; Mueller, Michael; Vandrovcova, Jana; Aitman, Timothy J; Rumsby, Gill

2015-01-01

Definitive diagnosis of primary hyperoxaluria (PH) currently utilizes sequential Sanger sequencing of the AGXT, GRPHR, and HOGA1 genes but efficacy is unproven. This analysis is time-consuming, relatively expensive, and delays in diagnosis and inappropriate treatment can occur if not pursued early in the diagnostic work-up. We reviewed testing outcomes of Sanger sequencing in 200 consecutive patient samples referred for analysis. In addition, the Illumina Truseq custom amplicon system was evaluated for paralleled next-generation sequencing (NGS) of AGXT,GRHPR, and HOGA1 in 90 known PH patients. AGXT sequencing was requested in all patients, permitting a diagnosis of PH1 in 50%. All remaining patients underwent targeted exon sequencing of GRHPR and HOGA1 with 8% diagnosed with PH2 and 8% with PH3. Complete sequencing of both GRHPR and HOGA1 was not requested in 25% of patients referred leaving their diagnosis in doubt. NGS analysis showed 98% agreement with Sanger sequencing and both approaches had 100% diagnostic specificity. Diagnostic sensitivity of Sanger sequencing was 98% and for NGS it was 97%. NGS has comparable diagnostic performance to Sanger sequencing for the diagnosis of PH and, if implemented, would screen for all forms of PH simultaneously ensuring prompt diagnosis at decreased cost. PMID:25629080

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease.

PubMed

Dilliott, Allison A; Farhan, Sali M K; Ghani, Mahdi; Sato, Christine; Liang, Eric; Zhang, Ming; McIntyre, Adam D; Cao, Henian; Racacho, Lemuel; Robinson, John F; Strong, Michael J; Masellis, Mario; Bulman, Dennis E; Rogaeva, Ekaterina; Lang, Anthony; Tartaglia, Carmela; Finger, Elizabeth; Zinman, Lorne; Turnbull, John; Freedman, Morris; Swartz, Rick; Black, Sandra E; Hegele, Robert A

2018-04-04

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Vacuolar H+-ATPase Protects Saccharomyces cerevisiae Cells against Ethanol-Induced Oxidative and Cell Wall Stresses

PubMed Central

Charoenbhakdi, Sirikarn; Dokpikul, Thanittra; Burphan, Thanawat; Techo, Todsapol

2016-01-01

ABSTRACT During fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H+-ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in Δvma2 and Δvma3 mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype of vma mutants resulted from severe cytosolic acidification. Interestingly, the vma mutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol. IMPORTANCE The yeast Saccharomyces cerevisiae has been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains

Vacuolar H+-ATPase Protects Saccharomyces cerevisiae Cells against Ethanol-Induced Oxidative and Cell Wall Stresses.

PubMed

Charoenbhakdi, Sirikarn; Dokpikul, Thanittra; Burphan, Thanawat; Techo, Todsapol; Auesukaree, Choowong

2016-05-15

During fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H(+)-ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in Δvma2 and Δvma3 mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype of vma mutants resulted from severe cytosolic acidification. Interestingly, the vma mutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol. The yeast Saccharomyces cerevisiae has been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains used in alcoholic

Targeted sequencing of plant genomes

Treesearch

Mark D. Huynh

2014-01-01

Next-generation sequencing (NGS) has revolutionized the field of genetics by providing a means for fast and relatively affordable sequencing. With the advancement of NGS, wholegenome sequencing (WGS) has become more commonplace. However, sequencing an entire genome is still not cost effective or even beneficial in all cases. In studies that do not require a whole-...

Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death1[OPEN

PubMed Central

Carmieli, Raanan; Mor, Avishai; Fluhr, Robert

2016-01-01

Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB. In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role. PMID:26884487

Non-Adjacent Consonant Sequence Patterns in English Target Words during the First-Word Period

ERIC Educational Resources Information Center

Aoyama, Katsura; Davis, Barbara L.

2017-01-01

The goal of this study was to investigate non-adjacent consonant sequence patterns in target words during the first-word period in infants learning American English. In the spontaneous speech of eighteen participants, target words with a Consonant-Vowel-Consonant (C[subscript 1]VC[subscript 2]) shape were analyzed. Target words were grouped into…

StarScan: a web server for scanning small RNA targets from degradome sequencing data.

PubMed

Liu, Shun; Li, Jun-Hao; Wu, Jie; Zhou, Ke-Ren; Zhou, Hui; Yang, Jian-Hua; Qu, Liang-Hu

2015-07-01

Endogenous small non-coding RNAs (sRNAs), including microRNAs, PIWI-interacting RNAs and small interfering RNAs, play important gene regulatory roles in animals and plants by pairing to the protein-coding and non-coding transcripts. However, computationally assigning these various sRNAs to their regulatory target genes remains technically challenging. Recently, a high-throughput degradome sequencing method was applied to identify biologically relevant sRNA cleavage sites. In this study, an integrated web-based tool, StarScan (sRNA target Scan), was developed for scanning sRNA targets using degradome sequencing data from 20 species. Given a sRNA sequence from plants or animals, our web server performs an ultrafast and exhaustive search for potential sRNA-target interactions in annotated and unannotated genomic regions. The interactions between small RNAs and target transcripts were further evaluated using a novel tool, alignScore. A novel tool, degradomeBinomTest, was developed to quantify the abundance of degradome fragments located at the 9-11th nucleotide from the sRNA 5' end. This is the first web server for discovering potential sRNA-mediated RNA cleavage events in plants and animals, which affords mechanistic insights into the regulatory roles of sRNAs. The StarScan web server is available at http://mirlab.sysu.edu.cn/starscan/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Vacuolar processing enzyme: an executor of plant cell death.

PubMed

Hara-Nishimura, Ikuko; Hatsugai, Noriyuki; Nakaune, Satoru; Kuroyanagi, Miwa; Nishimura, Mikio

2005-08-01

Apoptotic cell death in animals is regulated by cysteine proteinases called caspases. Recently, vacuolar processing enzyme (VPE) was identified as a plant caspase. VPE deficiency prevents cell death during hypersensitive response and cell death of limited cell layers at the early stage of embryogenesis. Because plants do not have macrophages, dying cells must degrade their materials by themselves. VPE plays an essential role in the regulation of the lytic system of plants during the processes of defense and development. VPE is localized in the vacuoles, unlike animal caspases, which are localized in the cytosol. Thus, plants might have evolved a regulated cellular suicide strategy that, unlike animal apoptosis, is mediated by VPE and the vacuoles.

Vacuolar status and water relations in embryonic axes of recalcitrant Aesculus hippocastanum seeds during stratification and early germination.

PubMed

Obroucheva, Natalie V; Lityagina, Snezhana V; Novikova, Galina V; Sin'kevich, Irina A

2012-01-01

In tropical recalcitrant seeds, their rapid transition from shedding to germination at high hydration level is of physiological interest but difficult to study because of the time constraint. In recalcitrant horse chestnut seeds produced in central Russia, this transition is much longer and extends through dormancy and dormancy release. This extended time period permits studies of the water relations in embryonic axes during the long recalcitrant period in terms of vacuolar status and water transport. Horse chestnut (Aesculus hippocastanum) seeds sampled in Moscow were stratified in cold wet sand for 4 months. Vacuole presence and development in embryonic axes were examined by vital staining, light and electron microscopy. Aquaporins and vacuolar H(+)-ATPase were identified immunochemically. Water channel operation was tested by water inflow rate. Vacuolar acid invertase was estimated in terms of activity and electrophoretic properties. Throughout the long recalcitrant period after seed shedding, cells of embryonic axes maintained active vacuoles and a high water content. Preservation of enzyme machinery in vacuoles was evident from retention of invertase activity, substrate specificity, molecular mass and subunit composition. Plasmalemma and tonoplast aquaporins and the E subunit of vacuolar H(+)-ATPase were also present. In non-dormant seeds prior to growth initiation, vacuoles enlarged at first in hypocotyls, and then in radicles, with their biogenesis being similar. Vacuolation was accompanied by increasing invertase activity, leading to sugar accumulation and active osmotic functioning. After growth initiation, vacuole enlargement was favoured by enhanced water inflow through water channels formed by aquaporins. Maintenance of high water content and desiccation sensitivity, as well as preservation of active vacuoles in embryonic axes after shedding, can be considered a specific feature of recalcitrant seeds, overlooked when studying tropical recalcitrants due

Vacuolar status and water relations in embryonic axes of recalcitrant Aesculus hippocastanum seeds during stratification and early germination

PubMed Central

Obroucheva, Natalie V.; Lityagina, Snezhana V.; Novikova, Galina V.; Sin'kevich, Irina A.

2012-01-01

Backgrounds and aims In tropical recalcitrant seeds, their rapid transition from shedding to germination at high hydration level is of physiological interest but difficult to study because of the time constraint. In recalcitrant horse chestnut seeds produced in central Russia, this transition is much longer and extends through dormancy and dormancy release. This extended time period permits studies of the water relations in embryonic axes during the long recalcitrant period in terms of vacuolar status and water transport. Methodology Horse chestnut (Aesculus hippocastanum) seeds sampled in Moscow were stratified in cold wet sand for 4 months. Vacuole presence and development in embryonic axes were examined by vital staining, light and electron microscopy. Aquaporins and vacuolar H+-ATPase were identified immunochemically. Water channel operation was tested by water inflow rate. Vacuolar acid invertase was estimated in terms of activity and electrophoretic properties. Principal results Throughout the long recalcitrant period after seed shedding, cells of embryonic axes maintained active vacuoles and a high water content. Preservation of enzyme machinery in vacuoles was evident from retention of invertase activity, substrate specificity, molecular mass and subunit composition. Plasmalemma and tonoplast aquaporins and the E subunit of vacuolar H+-ATPase were also present. In non-dormant seeds prior to growth initiation, vacuoles enlarged at first in hypocotyls, and then in radicles, with their biogenesis being similar. Vacuolation was accompanied by increasing invertase activity, leading to sugar accumulation and active osmotic functioning. After growth initiation, vacuole enlargement was favoured by enhanced water inflow through water channels formed by aquaporins. Conclusions Maintenance of high water content and desiccation sensitivity, as well as preservation of active vacuoles in embryonic axes after shedding, can be considered a specific feature of recalcitrant

Possible roles of vacuolar H+-ATPase and mitochondrial function in tolerance to air-drying stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains.

PubMed

Shima, Jun; Ando, Akira; Takagi, Hiroshi

2008-03-01

Yeasts used in bread making are exposed to air-drying stress during dried yeast production processes. To clarify the genes required for air-drying tolerance, we performed genome-wide screening using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 278 gene deletions responsible for air-drying sensitivity. These genes were classified based on their cellular function and on the localization of their gene products. The results showed that the genes required for air-drying tolerance were frequently involved in mitochondrial functions and in connection with vacuolar H(+)-ATPase, which plays a role in vacuolar acidification. To determine the role of vacuolar acidification in air-drying stress tolerance, we monitored intracellular pH. The results showed that intracellular acidification was induced during air-drying and that this acidification was amplified in a deletion mutant of the VMA2 gene encoding a component of vacuolar H(+)-ATPase, suggesting that vacuolar H(+)-ATPase helps maintain intracellular pH homeostasis, which is affected by air-drying stress. To determine the effects of air-drying stress on mitochondria, we analysed the mitochondrial membrane potential under air-drying stress conditions using MitoTracker. The results showed that mitochondria were extremely sensitive to air-drying stress, suggesting that a mitochondrial function is required for tolerance to air-drying stress. We also analysed the correlation between oxidative-stress sensitivity and air-drying-stress sensitivity. The results suggested that oxidative stress is a critical determinant of sensitivity to air-drying stress, although ROS-scavenging systems are not necessary for air-drying stress tolerance. (c) 2008 John Wiley & Sons, Ltd.

Targeted next generation sequencing for molecular diagnosis of Usher syndrome.

PubMed

Aparisi, María J; Aller, Elena; Fuster-García, Carla; García-García, Gema; Rodrigo, Regina; Vázquez-Manrique, Rafael P; Blanco-Kelly, Fiona; Ayuso, Carmen; Roux, Anne-Françoise; Jaijo, Teresa; Millán, José M

2014-11-18

Usher syndrome is an autosomal recessive disease that associates sensorineural hearing loss, retinitis pigmentosa and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous. To date, 10 genes have been associated with the disease, making its molecular diagnosis based on Sanger sequencing, expensive and time-consuming. Consequently, the aim of the present study was to develop a molecular diagnostics method for Usher syndrome, based on targeted next generation sequencing. A custom HaloPlex panel for Illumina platforms was designed to capture all exons of the 10 known causative Usher syndrome genes (MYO7A, USH1C, CDH23, PCDH15, USH1G, CIB2, USH2A, GPR98, DFNB31 and CLRN1), the two Usher syndrome-related genes (HARS and PDZD7) and the two candidate genes VEZT and MYO15A. A cohort of 44 patients suffering from Usher syndrome was selected for this study. This cohort was divided into two groups: a test group of 11 patients with known mutations and another group of 33 patients with unknown mutations. Forty USH patients were successfully sequenced, 8 USH patients from the test group and 32 patients from the group composed of USH patients without genetic diagnosis. We were able to detect biallelic mutations in one USH gene in 22 out of 32 USH patients (68.75%) and to identify 79.7% of the expected mutated alleles. Fifty-three different mutations were detected. These mutations included 21 missense, 8 nonsense, 9 frameshifts, 9 intronic mutations and 6 large rearrangements. Targeted next generation sequencing allowed us to detect both point mutations and large rearrangements in a single experiment, minimizing the economic cost of the study, increasing the detection ratio of the genetic cause of the disease and improving the genetic diagnosis of Usher syndrome patients.

The Malarial Host-Targeting Signal Is Conserved in the Irish Potato Famine Pathogen

PubMed Central

Liolios, Konstantinos; Win, Joe; Kanneganti, Thirumala-Devi; Young, Carolyn; Kamoun, Sophien; Haldar, Kasturi

2006-01-01

Animal and plant eukaryotic pathogens, such as the human malaria parasite Plasmodium falciparum and the potato late blight agent Phytophthora infestans, are widely divergent eukaryotic microbes. Yet they both produce secretory virulence and pathogenic proteins that alter host cell functions. In P. falciparum, export of parasite proteins to the host erythrocyte is mediated by leader sequences shown to contain a host-targeting (HT) motif centered on an RxLx (E, D, or Q) core: this motif appears to signify a major pathogenic export pathway with hundreds of putative effectors. Here we show that a secretory protein of P. infestans, which is perceived by plant disease resistance proteins and induces hypersensitive plant cell death, contains a leader sequence that is equivalent to the Plasmodium HT-leader in its ability to export fusion of green fluorescent protein (GFP) from the P. falciparum parasite to the host erythrocyte. This export is dependent on an RxLR sequence conserved in P. infestans leaders, as well as in leaders of all ten secretory oomycete proteins shown to function inside plant cells. The RxLR motif is also detected in hundreds of secretory proteins of P. infestans, Phytophthora sojae, and Phytophthora ramorum and has high value in predicting host-targeted leaders. A consensus motif further reveals E/D residues enriched within ~25 amino acids downstream of the RxLR, which are also needed for export. Together the data suggest that in these plant pathogenic oomycetes, a consensus HT motif may reside in an extended sequence of ~25–30 amino acids, rather than in a short linear sequence. Evidence is presented that although the consensus is much shorter in P. falciparum, information sufficient for vacuolar export is contained in a region of ~30 amino acids, which includes sequences flanking the HT core. Finally, positional conservation between Phytophthora RxLR and P. falciparum RxLx (E, D, Q) is consistent with the idea that the context of their

A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes

PubMed Central

Herbold, Craig W.; Pelikan, Claus; Kuzyk, Orest; Hausmann, Bela; Angel, Roey; Berry, David; Loy, Alexander

2015-01-01

High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse, and high quality sets of amplicon sequence data for modern studies in microbial ecology. PMID:26236305

Choline but not its derivative betaine blocks slow vacuolar channels in the halophyte Chenopodium quinoa: implications for salinity stress responses.

PubMed

Pottosin, Igor; Bonales-Alatorre, Edgar; Shabala, Sergey

2014-11-03

Activity of tonoplast slow vacuolar (SV, or TPC1) channels has to be under a tight control, to avoid undesirable leak of cations stored in the vacuole. This is particularly important for salt-grown plants, to ensure efficient vacuolar Na(+) sequestration. In this study we show that choline, a cationic precursor of glycine betaine, efficiently blocks SV channels in leaf and root vacuoles of the two chenopods, Chenopodium quinoa (halophyte) and Beta vulgaris (glycophyte). At the same time, betaine and proline, two major cytosolic organic osmolytes, have no significant effect on SV channel activity. Physiological implications of these findings are discussed. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes.

PubMed

Petrezselyova, Silvia; Dominguez, Angel; Herynkova, Pavla; Macias, Juan F; Sychrova, Hana

2013-10-01

Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters. Copyright © 2013 John Wiley & Sons, Ltd.

Method to amplify variable sequences without imposing primer sequences

DOEpatents

Bradbury, Andrew M.; Zeytun, Ahmet

2006-11-14

The present invention provides methods of amplifying target sequences without including regions flanking the target sequence in the amplified product or imposing amplification primer sequences on the amplified product. Also provided are methods of preparing a library from such amplified target sequences.

Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

PubMed Central

Stransky, Laura A.; Forgac, Michael

2015-01-01

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly. PMID:26378229

Allelic differences in a vacuolar invertase affect Arabidopsis growth at early plant development.

PubMed

Leskow, Carla Coluccio; Kamenetzky, Laura; Dominguez, Pia Guadalupe; Díaz Zirpolo, José Antonio; Obata, Toshihiro; Costa, Hernán; Martí, Marcelo; Taboga, Oscar; Keurentjes, Joost; Sulpice, Ronan; Ishihara, Hirofumi; Stitt, Mark; Fernie, Alisdair Robert; Carrari, Fernando

2016-07-01

Improving carbon fixation in order to enhance crop yield is a major goal in plant sciences. By quantitative trait locus (QTL) mapping, it has been demonstrated that a vacuolar invertase (vac-Inv) plays a key role in determining the radical length in Arabidopsis. In this model, variation in vac-Inv activity was detected in a near isogenic line (NIL) population derived from a cross between two divergent accessions: Landsberg erecta (Ler) and Cape Verde Island (CVI), with the CVI allele conferring both higher Inv activity and longer radicles. The aim of the current work is to understand the mechanism(s) underlying this QTL by analyzing structural and functional differences of vac-Inv from both accessions. Relative transcript abundance analyzed by quantitative real-time PCR (qRT-PCR) showed similar expression patterns in both accessions; however, DNA sequence analyses revealed several polymorphisms that lead to changes in the corresponding protein sequence. Moreover, activity assays revealed higher vac-Inv activity in genotypes carrying the CVI allele than in those carrying the Ler allele. Analyses of purified recombinant proteins showed a similar K m for both alleles and a slightly higher V max for that of Ler. Treatment of plant extracts with foaming to release possible interacting Inv inhibitory protein(s) led to a large increase in activity for the Ler allele, but no changes for genotypes carrying the CVI allele. qRT-PCR analyses of two vac-Inv inhibitors in seedlings from parental and NIL genotypes revealed different expression patterns. Taken together, these results demonstrate that the vac-Inv QTL affects root biomass accumulation and also carbon partitioning through a differential regulation of vac-Inv inhibitors at the mRNA level. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Computer program for the IBM personal computer which searches for approximate matches to short oligonucleotide sequences in long target DNA sequences.

PubMed Central

Myers, E W; Mount, D W

1986-01-01

We describe a program which may be used to find approximate matches to a short predefined DNA sequence in a larger target DNA sequence. The program predicts the usefulness of specific DNA probes and sequencing primers and finds nearly identical sequences that might represent the same regulatory signal. The program is written in the C programming language and will run on virtually any computer system with a C compiler, such as the IBM/PC and other computers running under the MS/DOS and UNIX operating systems. The program has been integrated into an existing software package for the IBM personal computer (see article by Mount and Conrad, this volume). Some examples of its use are given. PMID:3753785

Identifying mRNA sequence elements for target recognition by human Argonaute proteins

PubMed Central

Li, Jingjing; Kim, TaeHyung; Nutiu, Razvan; Ray, Debashish; Hughes, Timothy R.; Zhang, Zhaolei

2014-01-01

It is commonly known that mammalian microRNAs (miRNAs) guide the RNA-induced silencing complex (RISC) to target mRNAs through the seed-pairing rule. However, recent experiments that coimmunoprecipitate the Argonaute proteins (AGOs), the central catalytic component of RISC, have consistently revealed extensive AGO-associated mRNAs that lack seed complementarity with miRNAs. We herein test the hypothesis that AGO has its own binding preference within target mRNAs, independent of guide miRNAs. By systematically analyzing the data from in vivo cross-linking experiments with human AGOs, we have identified a structurally accessible and evolutionarily conserved region (∼10 nucleotides in length) that alone can accurately predict AGO–mRNA associations, independent of the presence of miRNA binding sites. Within this region, we further identified an enriched motif that was replicable on independent AGO-immunoprecipitation data sets. We used RNAcompete to enumerate the RNA-binding preference of human AGO2 to all possible 7-mer RNA sequences and validated the AGO motif in vitro. These findings reveal a novel function of AGOs as sequence-specific RNA-binding proteins, which may aid miRNAs in recognizing their targets with high specificity. PMID:24663241

Clinical Validation of Copy Number Variant Detection from Targeted Next-Generation Sequencing Panels.

PubMed

Kerkhof, Jennifer; Schenkel, Laila C; Reilly, Jack; McRobbie, Sheri; Aref-Eshghi, Erfan; Stuart, Alan; Rupar, C Anthony; Adams, Paul; Hegele, Robert A; Lin, Hanxin; Rodenhiser, David; Knoll, Joan; Ainsworth, Peter J; Sadikovic, Bekim

2017-11-01

Next-generation sequencing (NGS) technology has rapidly replaced Sanger sequencing in the assessment of sequence variations in clinical genetics laboratories. One major limitation of current NGS approaches is the ability to detect copy number variations (CNVs) approximately >50 bp. Because these represent a major mutational burden in many genetic disorders, parallel CNV assessment using alternate supplemental methods, along with the NGS analysis, is normally required, resulting in increased labor, costs, and turnaround times. The objective of this study was to clinically validate a novel CNV detection algorithm using targeted clinical NGS gene panel data. We have applied this approach in a retrospective cohort of 391 samples and a prospective cohort of 2375 samples and found a 100% sensitivity (95% CI, 89%-100%) for 37 unique events and a high degree of specificity to detect CNVs across nine distinct targeted NGS gene panels. This NGS CNV pipeline enables stand-alone first-tier assessment for CNV and sequence variants in a clinical laboratory setting, dispensing with the need for parallel CNV analysis using classic techniques, such as microarray, long-range PCR, or multiplex ligation-dependent probe amplification. This NGS CNV pipeline can also be applied to the assessment of complex genomic regions, including pseudogenic DNA sequences, such as the PMS2CL gene, and to mitochondrial genome heteroplasmy detection. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Integration of targeted sequencing and NIPT into clinical practice in a Chinese family with maple syrup urine disease.

PubMed

You, Yanqin; Sun, Yan; Li, Xuchao; Li, Yali; Wei, Xiaoming; Chen, Fang; Ge, Huijuan; Lan, Zhangzhang; Zhu, Qian; Tang, Ying; Wang, Shujuan; Gao, Ya; Jiang, Fuman; Song, Jiaping; Shi, Quan; Zhu, Xuan; Mu, Feng; Dong, Wei; Gao, Vince; Jiang, Hui; Yi, Xin; Wang, Wei; Gao, Zhiying

2014-08-01

This article demonstrates a prominent noninvasive prenatal approach to assist the clinical diagnosis of a single-gene disorder disease, maple syrup urine disease, using targeted sequencing knowledge from the affected family. The method reported here combines novel mutant discovery in known genes by targeted massively parallel sequencing with noninvasive prenatal testing. By applying this new strategy, we successfully revealed novel mutations in the gene BCKDHA (Ex2_4dup and c.392A>G) in this Chinese family and developed a prenatal haplotype-assisted approach to noninvasively detect the genotype of the fetus (transmitted from both parents). This is the first report of integration of targeted sequencing and noninvasive prenatal testing into clinical practice. Our study has demonstrated that this massively parallel sequencing-based strategy can potentially be used for single-gene disorder diagnosis in the future.

Genetic diagnosis of familial hypercholesterolaemia by targeted next-generation sequencing

PubMed Central

Maglio, C; Mancina, R M; Motta, B M; Stef, M; Pirazzi, C; Palacios, L; Askaryar, N; Borén, J; Wiklund, O; Romeo, S

2014-01-01

Maglio C., Mancina R. M., Motta B. M., Stef M., Pirazzi C., Palacios L., Askaryar N., Borén J., Wiklund O., Romeo S. (University of Gothenburg, Gothenburg, Sweden; University Magna Graecia of Catanzaro, Italy; University of Milan, Italy; Progenika Biopharma SA, Derio, Spain). Genetic diagnosis of familial hypercholesterolaemia by targeted next-generation sequencing. Objectives The aim of this study was to combine clinical criteria and next-generation sequencing (pyrosequencing) to establish a diagnosis of familial hypercholesterolaemia (FH). Design, setting and subjects A total of 77 subjects with a Dutch Lipid Clinic Network score of ≥3 (possible, probable or definite FH clinical diagnosis) were recruited from the Lipid Clinic at Sahlgrenska Hospital, Gothenburg, Sweden. Next-generation sequencing was performed in all subjects using SEQPRO LIPO RS, a kit that detects mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9) and LDLR adapter protein 1 (LDLRAP1) genes; copy-number variations in the LDLR gene were also examined. Results A total of 26 mutations were detected in 50 subjects (65% success rate). Amongst these, 23 mutations were in the LDLR gene, two in the APOB gene and one in the PCSK9 gene. Four mutations with unknown pathogenicity were detected in LDLR. Of these, three mutations (Gly505Asp, Ile585Thr and Gln660Arg) have been previously reported in subjects with FH, but their pathogenicity has not been proved. The fourth, a mutation in LDLR affecting a splicing site (exon 6–intron 6) has not previously been reported; it was found to segregate with high cholesterol levels in the family of the proband. Conclusions Using a combination of clinical criteria and targeted next-generation sequencing, we have achieved FH diagnosis with a high success rate. Furthermore, we identified a new splicing-site mutation in the LDLR gene. PMID:24785115

A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape.

PubMed

Quereda, Juan J; Pizarro-Cerdá, Javier; Balestrino, Damien; Bobard, Alexandre; Danckaert, Anne; Aulner, Nathalie; Shorte, Spencer; Enninga, Jost; Cossart, Pascale

2016-01-01

Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Colorimetric biosensing of targeted gene sequence using dual nanoparticle platforms

PubMed Central

Thavanathan, Jeevan; Huang, Nay Ming; Thong, Kwai Lin

2015-01-01

We have developed a colorimetric biosensor using a dual platform of gold nanoparticles and graphene oxide sheets for the detection of Salmonella enterica. The presence of the invA gene in S. enterica causes a change in color of the biosensor from its original pinkish-red to a light purplish solution. This occurs through the aggregation of the primary gold nanoparticles–conjugated DNA probe onto the surface of the secondary graphene oxide–conjugated DNA probe through DNA hybridization with the targeted DNA sequence. Spectrophotometry analysis showed a shift in wavelength from 525 nm to 600 nm with 1 μM of DNA target. Specificity testing revealed that the biosensor was able to detect various serovars of the S. enterica while no color change was observed with the other bacterial species. Sensitivity testing revealed the limit of detection was at 1 nM of DNA target. This proves the effectiveness of the biosensor in the detection of S. enterica through DNA hybridization. PMID:25897217

Sequence-based design of bioactive small molecules that target precursor microRNAs.

PubMed

Velagapudi, Sai Pradeep; Gallo, Steven M; Disney, Matthew D

2014-04-01

Oligonucleotides are designed to target RNA using base pairing rules, but they can be hampered by poor cellular delivery and nonspecific stimulation of the immune system. Small molecules are preferred as lead drugs or probes but cannot be designed from sequence. Herein, we describe an approach termed Inforna that designs lead small molecules for RNA from solely sequence. Inforna was applied to all human microRNA hairpin precursors, and it identified bioactive small molecules that inhibit biogenesis by binding nuclease-processing sites (44% hit rate). Among 27 lead interactions, the most avid interaction is between a benzimidazole (1) and precursor microRNA-96. Compound 1 selectively inhibits biogenesis of microRNA-96, upregulating a protein target (FOXO1) and inducing apoptosis in cancer cells. Apoptosis is ablated when FOXO1 mRNA expression is knocked down by an siRNA, validating compound selectivity. Markedly, microRNA profiling shows that 1 only affects microRNA-96 biogenesis and is at least as selective as an oligonucleotide.

Sequence-based design of bioactive small molecules that target precursor microRNAs

PubMed Central

Velagapudi, Sai Pradeep; Gallo, Steven M.; Disney, Matthew D.

2014-01-01

Oligonucleotides are designed to target RNA using base pairing rules, however, they are hampered by poor cellular delivery and non-specific stimulation of the immune system. Small molecules are preferred as lead drugs or probes, but cannot be designed from sequence. Herein, we describe an approach termed Inforna that designs lead small molecules for RNA from solely sequence. Inforna was applied to all human microRNA precursors and identified bioactive small molecules that inhibit biogenesis by binding to nuclease processing sites (41% hit rate). Amongst 29 lead interactions, the most avid interaction is between a benzimidazole (1) and precursor microRNA-96. Compound 1 selectively inhibits biogenesis of microRNA-96, upregulating a protein target (FOXO1) and inducing apoptosis in cancer cells. Apoptosis is ablated when FOXO1 mRNA expression is knocked down by an siRNA, validating compound selectivity. Importantly, microRNA profiling shows that 1 only significantly effects microRNA-96 biogenesis and is more selective than an oligonucleotide. PMID:24509821

Two-Way Gold Nanoparticle Label-Free Sensing of Specific Sequence and Small Molecule Targets Using Switchable Concatemers.

PubMed

Zhu, Longjiao; Shao, Xiangli; Luo, Yunbo; Huang, Kunlung; Xu, Wentao

2017-05-19

A two-way colorimetric biosensor based on unmodified gold nanoparticles (GNPs) and a switchable double-stranded DNA (dsDNA) concatemer have been demonstrated. Two hairpin probes (H1 and H2) were first designed that provided the fuels to assemble the dsDNA concatemers via hybridization chain reaction (HCR). A functional hairpin (FH) was rationally designed to recognize the target sequences. All the hairpins contained a single-stranded DNA (ssDNA) loop and sticky end to prevent GNPs from salt-induced aggregation. In the presence of target sequence, the capture probe blocked in the FH recognizes the target to form a duplex DNA, which causes the release of the initiator probe by FH conformational change. This process then starts the alternate-opening of H1 and H2 through HCR, and dsDNA concatemers grow from the target sequence. As a result, unmodified GNPs undergo salt-induced aggregation because the formed dsDNA concatemers are stiffer and provide less stabilization. A light purple-to-blue color variation was observed in the bulk solution, termed the light-off sensing way. Furthermore, H1 ingeniously inserted an aptamer sequence to generate dsDNA concatemers with multiple small molecule binding sites. In the presence of small molecule targets, concatemers can be disassembled into mixtures with ssDNA sticky ends. A blue-to-purple reverse color variation was observed due to the regeneration of the ssDNA, termed the light-on way. The two-way biosensor can detect both nucleic acids and small molecule targets with one sensing device. This switchable sensing element is label-free, enzyme-free, and sophisticated-instrumentation-free. The detection limits of both targets were below nanomolar.

Captured metagenomics: large-scale targeting of genes based on ‘sequence capture’ reveals functional diversity in soils

PubMed Central

Manoharan, Lokeshwaran; Kushwaha, Sandeep K.; Hedlund, Katarina; Ahrén, Dag

2015-01-01

Microbial enzyme diversity is a key to understand many ecosystem processes. Whole metagenome sequencing (WMG) obtains information on functional genes, but it is costly and inefficient due to large amount of sequencing that is required. In this study, we have applied a captured metagenomics technique for functional genes in soil microorganisms, as an alternative to WMG. Large-scale targeting of functional genes, coding for enzymes related to organic matter degradation, was applied to two agricultural soil communities through captured metagenomics. Captured metagenomics uses custom-designed, hybridization-based oligonucleotide probes that enrich functional genes of interest in metagenomic libraries where only probe-bound DNA fragments are sequenced. The captured metagenomes were highly enriched with targeted genes while maintaining their target diversity and their taxonomic distribution correlated well with the traditional ribosomal sequencing. The captured metagenomes were highly enriched with genes related to organic matter degradation; at least five times more than similar, publicly available soil WMG projects. This target enrichment technique also preserves the functional representation of the soils, thereby facilitating comparative metagenomics projects. Here, we present the first study that applies the captured metagenomics approach in large scale, and this novel method allows deep investigations of central ecosystem processes by studying functional gene abundances. PMID:26490729

Heterologous mitochondrial targeting sequences can deliver functional proteins into mitochondria.

PubMed

Marcus, Dana; Lichtenstein, Michal; Cohen, Natali; Hadad, Rita; Erlich-Hadad, Tal; Greif, Hagar; Lorberboum-Galski, Haya

2016-12-01

Mitochondrial Targeting Sequences (MTSs) are responsible for trafficking nuclear-encoded proteins into mitochondria. Once entering the mitochondria, the MTS is recognized and cleaved off. Some MTSs are long and undergo two-step processing, as in the case of the human frataxin (FXN) protein (80aa), implicated in Friedreich's ataxia (FA). Therefore, we chose the FXN protein to examine whether nuclear-encoded mitochondrial proteins can efficiently be targeted via a heterologous MTS (hMTS) and deliver a functional protein into mitochondria. We examined three hMTSs; that of citrate synthase (cs), lipoamide deydrogenase (LAD) and C6ORF66 (ORF), as classically MTS sequences, known to be removed by one-step processing, to deliver FXN into mitochondria, in the form of fusion proteins. We demonstrate that using hMTSs for delivering FXN results in the production of 4-5-fold larger amounts of the fusion proteins, and at 4-5-fold higher concentrations. Moreover, hMTSs delivered a functional FXN protein into the mitochondria even more efficiently than the native MTSfxn, as evidenced by the rescue of FA patients' cells from oxidative stress; demonstrating a 18%-54% increase in cell survival; and a 13%-33% increase in ATP levels, as compared to the fusion protein carrying the native MTS. One fusion protein with MTScs increased aconitase activity within patients' cells, by 400-fold. The implications form our studies are of vast importance for both basic and translational research of mitochondrial proteins as any mitochondrial protein can be delivered efficiently by an hMTS. Moreover, effective targeting of functional proteins is important for restoration of mitochondrial function and treatment of related disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

Relationship of the Membrane ATPase from Halobacterium saccharovorum to Vacuolar ATPases

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga; Bowman, Emma J.; Hochstein, Lawrence I.

1991-01-01

Polyclonal antiserum against subunit A (67 kDa) of the vacuolar ATPase from Neurospora crassa reacted with subunit I (87 kDa) from a membrane ATPase of the extremely halophilic archaebacterium Halobacterium saccharovorum. The halobacterial ATPase was inhibited by nitrate and N-ethylmaleimide; the extent of the latter inhibition was diminished in the presence of adenosine di- or triphosphates. 4-Chloro-7-nitrobenzofurazan in- hibited the hatobacterial ATPase also in a nucleotide- protectable manner; the bulk of inhibitor was associated with subunit II (60 kDa). The data suggested that this halobacterial ATPase may have conserved structural features from both the vacuotar and the F-type ATPases.

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis

PubMed Central

de Vries, Michel

2016-01-01

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development. PMID:26977085

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis.

PubMed

Verweij, Walter; Spelt, Cornelis E; Bliek, Mattijs; de Vries, Michel; Wit, Niek; Faraco, Marianna; Koes, Ronald; Quattrocchio, Francesca M

2016-03-01

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development. © 2016 American Society of Plant Biologists. All rights reserved.

SCFTIR1/AFB-auxin signalling regulates PIN vacuolar trafficking and auxin fluxes during root gravitropism

PubMed Central

Baster, Paweł; Robert, Stéphanie; Kleine-Vehn, Jürgen; Vanneste, Steffen; Kania, Urszula; Grunewald, Wim; De Rybel, Bert; Beeckman, Tom; Friml, Jiří

2013-01-01

The distribution of the phytohormone auxin regulates many aspects of plant development including growth response to gravity. Gravitropic root curvature involves coordinated and asymmetric cell elongation between the lower and upper side of the root, mediated by differential cellular auxin levels. The asymmetry in the auxin distribution is established and maintained by a spatio-temporal regulation of the PIN-FORMED (PIN) auxin transporter activity. We provide novel insights into the complex regulation of PIN abundance and activity during root gravitropism. We show that PIN2 turnover is differentially regulated on the upper and lower side of gravistimulated roots by distinct but partially overlapping auxin feedback mechanisms. In addition to regulating transcription and clathrin-mediated internalization, auxin also controls PIN abundance at the plasma membrane by promoting their vacuolar targeting and degradation. This effect of elevated auxin levels requires the activity of SKP-Cullin-F-boxTIR1/AFB (SCFTIR1/AFB)-dependent pathway. Importantly, also suboptimal auxin levels mediate PIN degradation utilizing the same signalling pathway. These feedback mechanisms are functionally important during gravitropic response and ensure fine-tuning of auxin fluxes for maintaining as well as terminating asymmetric growth. PMID:23211744

The Bimodal Lifestyle of Intracellular Salmonella in Epithelial Cells: Replication in the Cytosol Obscures Defects in Vacuolar Replication

PubMed Central

Steele-Mortimer, Olivia

2012-01-01

Salmonella enterica serovar Typhimurium invades and proliferates within epithelial cells. Intracellular bacteria replicate within a membrane bound vacuole known as the Salmonella containing vacuole. However, this bacterium can also replicate efficiently in the cytosol of epithelial cells and net intracellular growth is a product of both vacuolar and cytosolic replication. Here we have used semi-quantitative single-cell analyses to investigate the contribution of each of these replicative niches to intracellular proliferation in cultured epithelial cells. We show that cytosolic replication can account for the majority of net replication even though it occurs in less than 20% of infected cells. Consequently, assays for net growth in a population of infected cells, for example by recovery of colony forming units, are not good indicators of vacuolar proliferation. We also show that the Salmonella Type III Secretion System 2, which is required for SCV biogenesis, is not required for cytosolic replication. Altogether this study illustrates the value of single cell analyses when studying intracellular pathogens. PMID:22719929

Activities of Vacuolar Cysteine Proteases in Plant Senescence.

PubMed

Martínez, Dana E; Costa, Lorenza; Guiamét, Juan José

2018-01-01

Plant senescence is accompanied by a marked increase in proteolytic activities, and cysteine proteases (Cys-protease) represent the prevailing class among the responsible proteases. Cys-proteases predominantly locate to lytic compartments, i.e., to the central vacuole (CV) and to senescence-associated vacuoles (SAVs), the latter being specific to the photosynthetic cells of senescing leaves. Cellular fractionation of vacuolar compartments may facilitate Cys-proteases purification and their concentration for further analysis. Active Cys-proteases may be analyzed by different, albeit complementary approaches: (1) in vivo examination of proteolytic activity by fluorescence microscopy using specific substrates which become fluorescent upon cleavage by Cys-proteases, (2) protease labeling with specific probes that react irreversibly with the active enzymes, and (3) zymography, whereby protease activities are detected in polyacrylamide gels copolymerized with a substrate for proteases. Here we describe the three methods mentioned above for detection of active Cys-proteases and a cellular fractionation technique to isolate SAVs.

Computational optimisation of targeted DNA sequencing for cancer detection

NASA Astrophysics Data System (ADS)

Martinez, Pierre; McGranahan, Nicholas; Birkbak, Nicolai Juul; Gerlinger, Marco; Swanton, Charles

2013-12-01

Despite recent progress thanks to next-generation sequencing technologies, personalised cancer medicine is still hampered by intra-tumour heterogeneity and drug resistance. As most patients with advanced metastatic disease face poor survival, there is need to improve early diagnosis. Analysing circulating tumour DNA (ctDNA) might represent a non-invasive method to detect mutations in patients, facilitating early detection. In this article, we define reduced gene panels from publicly available datasets as a first step to assess and optimise the potential of targeted ctDNA scans for early tumour detection. Dividing 4,467 samples into one discovery and two independent validation cohorts, we show that up to 76% of 10 cancer types harbour at least one mutation in a panel of only 25 genes, with high sensitivity across most tumour types. Our analyses demonstrate that targeting ``hotspot'' regions would introduce biases towards in-frame mutations and would compromise the reproducibility of tumour detection.

Targeted cancer exome sequencing reveals recurrent mutations in myeloproliferative neoplasms

PubMed Central

Tenedini, E; Bernardis, I; Artusi, V; Artuso, L; Roncaglia, E; Guglielmelli, P; Pieri, L; Bogani, C; Biamonte, F; Rotunno, G; Mannarelli, C; Bianchi, E; Pancrazzi, A; Fanelli, T; Malagoli Tagliazucchi, G; Ferrari, S; Manfredini, R; Vannucchi, A M; Tagliafico, E

2014-01-01

With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in vitro-expanded CD3+T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG and NRAS), we demonstrated a mutation frequency between 3 and 8%. We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest dynamic international prognostic scoring system (DIPSS)-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing a NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score. PMID:24150215

Engineering salt-tolerant Brassica plants: characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation.

PubMed

Zhang, H X; Hodson, J N; Williams, J P; Blumwald, E

2001-10-23

Transgenic Brassica napus plants overexpressing AtNHX1, a vacuolar Na(+)/H(+) antiport from Arabidopsis thaliana, were able to grow, flower, and produce seeds in the presence of 200 mM sodium chloride. Although the transgenic plants grown in high salinity accumulated sodium up to 6% of their dry weight, growth of the these plants was only marginally affected by the high salt concentration. Moreover, seed yields and the seed oil quality were not affected by the high salinity of the soil. Our results demonstrate the potential use of these transgenic plants for agricultural use in saline soils. Our findings, showing that the modification of a single trait significantly improved the salinity tolerance of this crop plant, suggest that with a combination of breeding and transgenic plants it could be possible to produce salt-tolerant crops with far fewer target traits than had been anticipated.

Hereditary vacuolar internal anal sphincter myopathy causing proctalgia fugax and constipation: a new case contribution.

PubMed

de la Portilla, Fernando; Borrero, Juan José; Rafel, Enrique

2005-03-01

Hereditary anal sphincter myopathy is rare. We present a family with one affected member with proctalgia fugax, constipation and internal anal sphincter hypertrophy. Ultrastructural findings show vacuolization of smooth muscle cells without the characteristic polyglucosan inclusion. Further relief of symptoms was obtained using an oral calcium antagonist. Based on clinical presentation, endosonography and morphological findings, we consider our case is a histological variant of the vacuolar myopathy originally described.

Retrotransposon insertion targeting: a mechanism for homogenization of centromere sequences on nonhomologous chromosomes.

PubMed

Birchler, James A; Presting, Gernot G

2012-04-01

The centromeres of most eukaryotic organisms consist of highly repetitive arrays that are similar across nonhomologous chromosomes. These sequences evolve rapidly, thus posing a mystery as to how such arrays can be homogenized. Recent work in species in which centromere-enriched retrotransposons occur indicates that these elements preferentially insert into the centromeric regions. In two different Arabidopsis species, a related element was recognized in which the specificity for such targeting was altered. These observations provide a partial explanation for how homogenization of centromere DNA sequences occurs.

In-depth glycoproteomic characterisation of grape berry vacuolar invertase using a combination of mass spectrometry-based approaches.

PubMed

Hovasse, Agnès; Alayi, Tchilabalo Dilezitoko; Van Dorsselaer, Alain; Marchal, Richard; Jégou, Sandrine; Schaeffer-Reiss, Christine

2016-06-01

Vacuolar invertase is a key enzyme of sugar metabolism in grape berries. A full characterisation of this highly N-glycosylated protein is required to help understand its biological and biochemical significance in grapes. We have developed a mass spectrometry (MS)-based glycoproteomic approach wherein deglycosylated peptides are analysed by LC-MS/MS, while intact glycopeptides are characterised using a dedicated MS method to determine the attachment sites and micro-heterogeneity. For grape invertase, in parallel with deglycosylated peptides analysis, different enzymatic digestions were performed and glycopeptide detection was improved by enrichment method, nanoLC-MS and oxonium glycan ions. This MS-based glycoproteomic approach demonstrates that vacuolar invertase is glycosylated at all twelve potential N-glycosylation sites. Glycosylation is heterogeneous, with twelve glycoforms identified at six of the sites. The identification of several types of N-glycans is a major result to correlate with the surface and foaming properties of wine, the solubility, allergenicity, and protease resistance of wine proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sensitive and Specific Target Sequences Selected from Retrotransposons of Schistosoma japonicum for the Diagnosis of Schistosomiasis

PubMed Central

Xu, Jing; Zhu, Xing-Quan; Wang, Sheng-Yue; Xia, Chao-Ming

2012-01-01

Background Schistosomiasis japonica is a serious debilitating and sometimes fatal disease. Accurate diagnostic tests play a key role in patient management and control of the disease. However, currently available diagnostic methods are not ideal, and the detection of the parasite DNA in blood samples has turned out to be one of the most promising tools for the diagnosis of schistosomiasis. In our previous investigations, a 230-bp sequence from the highly repetitive retrotransposon SjR2 was identified and it showed high sensitivity and specificity for detecting Schistosoma japonicum DNA in the sera of rabbit model and patients. Recently, 29 retrotransposons were found in S. japonicum genome by our group. The present study highlighted the key factors for selecting a new perspective sensitive target DNA sequence for the diagnosis of schistosomiasis, which can serve as example for other parasitic pathogens. Methodology/Principal Findings In this study, we demonstrated that the key factors based on the bioinformatic analysis for selecting target sequence are the higher genome proportion, repetitive complete copies and partial copies, and active ESTs than the others in the chromosome genome. New primers based on 25 novel retrotransposons and SjR2 were designed and their sensitivity and specificity for detecting S. japonicum DNA were compared. The results showed that a new 303-bp sequence from non-long terminal repeat (LTR) retrotransposon (SjCHGCS19) had high sensitivity and specificity. The 303-bp target sequence was amplified from the sera of rabbit model at 3 d post-infection by nested-PCR and it became negative at 17 weeks post-treatment. Furthermore, the percentage sensitivity of the nested-PCR was 97.67% in 43 serum samples of S. japonicum-infected patients. Conclusions/Significance Our findings highlighted the key factors based on the bioinformatic analysis for selecting target sequence from S. japonicum genome, which provide basis for establishing powerful

Functional characterization of a vacuolar invertase from Solanum lycopersicum: post-translational regulation by N-glycosylation and a proteinaceous inhibitor.

PubMed

Tauzin, Alexandra S; Sulzenbacher, Gerlind; Lafond, Mickael; Desseaux, Véronique; Reca, Ida Barbara; Perrier, Josette; Bellincampi, Daniela; Fourquet, Patrick; Lévêque, Christian; Giardina, Thierry

2014-06-01

Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Regulation of Vacuolar H+-ATPase (V-ATPase) Reassembly by Glycolysis Flow in 6-Phosphofructo-1-kinase (PFK-1)-deficient Yeast Cells*

PubMed Central

Chan, Chun-Yuan; Dominguez, Dennis; Parra, Karlett J.

2016-01-01

Yeast 6-phosphofructo-1-kinase (PFK-1) has two subunits, Pfk1p and Pfk2p. Deletion of Pfk2p alters glucose-dependent V-ATPase reassembly and vacuolar acidification (Chan, C. Y., and Parra, K. J. (2014) Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly. J. Biol. Chem. 289, 19448–19457). This study capitalized on the mechanisms suppressing vacuolar H+-ATPase (V-ATPase) in pfk2Δ to gain new knowledge of the mechanisms underlying glucose-dependent V-ATPase regulation. Because V-ATPase is fully assembled in pfk2Δ, and glycolysis partially suppressed at steady state, we manipulated glycolysis and assessed its direct involvement on V-ATPase function. At steady state, the ratio of proton transport to ATP hydrolysis increased 24% after increasing the glucose concentration from 2% to 4% to enhance the glycolysis flow in pfk2Δ. Tighter coupling restored vacuolar pH when glucose was abundant and glycolysis operated below capacity. After readdition of glucose to glucose-deprived cells, glucose-dependent V1Vo reassembly was proportional to the glycolysis flow. Readdition of 2% glucose to pfk2Δ cells, which restored 62% of ethanol concentration, led to equivalent 60% V1Vo reassembly levels. Steady-state level of assembly (100% reassembly) was reached at 4% glucose when glycolysis reached a threshold in pfk2Δ (≥40% the wild-type flow). At 4% glucose, the level of Pfk1p co-immunoprecipitated with V-ATPase decreased 58% in pfk2Δ, suggesting that Pfk1p binding to V-ATPase may be inhibitory in the mutant. We concluded that V-ATPase activity at steady state and V-ATPase reassembly after readdition of glucose to glucose-deprived cells are controlled by the glycolysis flow. We propose a new mechanism by which glucose regulates V-ATPase catalytic activity that occurs at steady state without changing V1Vo assembly. PMID:27226568

Clinical Utility of LC3 and p62 Immunohistochemistry in Diagnosis of Drug-Induced Autophagic Vacuolar Myopathies: A Case-Control Study

PubMed Central

Lee, Han S.; Daniels, Brianne H.; Salas, Eduardo; Bollen, Andrew W.; Debnath, Jayanta; Margeta, Marta

2012-01-01

Background Some patients treated with chloroquine, hydroxychloroquine, or colchicine develop autophagic vacuolar myopathy, the diagnosis of which currently requires electron microscopy. The goal of the current study was to develop an immunohistochemical diagnostic marker for this pathologic entity. Methodology Microtubule-associated protein light chain 3 (LC3) has emerged as a robust marker of autophagosomes. LC3 binds p62/SQSTM1, an adapter protein that is selectively degraded via autophagy. In this study, we evaluated the utility of immunohistochemical stains for LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar myopathy. The staining was performed on archival muscle biopsy material, with subject assignment to normal control, drug-treated control, and autophagic myopathy groups based on history of drug use and morphologic criteria. Principal Findings In all drug-treated subjects, but not in normal controls, LC3 and p62 showed punctate staining characteristic of autophagosome buildup. In the autophagic myopathy subjects, puncta were coarser and tended to coalesce into linear structures aligned with the longitudinal axis of the fiber, often in the vicinity of vacuoles. The percentage of LC3- and p62-positive fibers was significantly higher in the autophagic myopathy group compared to either the normal control (p<0.001) or the drug-treated control group (p<0.05). With the diagnostic threshold set between 8% and 15% positive fibers (depending on the desired level of sensitivity and specificity), immunohistochemical staining for either LC3 or p62 could be used to identify subjects with autophagic vacuolar myopathy within the drug-treated subject group (p≤0.001). Significance Immunohistochemistry for LC3 and p62 can facilitate tissue-based diagnosis of drug-induced autophagic vacuolar myopathies. By limiting the need for electron microscopy (a time consuming and costly technique with high specificity, but low sensitivity), clinical use of these

Clinical utility of LC3 and p62 immunohistochemistry in diagnosis of drug-induced autophagic vacuolar myopathies: a case-control study.

PubMed

Lee, Han S; Daniels, Brianne H; Salas, Eduardo; Bollen, Andrew W; Debnath, Jayanta; Margeta, Marta

2012-01-01

Some patients treated with chloroquine, hydroxychloroquine, or colchicine develop autophagic vacuolar myopathy, the diagnosis of which currently requires electron microscopy. The goal of the current study was to develop an immunohistochemical diagnostic marker for this pathologic entity. Microtubule-associated protein light chain 3 (LC3) has emerged as a robust marker of autophagosomes. LC3 binds p62/SQSTM1, an adapter protein that is selectively degraded via autophagy. In this study, we evaluated the utility of immunohistochemical stains for LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar myopathy. The staining was performed on archival muscle biopsy material, with subject assignment to normal control, drug-treated control, and autophagic myopathy groups based on history of drug use and morphologic criteria. In all drug-treated subjects, but not in normal controls, LC3 and p62 showed punctate staining characteristic of autophagosome buildup. In the autophagic myopathy subjects, puncta were coarser and tended to coalesce into linear structures aligned with the longitudinal axis of the fiber, often in the vicinity of vacuoles. The percentage of LC3- and p62-positive fibers was significantly higher in the autophagic myopathy group compared to either the normal control (p<0.001) or the drug-treated control group (p<0.05). With the diagnostic threshold set between 8% and 15% positive fibers (depending on the desired level of sensitivity and specificity), immunohistochemical staining for either LC3 or p62 could be used to identify subjects with autophagic vacuolar myopathy within the drug-treated subject group (p ≤ 0.001). Immunohistochemistry for LC3 and p62 can facilitate tissue-based diagnosis of drug-induced autophagic vacuolar myopathies. By limiting the need for electron microscopy (a time consuming and costly technique with high specificity, but low sensitivity), clinical use of these markers will improve the speed and accuracy of

Targeted next generation sequencing for the detection of ciprofloxacin resistance markers using molecular inversion probes

DTIC Science & Technology

2016-07-06

1 Targeted next-generation sequencing for the detection of ciprofloxacin resistance markers using molecular inversion probes Christopher P...development and evaluation of a panel of 44 single-stranded molecular inversion probes (MIPs) coupled to next-generation sequencing (NGS) for the...padlock and molecular inversion probes as upfront enrichment steps for use with NGS showed the specificity and multiplexability of these techniques

RhVI1 is a membrane-anchored vacuolar invertase highly expressed in Rosa hybrida L. petals

PubMed Central

Farci, Domenica; Collu, Gabriella; Kirkpatrick, Joanna; Esposito, Francesca; Piano, Dario

2016-01-01

Invertases are a widespread group of enzymes that catalyse the conversion of sucrose into fructose and glucose. Plants invertases and their substrates are essential factors that play an active role in primary metabolism and in cellular differentiation and by these activities they sustain development and growth. Being naturally present in multiple isoforms, invertases are known to be highly differentiated and tissue specific in such a way that every isoform is characteristic of a specific part of the plant. In this work, we report the identification of the invertase RhVI1 that was found to be highly expressed in rose petals. A characterization of this protein revealed that RhVI1 is a glycosylated membrane-anchored protein associated with the cytosolic side of the vacuolar membrane which occurs in vivo in a monomeric form. Purification yields have shown that the levels of expression decreased during the passage of petals from buds to mature and pre-senescent flowers. Moreover, the activity assay indicates RhVI1 to be an acidic vacuolar invertase. The physiological implications of these findings are discussed, suggesting a possible role of this protein during anthesis. PMID:27083698

Abscisic acid induction of vacuolar H+-ATPase activity in mesembryanthemum crystallinum is developmentally regulated

PubMed

Barkla; Vera-Estrella; Maldonado-Gama; Pantoja

1999-07-01

Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways.

Plastoglobule-Targeting Competence of a Putative Transit Peptide Sequence from Rice Phytoene Synthase 2 in Plastids.

PubMed

You, Min Kyoung; Kim, Jin Hwa; Lee, Yeo Jin; Jeong, Ye Sol; Ha, Sun-Hwa

2016-12-22

Plastoglobules (PGs) are thylakoid membrane microdomains within plastids that are known as specialized locations of carotenogenesis. Three rice phytoene synthase proteins (OsPSYs) involved in carotenoid biosynthesis have been identified. Here, the N-terminal 80-amino-acid portion of OsPSY2 (PTp) was demonstrated to be a chloroplast-targeting peptide by displaying cytosolic localization of OsPSY2(ΔPTp):mCherry in rice protoplast, in contrast to chloroplast localization of OsPSY2:mCherry in a punctate pattern. The peptide sequence of a PTp was predicted to harbor two transmembrane domains eligible for a putative PG-targeting signal. To assess and enhance the PG-targeting ability of PTp, the original PTp DNA sequence ( PTp ) was modified to a synthetic DNA sequence ( stPTp ), which had 84.4% similarity to the original sequence. The motivation of this modification was to reduce the GC ratio from 75% to 65% and to disentangle the hairpin loop structures of PTp . These two DNA sequences were fused to the sequence of the synthetic green fluorescent protein (sGFP) and drove GFP expression with different efficiencies. In particular, the RNA and protein levels of stPTp-sGFP were slightly improved to 1.4-fold and 1.3-fold more than those of sGFP, respectively. The green fluorescent signals of their mature proteins were all observed as speckle-like patterns with slightly blurred stromal signals in chloroplasts. These discrete green speckles of PTp - sGFP and stPTp - sGFP corresponded exactly to the red fluorescent signal displayed by OsPSY2:mCherry in both etiolated and greening protoplasts and it is presumed to correspond to distinct PGs. In conclusion, we identified PTp as a transit peptide sequence facilitating preferential translocation of foreign proteins to PGs, and developed an improved PTp sequence, a s tPTp , which is expected to be very useful for applications in plant biotechnologies requiring precise micro-compartmental localization in plastids.

Vacuolar processing enzyme plays an essential role in the crystalline structure of glutelin in rice seed.

PubMed

Kumamaru, Toshihiro; Uemura, Yuji; Inoue, Yoshimi; Takemoto, Yoko; Siddiqui, Sadar Uddin; Ogawa, Masahiro; Hara-Nishimura, Ikuko; Satoh, Hikaru

2010-01-01

To identify the function of genes that regulate the processing of proglutelin, we performed an analysis of glup3 mutants, which accumulates excess amounts of proglutelin and lack the vacuolar processing enzyme (VPE). VPE activity in developing seeds from glup3 lines was reduced remarkably compared with the wild type. DNA sequencing of the VPE gene in glup3 mutants revealed either amino acid substitutions or the appearance of a stop codon within the coding region. Microscopic observations showed that alpha-globulin and proglutelin were distributed homogeneously within glup3 protein storage vacuoles (PSVs), and that glup3 PSVs lacked the crystalline lattice structure typical of wild-type PSVs. This suggests that the processing of proglutelin by VPE in rice is essential for proper PSV structure and compartmentalization of storage proteins. Growth retardation in glup3 seedlings was also observed, indicating that the processing of proglutelin influences early seedling development. These findings indicate that storage of glutelin in its mature form as a crystalline structure in PSVs is required for the rapid use of glutelin as a source of amino acids during early seedling development. In conclusion, VPE plays an important role in the formation of protein crystalline structures in PSVs.

The Na+(K+)/H+ exchanger Nhx1 controls multivesicular body-vacuolar lysosome fusion.

PubMed

Karim, Mahmoud Abdul; Brett, Christopher Leonard

2018-02-01

Loss-of-function mutations in human endosomal Na + (K + )/H + exchangers (NHEs) NHE6 and NHE9 are implicated in neurological disorders including Christianson syndrome, autism, and attention deficit and hyperactivity disorder. These mutations disrupt retention of surface receptors within neurons and glial cells by affecting their delivery to lysosomes for degradation. However, the molecular basis of how these endosomal NHEs control endocytic trafficking is unclear. Using Saccharomyces cerevisiae as a model, we conducted cell-free organelle fusion assays to show that transport activity of the orthologous endosomal NHE Nhx1 is important for multivesicular body (MVB)-vacuolar lysosome fusion, the last step of endocytosis required for surface protein degradation. We find that deleting Nhx1 disrupts the fusogenicity of the MVB, not the vacuole, by targeting pH-sensitive machinery downstream of the Rab-GTPase Ypt7 needed for SNARE-mediated lipid bilayer merger. All contributing mechanisms are evolutionarily conserved offering new insight into the etiology of human disorders linked to loss of endosomal NHE function. © 2018 Karim and Brett. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Vacuolar CAX1 and CAX3 influence auxin transport in guard cells via regulation of apoplastic pH

USDA-ARS?s Scientific Manuscript database

Cation exchangers CAX1 and CAX3 are vacuolar ion transporters involved in ion homeostasis in plants. Widely expressed in the plant, they mediate calcium transport from the cytosol to the vacuole lumen using the proton gradient across the tonoplast. Here, we report an unexpected role of CAX1 and CAX3...

Massively Parallel Sequencing of Patients with Intellectual Disability, Congenital Anomalies and/or Autism Spectrum Disorders with a Targeted Gene Panel

PubMed Central

Brett, Maggie; McPherson, John; Zang, Zhi Jiang; Lai, Angeline; Tan, Ee-Shien; Ng, Ivy; Ong, Lai-Choo; Cham, Breana; Tan, Patrick; Rozen, Steve; Tan, Ene-Choo

2014-01-01

Developmental delay and/or intellectual disability (DD/ID) affects 1–3% of all children. At least half of these are thought to have a genetic etiology. Recent studies have shown that massively parallel sequencing (MPS) using a targeted gene panel is particularly suited for diagnostic testing for genetically heterogeneous conditions. We report on our experiences with using massively parallel sequencing of a targeted gene panel of 355 genes for investigating the genetic etiology of eight patients with a wide range of phenotypes including DD/ID, congenital anomalies and/or autism spectrum disorder. Targeted sequence enrichment was performed using the Agilent SureSelect Target Enrichment Kit and sequenced on the Illumina HiSeq2000 using paired-end reads. For all eight patients, 81–84% of the targeted regions achieved read depths of at least 20×, with average read depths overlapping targets ranging from 322× to 798×. Causative variants were successfully identified in two of the eight patients: a nonsense mutation in the ATRX gene and a canonical splice site mutation in the L1CAM gene. In a third patient, a canonical splice site variant in the USP9X gene could likely explain all or some of her clinical phenotypes. These results confirm the value of targeted MPS for investigating DD/ID in children for diagnostic purposes. However, targeted gene MPS was less likely to provide a genetic diagnosis for children whose phenotype includes autism. PMID:24690944

Glucose Starvation Inhibits Autophagy via Vacuolar Hydrolysis and Induces Plasma Membrane Internalization by Down-regulating Recycling*

PubMed Central

Lang, Michael J.; Martinez-Marquez, Jorge Y.; Prosser, Derek C.; Ganser, Laura R.; Buelto, Destiney; Wendland, Beverly; Duncan, Mara C.

2014-01-01

Cellular energy influences all aspects of cellular function. Although cells can adapt to a gradual reduction in energy, acute energy depletion poses a unique challenge. Because acute depletion hampers the transport of new energy sources into the cell, the cell must use endogenous substrates to replenish energy after acute depletion. In the yeast Saccharomyces cerevisiae, glucose starvation causes an acute depletion of intracellular energy that recovers during continued glucose starvation. However, how the cell replenishes energy during the early phase of glucose starvation is unknown. In this study, we investigated the role of pathways that deliver proteins and lipids to the vacuole during glucose starvation. We report that in response to glucose starvation, plasma membrane proteins are directed to the vacuole through reduced recycling at the endosomes. Furthermore, we found that vacuolar hydrolysis inhibits macroautophagy in a target of rapamycin complex 1-dependent manner. Accordingly, we found that endocytosis and hydrolysis are required for survival in glucose starvation, whereas macroautophagy is dispensable. Together, these results suggest that hydrolysis of components delivered to the vacuole independent of autophagy is the cell survival mechanism used by S. cerevisiae in response to glucose starvation. PMID:24753258

SCF(TIR1/AFB)-auxin signalling regulates PIN vacuolar trafficking and auxin fluxes during root gravitropism.

PubMed

Baster, Paweł; Robert, Stéphanie; Kleine-Vehn, Jürgen; Vanneste, Steffen; Kania, Urszula; Grunewald, Wim; De Rybel, Bert; Beeckman, Tom; Friml, Jiří

2013-01-23

The distribution of the phytohormone auxin regulates many aspects of plant development including growth response to gravity. Gravitropic root curvature involves coordinated and asymmetric cell elongation between the lower and upper side of the root, mediated by differential cellular auxin levels. The asymmetry in the auxin distribution is established and maintained by a spatio-temporal regulation of the PIN-FORMED (PIN) auxin transporter activity. We provide novel insights into the complex regulation of PIN abundance and activity during root gravitropism. We show that PIN2 turnover is differentially regulated on the upper and lower side of gravistimulated roots by distinct but partially overlapping auxin feedback mechanisms. In addition to regulating transcription and clathrin-mediated internalization, auxin also controls PIN abundance at the plasma membrane by promoting their vacuolar targeting and degradation. This effect of elevated auxin levels requires the activity of SKP-Cullin-F-box(TIR1/AFB) (SCF(TIR1/AFB))-dependent pathway. Importantly, also suboptimal auxin levels mediate PIN degradation utilizing the same signalling pathway. These feedback mechanisms are functionally important during gravitropic response and ensure fine-tuning of auxin fluxes for maintaining as well as terminating asymmetric growth.

Atypical case of Wolfram syndrome revealed through targeted exome sequencing in a patient with suspected mitochondrial disease

PubMed Central

2012-01-01

Background Mitochondrial diseases comprise a diverse set of clinical disorders that affect multiple organ systems with varying severity and age of onset. Due to their clinical and genetic heterogeneity, these diseases are difficult to diagnose. We have developed a targeted exome sequencing approach to improve our ability to properly diagnose mitochondrial diseases and apply it here to an individual patient. Our method targets mitochondrial DNA (mtDNA) and the exons of 1,600 nuclear genes involved in mitochondrial biology or Mendelian disorders with multi-system phenotypes, thereby allowing for simultaneous evaluation of multiple disease loci. Case Presentation Targeted exome sequencing was performed on a patient initially suspected to have a mitochondrial disorder. The patient presented with diabetes mellitus, diffuse brain atrophy, autonomic neuropathy, optic nerve atrophy, and a severe amnestic syndrome. Further work-up revealed multiple heteroplasmic mtDNA deletions as well as profound thiamine deficiency without a clear nutritional cause. Targeted exome sequencing revealed a homozygous c.1672C > T (p.R558C) missense mutation in exon 8 of WFS1 that has previously been reported in a patient with Wolfram syndrome. Conclusion This case demonstrates how clinical application of next-generation sequencing technology can enhance the diagnosis of patients suspected to have rare genetic disorders. Furthermore, the finding of unexplained thiamine deficiency in a patient with Wolfram syndrome suggests a potential link between WFS1 biology and thiamine metabolism that has implications for the clinical management of Wolfram syndrome patients. PMID:22226368

Performance Comparison of Bench-Top Next Generation Sequencers Using Microdroplet PCR-Based Enrichment for Targeted Sequencing in Patients with Autism Spectrum Disorder

PubMed Central

Okamoto, Nobuhiko; Nakashima, Mitsuko; Tsurusaki, Yoshinori; Miyake, Noriko; Saitsu, Hirotomo; Matsumoto, Naomichi

2013-01-01

Next-generation sequencing (NGS) combined with enrichment of target genes enables highly efficient and low-cost sequencing of multiple genes for genetic diseases. The aim of this study was to validate the accuracy and sensitivity of our method for comprehensive mutation detection in autism spectrum disorder (ASD). We assessed the performance of the bench-top Ion Torrent PGM and Illumina MiSeq platforms as optimized solutions for mutation detection, using microdroplet PCR-based enrichment of 62 ASD associated genes. Ten patients with known mutations were sequenced using NGS to validate the sensitivity of our method. The overall read quality was better with MiSeq, largely because of the increased indel-related error associated with PGM. The sensitivity of SNV detection was similar between the two platforms, suggesting they are both suitable for SNV detection in the human genome. Next, we used these methods to analyze 28 patients with ASD, and identified 22 novel variants in genes associated with ASD, with one mutation detected by MiSeq only. Thus, our results support the combination of target gene enrichment and NGS as a valuable molecular method for investigating rare variants in ASD. PMID:24066114

Rapid molecular diagnostics of severe primary immunodeficiency determined by using targeted next-generation sequencing.

PubMed

Yu, Hui; Zhang, Victor Wei; Stray-Pedersen, Asbjørg; Hanson, Imelda Celine; Forbes, Lisa R; de la Morena, M Teresa; Chinn, Ivan K; Gorman, Elizabeth; Mendelsohn, Nancy J; Pozos, Tamara; Wiszniewski, Wojciech; Nicholas, Sarah K; Yates, Anne B; Moore, Lindsey E; Berge, Knut Erik; Sorte, Hanne; Bayer, Diana K; ALZahrani, Daifulah; Geha, Raif S; Feng, Yanming; Wang, Guoli; Orange, Jordan S; Lupski, James R; Wang, Jing; Wong, Lee-Jun

2016-10-01

Primary immunodeficiency diseases (PIDDs) are inherited disorders of the immune system. The most severe form, severe combined immunodeficiency (SCID), presents with profound deficiencies of T cells, B cells, or both at birth. If not treated promptly, affected patients usually do not live beyond infancy because of infections. Genetic heterogeneity of SCID frequently delays the diagnosis; a specific diagnosis is crucial for life-saving treatment and optimal management. We developed a next-generation sequencing (NGS)-based multigene-targeted panel for SCID and other severe PIDDs requiring rapid therapeutic actions in a clinical laboratory setting. The target gene capture/NGS assay provides an average read depth of approximately 1000×. The deep coverage facilitates simultaneous detection of single nucleotide variants and exonic copy number variants in one comprehensive assessment. Exons with insufficient coverage (<20× read depth) or high sequence homology (pseudogenes) are complemented by amplicon-based sequencing with specific primers to ensure 100% coverage of all targeted regions. Analysis of 20 patient samples with low T-cell receptor excision circle numbers on newborn screening or a positive family history or clinical suspicion of SCID or other severe PIDD identified deleterious mutations in 14 of them. Identified pathogenic variants included both single nucleotide variants and exonic copy number variants, such as hemizygous nonsense, frameshift, and missense changes in IL2RG; compound heterozygous changes in ATM, RAG1, and CIITA; homozygous changes in DCLRE1C and IL7R; and a heterozygous nonsense mutation in CHD7. High-throughput deep sequencing analysis with complete clinical validation greatly increases the diagnostic yield of severe primary immunodeficiency. Establishing a molecular diagnosis enables early immune reconstitution through prompt therapeutic intervention and guides management for improved long-term quality of life. Copyright © 2016 American

Loss of G2 subunit of vacuolar-type proton transporting ATPase leads to G1 subunit upregulation in the brain

PubMed Central

Kawamura, Nobuyuki; Sun-Wada, Ge-Hong; Wada, Yoh

2015-01-01

Vacuolar-type ATPase (V-ATPase) is a primary proton pump with versatile functions in various tissues. In nerve cells, V-ATPase is required for accumulation of neurotransmitters into secretory vesicles and subsequent release at the synapse. Neurons express a specific isoform (G2) of the G subunit of V-ATPase constituting the catalytic sector of the enzyme complex. Using gene targeting, we generated a mouse lacking functional G2 (G2 null), which showed no apparent disorders in architecture and behavior. In the G2-null mouse brain, a G1 subunit isoform, which is ubiquitously expressed in neuronal and non-neuronal tissues, accumulated more abundantly than in wild-type animals. This G1 upregulation was not accompanied by an increase in mRNA. These results indicate that loss of function of neuron-specific G2 isoform was compensated by an increase in levels of the G1 isoform without apparent upregulation of the G1 mRNA. PMID:26353914

Rational Design of Small Molecules Targeting Oncogenic Noncoding RNAs from Sequence.

PubMed

Disney, Matthew D; Angelbello, Alicia J

2016-12-20

The discovery of RNA catalysis in the 1980s and the dissemination of the human genome sequence at the start of this century inspired investigations of the regulatory roles of noncoding RNAs in biology. In fact, the Encyclopedia of DNA Elements (ENCODE) project has shown that only 1-2% of the human genome encodes protein, yet 75% is transcribed into RNA. Functional studies both preceding and following the ENCODE project have shown that these noncoding RNAs have important roles in regulating gene expression, developmental timing, and other critical functions. RNA's diverse roles are often a consequence of the various folds that it adopts. The single-stranded nature of the biopolymer enables it to adopt intramolecular folds with noncanonical pairings to lower its free energy. These folds can be scaffolds to bind proteins or to form frameworks to interact with other RNAs. Not surprisingly, dysregulation of certain noncoding RNAs has been shown to be causative of disease. Given this as the background, it is easy to see why it would be useful to develop methods that target RNA and manipulate its biology in rational and predictable ways. The antisense approach has afforded strategies to target RNAs via Watson-Crick base pairing and has typically focused on targeting partially unstructured regions of RNA. Small molecule strategies to target RNA would be desirable not only because compounds could be lead optimized via medicinal chemistry but also because structured regions within an RNA of interest could be targeted to directly interfere with RNA folds that contribute to disease. Additionally, small molecules have historically been the most successful drug candidates. Until recently, the ability to design small molecules that target non-ribosomal RNAs has been elusive, creating the perception that they are "undruggable". In this Account, approaches to demystify targeting RNA with small molecules are described. Rather than bulk screening for compounds that bind to singular

Salinity Tolerance of Two Potato Cultivars (Solanum tuberosum) Correlates With Differences in Vacuolar Transport Activity

PubMed Central

Jaarsma, Rinse; de Boer, Albertus H.

2018-01-01

Potato is an important cultivated crop species and since it is moderately salt sensitive there is a need to develop more salt tolerant cultivars. A high activity of Na+ transport across the tonoplast in exchange for H+ is essential to reduce Na+ toxicity. The proton motive force (PMF) generated by the V-H+-ATPase and the V-H+-PPase energizes the Na+(K+)/H+ antiport. We compared the activity, gene expression, and protein levels of the vacuolar proton pumps and the Na+/H+ antiporters in two potato cultivars (Solanum tuberosum) contrasting in their salt tolerance (cv. Desiree; tolerant and Mozart; sensitive) grown at 0 and 60 mM NaCl. Tonoplast-enriched vesicles were used to study the pump activity and protein levels of the V-H+-ATPase and the V-H+-PPase and the activity of the Na+/H+ antiporter. Although salt stress reduced the V-H+-ATPase and the V-H+-PPase activity in both cultivars, the decline in H+ pump activity was more severe in the salt-sensitive cultivar Mozart. After salt treatment, protein amounts of the vacuolar H+ pumps decreased in Mozart but remained unchanged in the cultivar Desiree. Decreased protein amounts of the V-H+-PPase found in Mozart may explain the reduced V-H+-PPase activity found for Mozart after salt stress. Under non-stress conditions, protein amounts of V-H+-PPase were equal in both cultivars while the V-H+-PPase activity was already twice as high and remained higher after salt treatment in the cultivar Desiree as compared to Mozart. This cultivar-dependent V-H+-PPase activity may explain the higher salt tolerance of Desiree. Moreover, combined with reduced vacuolar H+ pump activity, Mozart showed a lower Na+/H+ exchange activity and the Km for Na+ is at least twofold lower in tonoplast vesicles from Desiree, what suggests that NHXs from Desiree have a higher affinity for Na+ as compared to Mozart. From these results, we conclude that the higher capacity in combination with the higher affinity for Na+ uptake can be an important factor

Salinity Tolerance of Two Potato Cultivars (Solanum tuberosum) Correlates With Differences in Vacuolar Transport Activity.

PubMed

Jaarsma, Rinse; de Boer, Albertus H

2018-01-01

Potato is an important cultivated crop species and since it is moderately salt sensitive there is a need to develop more salt tolerant cultivars. A high activity of Na + transport across the tonoplast in exchange for H + is essential to reduce Na + toxicity. The proton motive force (PMF) generated by the V-H + -ATPase and the V-H + -PPase energizes the Na + (K + )/H + antiport. We compared the activity, gene expression, and protein levels of the vacuolar proton pumps and the Na + /H + antiporters in two potato cultivars ( Solanum tuberosum ) contrasting in their salt tolerance (cv. Desiree; tolerant and Mozart; sensitive) grown at 0 and 60 mM NaCl. Tonoplast-enriched vesicles were used to study the pump activity and protein levels of the V-H + -ATPase and the V-H + -PPase and the activity of the Na + /H + antiporter. Although salt stress reduced the V-H + -ATPase and the V-H + -PPase activity in both cultivars, the decline in H + pump activity was more severe in the salt-sensitive cultivar Mozart. After salt treatment, protein amounts of the vacuolar H + pumps decreased in Mozart but remained unchanged in the cultivar Desiree. Decreased protein amounts of the V-H + -PPase found in Mozart may explain the reduced V-H + -PPase activity found for Mozart after salt stress. Under non-stress conditions, protein amounts of V-H + -PPase were equal in both cultivars while the V-H + -PPase activity was already twice as high and remained higher after salt treatment in the cultivar Desiree as compared to Mozart. This cultivar-dependent V-H + -PPase activity may explain the higher salt tolerance of Desiree. Moreover, combined with reduced vacuolar H + pump activity, Mozart showed a lower Na + /H + exchange activity and the K m for Na + is at least twofold lower in tonoplast vesicles from Desiree, what suggests that NHXs from Desiree have a higher affinity for Na + as compared to Mozart. From these results, we conclude that the higher capacity in combination with the higher

Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data.

PubMed

Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A; Larsen, Martin Jakob

2016-01-01

Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths.

Silent genetic alterations identified by targeted next-generation sequencing in pheochromocytoma/paraganglioma: A clinicopathological correlations.

PubMed

Pillai, Suja; Gopalan, Vinod; Lo, Chung Y; Liew, Victor; Smith, Robert A; Lam, Alfred King Y

2017-02-01

The goal of this pilot study was to develop a customized, cost-effective amplicon panel (Ampliseq) for target sequencing in a cohort of patients with sporadic phaeochromocytoma/paraganglioma. Phaeochromocytoma/paragangliomas from 25 patients were analysed by targeted next-generation sequencing approach using an Ion Torrent PGM instrument. Primers for 15 target genes (NF1, RET, VHL, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, MEN1, KIF1Bβ, EPAS1, CDKN2 & PHD2) were designed using ion ampliseq designer. Ion Reporter software and Ingenuity® Variant Analysis™ software (www.ingenuity.com/variants) from Ingenuity Systems were used to analysis these results. Overall, 713 variants were identified. The variants identified from the Ion Reporter ranged from 64 to 161 per patient. Single nucleotide variants (SNV) were the most common. Further annotation with the help of Ingenuity variant analysis revealed 29 of these 713variants were deletions. Of these, six variants were non-pathogenic and four were likely to be pathogenic. The remaining 19 variants were of uncertain significance. The most frequently altered gene in the cohort was KIF1B followed by NF1. Novel KIF1B pathogenic variant c.3375+1G>A was identified. The mutation was noted in a patient with clinically confirmed neurofibromatosis. Chromosome 1 showed the presence of maximum number of variants. Use of targeted next-generation sequencing is a sensitive method for the detecting genetic changes in patients with phaeochromocytoma/paraganglioma. The precise detection of these genetic changes helps in understanding the pathogenesis of these tumours. Copyright © 2016 Elsevier Inc. All rights reserved.

Classification of video sequences into chosen generalized use classes of target size and lighting level.

PubMed

Leszczuk, Mikołaj; Dudek, Łukasz; Witkowski, Marcin

The VQiPS (Video Quality in Public Safety) Working Group, supported by the U.S. Department of Homeland Security, has been developing a user guide for public safety video applications. According to VQiPS, five parameters have particular importance influencing the ability to achieve a recognition task. They are: usage time-frame, discrimination level, target size, lighting level, and level of motion. These parameters form what are referred to as Generalized Use Classes (GUCs). The aim of our research was to develop algorithms that would automatically assist classification of input sequences into one of the GUCs. Target size and lighting level parameters were approached. The experiment described reveals the experts' ambiguity and hesitation during the manual target size determination process. However, the automatic methods developed for target size classification make it possible to determine GUC parameters with 70 % compliance to the end-users' opinion. Lighting levels of the entire sequence can be classified with an efficiency reaching 93 %. To make the algorithms available for use, a test application has been developed. It is able to process video files and display classification results, the user interface being very simple and requiring only minimal user interaction.

Evaluating allopolyploid origins in strawberries (Fragaria) using haplotypes generated from target capture sequencing.

PubMed

Kamneva, Olga K; Syring, John; Liston, Aaron; Rosenberg, Noah A

2017-08-04

Hybridization is observed in many eukaryotic lineages and can lead to the formation of polyploid species. The study of hybridization and polyploidization faces challenges both in data generation and in accounting for population-level phenomena such as coalescence processes in phylogenetic analysis. Genus Fragaria is one example of a set of plant taxa in which a range of ploidy levels is observed across species, but phylogenetic origins are unknown. Here, using 20 diploid and polyploid Fragaria species, we combine approaches from NGS data analysis and phylogenetics to infer evolutionary origins of polyploid strawberries, taking into account coalescence processes. We generate haplotype sequences for 257 low-copy nuclear markers assembled from Illumina target capture sequence data. We then identify putative hybridization events by analyzing gene tree topologies, and further test predicted hybridizations in a coalescence framework. This approach confirms the allopolyploid ancestry of F. chiloensis and F. virginiana, and provides new allopolyploid ancestry hypotheses for F. iturupensis, F. moschata, and F. orientalis. Evidence of gene flow between diploids F. bucharica and F. vesca is also detected, suggesting that it might be appropriate to consider these groups as conspecifics. This study is one of the first in which target capture sequencing followed by computational deconvolution of individual haplotypes is used for tracing origins of polyploid taxa. The study also provides new perspectives on the evolutionary history of Fragaria.

Amyloplasts and Vacuolar Membrane Dynamics in the Living Graviperceptive Cell of the Arabidopsis Inflorescence StemW⃞

PubMed Central

Saito, Chieko; Morita, Miyo T.; Kato, Takehide; Tasaka, Masao

2005-01-01

We developed an adequate method for the in vivo analysis of organelle dynamics in the gravity-perceptive cell (endodermis) of the Arabidopsis thaliana inflorescence stem, revealing behavior of amyloplasts and vacuolar membranes in those cells. Amyloplasts in the endodermis showed saltatory movements even before gravistimulation by reorientation, and these movements were confirmed as microfilament dependent. From our quantitative analysis in the wild type, the gravity-oriented movement of amyloplasts mainly occurred during 0 to 3 min after gravistimulation by reorientation, supporting findings from our previous physiological study. Even after microfilament disruption, the gravity-oriented movement of amyloplasts remained. By contrast, in zig/sgr4 mutants, where a SNARE molecule functioning in vacuole biogenesis has been disrupted, the movement of amyloplasts in the endodermis is severely restricted both before and after gravistimulation by reorientation. Here, we describe vacuolar membrane behavior in these cells in the wild-type, actin filament–disrupted, and zig/sgr4 mutants and discuss its putatively important features for the perception of gravity. We also discuss the data on the two kinds of movements of amyloplasts that may play an important role in gravitropism: (1) the leading edge amyloplasts and (2) the en mass movement of amyloplasts. PMID:15689424

RhVI1 is a membrane-anchored vacuolar invertase highly expressed in Rosa hybrida L. petals.

PubMed

Farci, Domenica; Collu, Gabriella; Kirkpatrick, Joanna; Esposito, Francesca; Piano, Dario

2016-05-01

Invertases are a widespread group of enzymes that catalyse the conversion of sucrose into fructose and glucose. Plants invertases and their substrates are essential factors that play an active role in primary metabolism and in cellular differentiation and by these activities they sustain development and growth. Being naturally present in multiple isoforms, invertases are known to be highly differentiated and tissue specific in such a way that every isoform is characteristic of a specific part of the plant. In this work, we report the identification of the invertase RhVI1 that was found to be highly expressed in rose petals. A characterization of this protein revealed that RhVI1 is a glycosylated membrane-anchored protein associated with the cytosolic side of the vacuolar membrane which occurs in vivo in a monomeric form. Purification yields have shown that the levels of expression decreased during the passage of petals from buds to mature and pre-senescent flowers. Moreover, the activity assay indicates RhVI1 to be an acidic vacuolar invertase. The physiological implications of these findings are discussed, suggesting a possible role of this protein during anthesis. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Evolutionary Divergence of Plant Borate Exporters and Critical Amino Acid Residues for the Polar Localization and Boron-Dependent Vacuolar Sorting of AtBOR1.

PubMed

Wakuta, Shinji; Mineta, Katsuhiko; Amano, Taro; Toyoda, Atsushi; Fujiwara, Toru; Naito, Satoshi; Takano, Junpei

2015-05-01

Boron (B) is an essential micronutrient for plants but is toxic when accumulated in excess. The plant BOR family encodes plasma membrane-localized borate exporters (BORs) that control translocation and homeostasis of B under a wide range of conditions. In this study, we examined the evolutionary divergence of BORs among terrestrial plants and showed that the lycophyte Selaginella moellendorffii and angiosperms have evolved two types of BOR (clades I and II). Clade I includes AtBOR1 and homologs previously shown to be involved in efficient transport of B under conditions of limited B availability. AtBOR1 shows polar localization in the plasma membrane and high-B-induced vacuolar sorting, important features for efficient B transport under low-B conditions, and rapid down-regulation to avoid B toxicity. Clade II includes AtBOR4 and barley Bot1 involved in B exclusion for high-B tolerance. We showed, using yeast complementation and B transport assays, that three genes in S. moellendorffii, SmBOR1 in clade I and SmBOR3 and SmBOR4 in clade II, encode functional BORs. Furthermore, amino acid sequence alignments identified an acidic di-leucine motif unique in clade I BORs. Mutational analysis of AtBOR1 revealed that the acidic di-leucine motif is required for the polarity and high-B-induced vacuolar sorting of AtBOR1. Our data clearly indicated that the common ancestor of vascular plants had already acquired two types of BOR for low- and high-B tolerance, and that the BOR family evolved to establish B tolerance in each lineage by adapting to their environments. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Vacuolar invertase gene silencing in potato (Solanum tuberosum L.) improves processing quality by decreasing the frequency of sugar-end defects

USDA-ARS?s Scientific Manuscript database

Sugar-end defect is a tuber quality disorder that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form da...

Co-overexpressing a plasma membrane and a vacuolar membrane sodium/proton antiporter significantly improves salt tolerance in transgenic Arabidopsis plants.

USDA-ARS?s Scientific Manuscript database

The Arabidopsis gene AtNHX1 encodes a vacuolar membrane bound sodium/proton (Sodium/Hydrogen) antiporter that transports sodium into the vacuole and exports hydrogen into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane bound sodium/hydrogen antiporter that exports sodium to the ex...

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.

PubMed

Tomioku, Kan-Na; Shigekuni, Mikiko; Hayashi, Hiroki; Yoshida, Akane; Futagami, Taiki; Tamaki, Hisanori; Tanabe, Kenji; Fujita, Akikazu

2018-05-01

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100 nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane. Copyright © 2018 Elsevier GmbH. All rights reserved.

Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data

PubMed Central

Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A.; Larsen, Martin Jakob

2016-01-01

Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths. PMID:27002637

High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

PubMed

Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

2015-08-01

Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Experimental and statistical post-validation of positive example EST sequences carrying peroxisome targeting signals type 1 (PTS1).

PubMed

Lingner, Thomas; Kataya, Amr R A; Reumann, Sigrun

2012-02-01

We recently developed the first algorithms specifically for plants to predict proteins carrying peroxisome targeting signals type 1 (PTS1) from genome sequences. As validated experimentally, the prediction methods are able to correctly predict unknown peroxisomal Arabidopsis proteins and to infer novel PTS1 tripeptides. The high prediction performance is primarily determined by the large number and sequence diversity of the underlying positive example sequences, which mainly derived from EST databases. However, a few constructs remained cytosolic in experimental validation studies, indicating sequencing errors in some ESTs. To identify erroneous sequences, we validated subcellular targeting of additional positive example sequences in the present study. Moreover, we analyzed the distribution of prediction scores separately for each orthologous group of PTS1 proteins, which generally resembled normal distributions with group-specific mean values. The cytosolic sequences commonly represented outliers of low prediction scores and were located at the very tail of a fitted normal distribution. Three statistical methods for identifying outliers were compared in terms of sensitivity and specificity." Their combined application allows elimination of erroneous ESTs from positive example data sets. This new post-validation method will further improve the prediction accuracy of both PTS1 and PTS2 protein prediction models for plants, fungi, and mammals.

Experimental and statistical post-validation of positive example EST sequences carrying peroxisome targeting signals type 1 (PTS1)

PubMed Central

Lingner, Thomas; Kataya, Amr R. A.; Reumann, Sigrun

2012-01-01

We recently developed the first algorithms specifically for plants to predict proteins carrying peroxisome targeting signals type 1 (PTS1) from genome sequences.1 As validated experimentally, the prediction methods are able to correctly predict unknown peroxisomal Arabidopsis proteins and to infer novel PTS1 tripeptides. The high prediction performance is primarily determined by the large number and sequence diversity of the underlying positive example sequences, which mainly derived from EST databases. However, a few constructs remained cytosolic in experimental validation studies, indicating sequencing errors in some ESTs. To identify erroneous sequences, we validated subcellular targeting of additional positive example sequences in the present study. Moreover, we analyzed the distribution of prediction scores separately for each orthologous group of PTS1 proteins, which generally resembled normal distributions with group-specific mean values. The cytosolic sequences commonly represented outliers of low prediction scores and were located at the very tail of a fitted normal distribution. Three statistical methods for identifying outliers were compared in terms of sensitivity and specificity.” Their combined application allows elimination of erroneous ESTs from positive example data sets. This new post-validation method will further improve the prediction accuracy of both PTS1 and PTS2 protein prediction models for plants, fungi, and mammals. PMID:22415050

A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries

PubMed Central

2011-01-01

Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol. PMID:21205303

Disruption of the vacuolar calcium-ATPases in Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway.

PubMed

Boursiac, Yann; Lee, Sang Min; Romanowsky, Shawn; Blank, Robert; Sladek, Chris; Chung, Woo Sik; Harper, Jeffrey F

2010-11-01

Calcium (Ca(2+)) signals regulate many aspects of plant development, including a programmed cell death pathway that protects plants from pathogens (hypersensitive response). Cytosolic Ca(2+) signals result from a combined action of Ca(2+) influx through channels and Ca(2+) efflux through pumps and cotransporters. Plants utilize calmodulin-activated Ca(2+) pumps (autoinhibited Ca(2+)-ATPase [ACA]) at the plasma membrane, endoplasmic reticulum, and vacuole. Here, we show that a double knockout mutation of the vacuolar Ca(2+) pumps ACA4 and ACA11 in Arabidopsis (Arabidopsis thaliana) results in a high frequency of hypersensitive response-like lesions. The appearance of macrolesions could be suppressed by growing plants with increased levels (greater than 15 mm) of various anions, providing a method for conditional suppression. By removing plants from a conditional suppression, lesion initials were found to originate primarily in leaf mesophyll cells, as detected by aniline blue staining. Initiation and spread of lesions could also be suppressed by disrupting the production or accumulation of salicylic acid (SA), as shown by combining aca4/11 mutations with a sid 2 (for salicylic acid induction-deficient2) mutation or expression of the SA degradation enzyme NahG. This indicates that the loss of the vacuolar Ca(2+) pumps by itself does not cause a catastrophic defect in ion homeostasis but rather potentiates the activation of a SA-dependent programmed cell death pathway. Together, these results provide evidence linking the activity of the vacuolar Ca(2+) pumps to the control of a SA-dependent programmed cell death pathway in plants.

Whole-exome sequencing and targeted gene sequencing provide insights into the role of PALB2 as a male breast cancer susceptibility gene.

PubMed

Silvestri, Valentina; Zelli, Veronica; Valentini, Virginia; Rizzolo, Piera; Navazio, Anna Sara; Coppa, Anna; Agata, Simona; Oliani, Cristina; Barana, Daniela; Castrignanò, Tiziana; Viel, Alessandra; Russo, Antonio; Tibiletti, Maria Grazia; Zanna, Ines; Masala, Giovanna; Cortesi, Laura; Manoukian, Siranoush; Azzollini, Jacopo; Peissel, Bernard; Bonanni, Bernardo; Peterlongo, Paolo; Radice, Paolo; Palli, Domenico; Giannini, Giuseppe; Chillemi, Giovanni; Montagna, Marco; Ottini, Laura

2017-01-01

Male breast cancer (MBC) is a rare disease whose etiology appears to be largely associated with genetic factors. BRCA1 and BRCA2 mutations account for about 10% of all MBC cases. Thus, a fraction of MBC cases are expected to be due to genetic factors not yet identified. To further explain the genetic susceptibility for MBC, whole-exome sequencing (WES) and targeted gene sequencing were applied to high-risk, BRCA1/2 mutation-negative MBC cases. Germ-line DNA of 1 male and 2 female BRCA1/2 mutation-negative breast cancer (BC) cases from a pedigree showing a first-degree family history of MBC was analyzed with WES. Targeted gene sequencing for the validation of WES results was performed for 48 high-risk, BRCA1/2 mutation-negative MBC cases from an Italian multicenter study of MBC. A case-control series of 433 BRCA1/2 mutation-negative MBC and female breast cancer (FBC) cases and 849 male and female controls was included in the study. WES in the family identified the partner and localizer of BRCA2 (PALB2) c.419delA truncating mutation carried by the proband, her father, and her paternal uncle (all affected with BC) and the N-acetyltransferase 1 (NAT1) c.97C>T nonsense mutation carried by the proband's maternal aunt. Targeted PALB2 sequencing detected the c.1984A>T nonsense mutation in 1 of the 48 BRCA1/2 mutation-negative MBC cases. NAT1 c.97C>T was not found in the case-control series. These results add strength to the evidence showing that PALB2 is involved in BC risk for both sexes and indicate that consideration should be given to clinical testing of PALB2 for BRCA1/2 mutation-negative families with multiple MBC and FBC cases. Cancer 2017;123:210-218. © 2016 American Cancer Society. © 2016 American Cancer Society.

Vacuolar H(+)-Pyrophosphatase AVP1 is Involved in Amine Fungicide Tolerance in Arabidopsis thaliana and Provides Tridemorph Resistance in Yeast.

PubMed

Hernández, Agustín; Herrera-Palau, Rosana; Madroñal, Juan M; Albi, Tomás; López-Lluch, Guillermo; Perez-Castiñeira, José R; Navas, Plácido; Valverde, Federico; Serrano, Aurelio

2016-01-01

Amine fungicides are widely used as crop protectants. Their success is believed to be related to their ability to inhibit postlanosterol sterol biosynthesis in fungi, in particular sterol-Δ(8),Δ(7)-isomerases and sterol-Δ(14)-reductases, with a concomitant accumulation of toxic abnormal sterols. However, their actual cellular effects and mechanisms of death induction are still poorly understood. Paradoxically, plants exhibit a natural resistance to amine fungicides although they have similar enzymes in postcicloartenol sterol biosynthesis that are also susceptible to fungicide inhibition. A major difference in vacuolar ion homeostasis between plants and fungi is the presence of a dual set of primary proton pumps in the former (V-ATPase and H(+)-pyrophosphatase), but only the V-ATPase in the latter. Abnormal sterols affect the proton-pumping capacity of V-ATPases in fungi and this has been proposed as a major determinant in fungicide action. Using Saccharomyces cerevisiae as a model fungus, we provide evidence that amine fungicide treatment induced cell death by apoptosis. Cell death was concomitant with impaired H(+)-pumping capacity in vacuole vesicles and dependent on vacuolar proteases. Also, the heterologous expression of the Arabidopsis thaliana main H(+)-pyrophosphatase (AVP1) at the fungal vacuolar membrane reduced apoptosis levels in yeast and increased resistance to amine fungicides. Consistently, A. thaliana avp1 mutant seedlings showed increased susceptibility to this amine fungicide, particularly at the level of root development. This is in agreement with AVP1 being nearly the sole H(+)-pyrophosphatase gene expressed at the root elongation zones. All in all, the present data suggest that H(+)-pyrophosphatases are major determinants of plant tolerance to amine fungicides.

Individual sequences in large sets of gene sequences may be distinguished efficiently by combinations of shared sub-sequences

PubMed Central

Gibbs, Mark J; Armstrong, John S; Gibbs, Adrian J

2005-01-01

Background Most current DNA diagnostic tests for identifying organisms use specific oligonucleotide probes that are complementary in sequence to, and hence only hybridise with the DNA of one target species. By contrast, in traditional taxonomy, specimens are usually identified by 'dichotomous keys' that use combinations of characters shared by different members of the target set. Using one specific character for each target is the least efficient strategy for identification. Using combinations of shared bisectionally-distributed characters is much more efficient, and this strategy is most efficient when they separate the targets in a progressively binary way. Results We have developed a practical method for finding minimal sets of sub-sequences that identify individual sequences, and could be targeted by combinations of probes, so that the efficient strategy of traditional taxonomic identification could be used in DNA diagnosis. The sizes of minimal sub-sequence sets depended mostly on sequence diversity and sub-sequence length and interactions between these parameters. We found that 201 distinct cytochrome oxidase subunit-1 (CO1) genes from moths (Lepidoptera) were distinguished using only 15 sub-sequences 20 nucleotides long, whereas only 8–10 sub-sequences 6–10 nucleotides long were required to distinguish the CO1 genes of 92 species from the 9 largest orders of insects. Conclusion The presence/absence of sub-sequences in a set of gene sequences can be used like the questions in a traditional dichotomous taxonomic key; hybridisation probes complementary to such sub-sequences should provide a very efficient means for identifying individual species, subtypes or genotypes. Sequence diversity and sub-sequence length are the major factors that determine the numbers of distinguishing sub-sequences in any set of sequences. PMID:15817134

Molecular and functional characterization of vacuolar-ATPase from the American dog tick Dermacentor variabilis.

PubMed

Petchampai, N; Sunyakumthorn, P; Guillotte, M L; Thepparit, C; Kearney, M T; Mulenga, A; Azad, A F; Macaluso, K R

2014-02-01

Vacuolar (V)-ATPase is a proton-translocating enzyme that acidifies cellular compartments for various functions such as receptor-mediated endocytosis, intracellular trafficking and protein degradation. Previous studies in Dermacentor variabilis chronically infected with Rickettsia montanensis have identified V-ATPase as one of the tick-derived molecules transcribed in response to rickettsial infection. To examine the role of the tick V-ATPase in tick-Rickettsia interactions, a full-length 2887-bp cDNA (2532-bp open reading frame) clone corresponding to the transcript of the V0 domain subunit a of D. variabilis V-ATPase (DvVATPaseV0a) gene encoding an 843 amino acid protein with an estimated molecular weight of ~96 kDa was isolated from D. variabilis. Amino acid sequence analysis of DvVATPaseV0a showed the highest similarity to VATPaseV0a from Ixodes scapularis. A potential N-glycosylation site and eight putative transmembrane segments were identified in the sequence. Western blot analysis of tick tissues probed with polyclonal antibody raised against recombinant DvVATPaseV0a revealed the expression of V-ATPase in the tick ovary. Transcriptional profiles of DvVATPaseV0a demonstrated a greater mRNA expression in the tick ovary, compared with the midgut and salivary glands; however, the mRNA level in each of these tick tissues remained unchanged after infection with R. montanensis for 1 h. V-ATPase inhibition bioassays resulted in a significant decrease in the ability of R. montanensis to invade tick cells in vitro, suggesting a role of V-ATPase in rickettsial infection of tick cells. Characterization of tick-derived molecules involved in rickettsial infection is essential for a thorough understanding of rickettsial transmission within tick populations and the ecology of tick-borne rickettsial diseases. © 2013 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society.

A weighted sampling algorithm for the design of RNA sequences with targeted secondary structure and nucleotide distribution.

PubMed

Reinharz, Vladimir; Ponty, Yann; Waldispühl, Jérôme

2013-07-01

The design of RNA sequences folding into predefined secondary structures is a milestone for many synthetic biology and gene therapy studies. Most of the current software uses similar local search strategies (i.e. a random seed is progressively adapted to acquire the desired folding properties) and more importantly do not allow the user to control explicitly the nucleotide distribution such as the GC-content in their sequences. However, the latter is an important criterion for large-scale applications as it could presumably be used to design sequences with better transcription rates and/or structural plasticity. In this article, we introduce IncaRNAtion, a novel algorithm to design RNA sequences folding into target secondary structures with a predefined nucleotide distribution. IncaRNAtion uses a global sampling approach and weighted sampling techniques. We show that our approach is fast (i.e. running time comparable or better than local search methods), seedless (we remove the bias of the seed in local search heuristics) and successfully generates high-quality sequences (i.e. thermodynamically stable) for any GC-content. To complete this study, we develop a hybrid method combining our global sampling approach with local search strategies. Remarkably, our glocal methodology overcomes both local and global approaches for sampling sequences with a specific GC-content and target structure. IncaRNAtion is available at csb.cs.mcgill.ca/incarnation/. Supplementary data are available at Bioinformatics online.

Development of Genetic Markers in Eucalyptus Species by Target Enrichment and Exome Sequencing

PubMed Central

Dasgupta, Modhumita Ghosh; Dharanishanthi, Veeramuthu; Agarwal, Ishangi; Krutovsky, Konstantin V.

2015-01-01

The advent of next-generation sequencing has facilitated large-scale discovery, validation and assessment of genetic markers for high density genotyping. The present study was undertaken to identify markers in genes supposedly related to wood property traits in three Eucalyptus species. Ninety four genes involved in xylogenesis were selected for hybridization probe based nuclear genomic DNA target enrichment and exome sequencing. Genomic DNA was isolated from the leaf tissues and used for on-array probe hybridization followed by Illumina sequencing. The raw sequence reads were trimmed and high-quality reads were mapped to the E. grandis reference sequence and the presence of single nucleotide variants (SNVs) and insertions/ deletions (InDels) were identified across the three species. The average read coverage was 216X and a total of 2294 SNVs and 479 InDels were discovered in E. camaldulensis, 2383 SNVs and 518 InDels in E. tereticornis, and 1228 SNVs and 409 InDels in E. grandis. Additionally, SNV calling and InDel detection were conducted in pair-wise comparisons of E. tereticornis vs. E. grandis, E. camaldulensis vs. E. tereticornis and E. camaldulensis vs. E. grandis. This study presents an efficient and high throughput method on development of genetic markers for family– based QTL and association analysis in Eucalyptus. PMID:25602379

Fluorescence turn-on detection of target sequence DNA based on silicon nanodot-mediated quenching.

PubMed

Zhang, Yanan; Ning, Xinping; Mao, Guobin; Ji, Xinghu; He, Zhike

2018-05-01

We have developed a new enzyme-free method for target sequence DNA detection based on the dynamic quenching of fluorescent silicon nanodots (SiNDs) toward Cy5-tagged DNA probe. Fascinatingly, the water-soluble SiNDs can quench the fluorescence of cyanine (Cy5) in Cy5-tagged DNA probe in homogeneous solution, and the fluorescence of Cy5-tagged DNA probe can be restored in the presence of target sequence DNA (the synthetic target miRNA-27a). Based on this phenomenon, a SiND-featured fluorescent sensor has been constructed for "turn-on" detection of the synthetic target miRNA-27a for the first time. This newly developed approach possesses the merits of low cost, simple design, and convenient operation since no enzymatic reaction, toxic reagents, or separation procedures are involved. The established method achieves a detection limit of 0.16 nM, and the relative standard deviation of this method is 9% (1 nM, n = 5). The linear range is 0.5-20 nM, and the recoveries in spiked human fluids are in the range of 90-122%. This protocol provides a new tactic in the development of the nonenzymic miRNA biosensors and opens a promising avenue for early diagnosis of miRNA-associated disease. Graphical abstract The SiND-based fluorescent sensor for detection of S-miR-27a.

Identification of peptide sequences that target to the brain using in vivo phage display.

PubMed

Li, Jingwei; Zhang, Qizhi; Pang, Zhiqing; Wang, Yuchen; Liu, Qingfeng; Guo, Liangran; Jiang, Xinguo

2012-06-01

Phage display technology could provide a rapid means for the discovery of novel peptides. To find peptide ligands specific for the brain vascular receptors, we performed a modified phage display method. Phages were recovered from mice brain parenchyma after administrated with a random 7-mer peptide library intravenously. A longer circulation time was arranged according to the biodistributive brain/blood ratios of phage particles. Following sequential rounds of isolation, a number of phages were sequenced and a peptide sequence (CTSTSAPYC, denoted as PepC7) was identified. Clone 7-1, which encodes PepC7, exhibited translocation efficiency about 41-fold higher than the random library phage. Immunofluorescence analysis revealed that Clone 7-1 had a significant superiority on transport efficiency into the brain compared with native M13 phage. Clone 7-1 was inhibited from homing to the brain in a dose-dependent fashion when cyclic peptides of the same sequence were present in a competition assay. Interestingly, the linear peptide (ATSTSAPYA, Pep7) and a scrambled control peptide PepSC7 (CSPATSYTC) did not compete with the phage at the same tested concentration (0.2-200 pg). Labeled by Cy5.5, PepC7 exhibited significant brain-targeting capability in in vivo optical imaging analysis. The cyclic conformation of PepC7 formed by disulfide bond, and the correct structure itself play a critical role in maintaining the selectivity and affinity for the brain. In conclusion, PepC7 is a promising brain-target motif never been reported before and it could be applied to targeted drug delivery into the brain.

Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy

NASA Astrophysics Data System (ADS)

Kühnemund, Malte; Wei, Qingshan; Darai, Evangelia; Wang, Yingjie; Hernández-Neuta, Iván; Yang, Zhao; Tseng, Derek; Ahlford, Annika; Mathot, Lucy; Sjöblom, Tobias; Ozcan, Aydogan; Nilsson, Mats

2017-01-01

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies.

Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision.

PubMed

Denise, Hubert; Moschos, Sterghios A; Sidders, Benjamin; Burden, Frances; Perkins, Hannah; Carter, Nikki; Stroud, Tim; Kennedy, Michael; Fancy, Sally-Ann; Lapthorn, Cris; Lavender, Helen; Kinloch, Ross; Suhy, David; Corbau, Romu

2014-02-04

TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5' RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).Molecular Therapy-Nucleic Acids (2014) 3, e145; doi:10.1038/mtna.2013.73; published online 4 February 2014.

Implementing targeted region capture sequencing for the clinical detection of Alagille syndrome: An efficient and cost‑effective method.

PubMed

Huang, Tianhong; Yang, Guilin; Dang, Xiao; Ao, Feijian; Li, Jiankang; He, Yizhou; Tang, Qiyuan; He, Qing

2017-11-01

Alagille syndrome (AGS) is a highly variable, autosomal dominant disease that affects multiple structures including the liver, heart, eyes, bones and face. Targeted region capture sequencing focuses on a panel of known pathogenic genes and provides a rapid, cost‑effective and accurate method for molecular diagnosis. In a Chinese family, this method was used on the proband and Sanger sequencing was applied to validate the candidate mutation. A de novo heterozygous mutation (c.3254_3255insT p.Leu1085PhefsX24) of the jagged 1 gene was identified as the potential disease‑causing gene mutation. In conclusion, the present study suggested that target region capture sequencing is an efficient, reliable and accurate approach for the clinical diagnosis of AGS. Furthermore, these results expand on the understanding of the pathogenesis of AGS.

Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated1

PubMed Central

Barkla, Bronwyn J.; Vera-Estrella, Rosario; Maldonado-Gama, Minerva; Pantoja, Omar

1999-01-01

Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways. PMID:10398716

Mitochondrial targeting sequence variants of the CHCHD2 gene are a risk for Lewy body disorders

PubMed Central

Ogaki, Kotaro; Koga, Shunsuke; Heckman, Michael G.; Fiesel, Fabienne C.; Ando, Maya; Labbé, Catherine; Lorenzo-Betancor, Oswaldo; Moussaud-Lamodière, Elisabeth L.; Soto-Ortolaza, Alexandra I.; Walton, Ronald L.; Strongosky, Audrey J.; Uitti, Ryan J.; McCarthy, Allan; Lynch, Timothy; Siuda, Joanna; Opala, Grzegorz; Rudzinska, Monika; Krygowska-Wajs, Anna; Barcikowska, Maria; Czyzewski, Krzysztof; Puschmann, Andreas; Nishioka, Kenya; Funayama, Manabu; Hattori, Nobutaka; Parisi, Joseph E.; Petersen, Ronald C.; Graff-Radford, Neill R.; Boeve, Bradley F.; Springer, Wolfdieter; Wszolek, Zbigniew K.; Dickson, Dennis W.

2015-01-01

Objective: To assess the role of CHCHD2 variants in patients with Parkinson disease (PD) and Lewy body disease (LBD) in Caucasian populations. Methods: All exons of the CHCHD2 gene were sequenced in a US Caucasian patient-control series (878 PD, 610 LBD, and 717 controls). Subsequently, exons 1 and 2 were sequenced in an Irish series (355 PD and 365 controls) and a Polish series (394 PD and 350 controls). Immunohistochemistry and immunofluorescence studies were performed on pathologic LBD cases with rare CHCHD2 variants. Results: We identified 9 rare exonic variants of unknown significance. These variants were more frequent in the combined group of PD and LBD patients compared to controls (0.6% vs 0.1%, p = 0.013). In addition, the presence of any rare variant was more common in patients with LBD (2.5% vs 1.0%, p = 0.050) compared to controls. Eight of these 9 variants were located within the gene's mitochondrial targeting sequence. Conclusions: Although the role of variants of the CHCHD2 gene in PD and LBD remains to be further elucidated, the rare variants in the mitochondrial targeting sequence may be a risk factor for Lewy body disorders, which may link CHCHD2 to other genetic forms of parkinsonism with mitochondrial dysfunction. PMID:26561290

Targeted exome sequencing reveals novel USH2A mutations in Chinese patients with simplex Usher syndrome.

PubMed

Shu, Hai-Rong; Bi, Huai; Pan, Yang-Chun; Xu, Hang-Yu; Song, Jian-Xin; Hu, Jie

2015-09-16

Usher syndrome (USH) is an autosomal recessive disorder characterized by hearing impairment and vision dysfunction due to retinitis pigmentosa. Phenotypic and genetic heterogeneities of this disease make it impractical to obtain a genetic diagnosis by conventional Sanger sequencing. In this study, we applied a next-generation sequencing approach to detect genetic abnormalities in patients with USH. Two unrelated Chinese families were recruited, consisting of two USH afflicted patients and four unaffected relatives. We selected 199 genes related to inherited retinal diseases as targets for deep exome sequencing. Through systematic data analysis using an established bioinformatics pipeline, all variants that passed filter criteria were validated by Sanger sequencing and co-segregation analysis. A homozygous frameshift mutation (c.4382delA, p.T1462Lfs*2) was revealed in exon20 of gene USH2A in the F1 family. Two compound heterozygous mutations, IVS47 + 1G > A and c.13156A > T (p.I4386F), located in intron 48 and exon 63 respectively, of USH2A, were identified as causative mutations for the F2 family. Of note, the missense mutation c.13156A > T has not been reported so far. In conclusion, targeted exome sequencing precisely and rapidly identified the genetic defects in two Chinese USH families and this technique can be applied as a routine examination for these disorders with significant clinical and genetic heterogeneity.

Vacuolar amino acid transporter Avt5p is responsible for lithium uptake in Schizosaccharomyces pombe.

PubMed

Iwaki, Tomoko; Sekito, Takayuki; Kakinuma, Yoshimi

2010-01-01

The fission yeast Schizosaccharomyces pombe was sensitive to salinity; cell growth was stopped by 0.5 M NaCl and by 10 mM LiCl. The avt5+ gene encodes a vacuolar transporter with a broad specificity for amino acids. We found that the avt5Delta mutant became highly tolerant of Li+ and Na+ in growth. Concanamycin A-sensitive Li+ uptake as well as cellular Li+ content was lower in the avt5 mutant, suggesting a role of Avt5p in cellular uptake of toxic Li+.

MTV1 and MTV4 encode plant-specific ENTH and ARF GAP proteins that mediate clathrin-dependent trafficking of vacuolar cargo from the trans-Golgi network.

PubMed

Sauer, Michael; Delgadillo, M Otilia; Zouhar, Jan; Reynolds, Gregory D; Pennington, Janice G; Jiang, Liwen; Liljegren, Sarah J; Stierhof, York-Dieter; De Jaeger, Geert; Otegui, Marisa S; Bednarek, Sebastian Y; Rojo, Enrique

2013-06-01

Many soluble proteins transit through the trans-Golgi network (TGN) and the prevacuolar compartment (PVC) en route to the vacuole, but our mechanistic understanding of this vectorial trafficking step in plants is limited. In particular, it is unknown whether clathrin-coated vesicles (CCVs) participate in this transport step. Through a screen for modified transport to the vacuole (mtv) mutants that secrete the vacuolar protein VAC2, we identified MTV1, which encodes an epsin N-terminal homology protein, and MTV4, which encodes the ADP ribosylation factor GTPase-activating protein nevershed/AGD5. MTV1 and NEV/AGD5 have overlapping expression patterns and interact genetically to transport vacuolar cargo and promote plant growth, but they have no apparent roles in protein secretion or endocytosis. MTV1 and NEV/AGD5 colocalize with clathrin at the TGN and are incorporated into CCVs. Importantly, mtv1 nev/agd5 double mutants show altered subcellular distribution of CCV cargo exported from the TGN. Moreover, MTV1 binds clathrin in vitro, and NEV/AGD5 associates in vivo with clathrin, directly linking these proteins to CCV formation. These results indicate that MTV1 and NEV/AGD5 are key effectors for CCV-mediated trafficking of vacuolar proteins from the TGN to the PVC in plants.

Identification of Five Novel Variants in Chinese Oculocutaneous Albinism by Targeted Next-Generation Sequencing.

PubMed

Qiu, Biyuan; Ma, Tao; Peng, Chunyan; Zheng, Xiaoqin; Yang, Jiyun

2018-04-01

The diagnosis of oculocutaneous albinism (OCA) is established using clinical signs and symptoms. OCA is, however, a highly genetically heterogeneous disease with mutations identified in at least nineteen unique genes, many of which produce overlapping phenotypic traits. Thus, differentiating genetic OCA subtypes for diagnoses and genetic counseling is challenging, based on clinical presentation alone, and would benefit from a comprehensive molecular diagnostic. To develop and validate a more comprehensive, targeted, next-generation-sequencing-based diagnostic for the identification of OCA-causing variants. The genomic DNA samples from 28 OCA probands were analyzed by targeted next-generation sequencing (NGS), and the candidate variants were confirmed through Sanger sequencing. We observed mutations in the TYR, OCA2, and SLC45A2 genes in 25/28 (89%) patients with OCA. We identified 38 pathogenic variants among these three genes, including 5 novel variants: c.1970G>T (p.Gly657Val), c.1669A>C (p.Thr557Pro), c.2339-2A>C, and c.1349C>G (p.Thr450Arg) in OCA2; c.459_470delTTTTGCTGCCGA (p.Ala155_Phe158del) in SLC45A2. Our findings expand the mutational spectrum of OCA in the Chinese population, and the assay we developed should be broadly useful as a molecular diagnostic, and as an aid for genetic counseling for OCA patients.

Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy

PubMed Central

Kühnemund, Malte; Wei, Qingshan; Darai, Evangelia; Wang, Yingjie; Hernández-Neuta, Iván; Yang, Zhao; Tseng, Derek; Ahlford, Annika; Mathot, Lucy; Sjöblom, Tobias; Ozcan, Aydogan; Nilsson, Mats

2017-01-01

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies. PMID:28094784

TP53, PIK3CA, FBXW7 and KRAS Mutations in Esophageal Cancer Identified by Targeted Sequencing.

PubMed

Zheng, Huili; Wang, Yan; Tang, Chuanning; Jones, Lindsey; Ye, Hua; Zhang, Guangchun; Cao, Weihai; Li, Jingwen; Liu, Lifeng; Liu, Zhencong; Zhang, Chao; Lou, Feng; Liu, Zhiyuan; Li, Yangyang; Shi, Zhenfen; Zhang, Jingbo; Zhang, Dandan; Sun, Hong; Dong, Haichao; Dong, Zhishou; Guo, Baishuai; Yan, H E; Lu, Qingyu; Huang, Xue; Chen, Si-Yi

2016-01-01

Esophageal cancer (EC) is a common malignancy with significant morbidity and mortality. As individual cancers exhibit unique mutation patterns, identifying and characterizing gene mutations in EC that may serve as biomarkers might help predict patient outcome and guide treatment. Traditionally, personalized cancer DNA sequencing was impractical and expensive. Recent technological advancements have made targeted DNA sequencing more cost- and time-effective with reliable results. This technology may be useful for clinicians to direct patient treatment. The Ion PGM and AmpliSeq Cancer Panel was used to identify mutations at 737 hotspot loci of 45 cancer-related genes in 64 EC samples from Chinese patients. Frequent mutations were found in TP53 and less frequent mutations in PIK3CA, FBXW7 and KRAS. These results demonstrate that targeted sequencing can reliably identify mutations in individual tumors that make this technology a possibility for clinical use. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Systematic evaluation of a targeted gene capture sequencing panel for molecular diagnosis of retinitis pigmentosa.

PubMed

Huang, Hui; Chen, Yanhua; Chen, Huishuang; Ma, Yuanyuan; Chiang, Pei-Wen; Zhong, Jing; Liu, Xuyang; Asan; Wu, Jing; Su, Yan; Li, Xin; Deng, Jianlian; Huang, Yingping; Zhang, Xinxin; Li, Yang; Fan, Ning; Wang, Ying; Tang, Lihui; Shen, Jinting; Chen, Meiyan; Zhang, Xiuqing; Te, Deng; Banerjee, Santasree; Liu, Hui; Qi, Ming; Yi, Xin

2018-01-01

Inherited eye diseases are major causes of vision loss in both children and adults. Inherited eye diseases are characterized by clinical variability and pronounced genetic heterogeneity. Genetic testing may provide an accurate diagnosis for ophthalmic genetic disorders and allow gene therapy for specific diseases. A targeted gene capture panel was designed to capture exons of 283 inherited eye disease genes including 58 known causative retinitis pigmentosa (RP) genes. 180 samples were tested with this panel, 68 were previously tested by Sanger sequencing. Systematic evaluation of our method and comprehensive molecular diagnosis were carried on 99 RP patients. 96.85% targeted regions were covered by at least 20 folds, the accuracy of variants detection was 99.994%. In 4 of the 68 samples previously tested by Sanger sequencing, mutations of other diseases not consisting with the clinical diagnosis were detected by next-generation sequencing (NGS) not Sanger. Among the 99 RP patients, 64 (64.6%) were detected with pathogenic mutations, while in 3 patients, it was inconsistent between molecular diagnosis and their initial clinical diagnosis. After revisiting, one patient's clinical diagnosis was reclassified. In addition, 3 patients were found carrying large deletions. We have systematically evaluated our method and compared it with Sanger sequencing, and have identified a large number of novel mutations in a cohort of 99 RP patients. The results showed a sufficient accuracy of our method and suggested the importance of molecular diagnosis in clinical diagnosis.

Systematic evaluation of a targeted gene capture sequencing panel for molecular diagnosis of retinitis pigmentosa

PubMed Central

Ma, Yuanyuan; Chiang, Pei-Wen; Zhong, Jing; Liu, Xuyang; Asan; Wu, Jing; Su, Yan; Li, Xin; Deng, Jianlian; Huang, Yingping; Zhang, Xinxin; Li, Yang; Fan, Ning; Wang, Ying; Tang, Lihui; Shen, Jinting; Chen, Meiyan; Zhang, Xiuqing; Te, Deng; Banerjee, Santasree; Liu, Hui; Qi, Ming; Yi, Xin

2018-01-01

Background Inherited eye diseases are major causes of vision loss in both children and adults. Inherited eye diseases are characterized by clinical variability and pronounced genetic heterogeneity. Genetic testing may provide an accurate diagnosis for ophthalmic genetic disorders and allow gene therapy for specific diseases. Methods A targeted gene capture panel was designed to capture exons of 283 inherited eye disease genes including 58 known causative retinitis pigmentosa (RP) genes. 180 samples were tested with this panel, 68 were previously tested by Sanger sequencing. Systematic evaluation of our method and comprehensive molecular diagnosis were carried on 99 RP patients. Results 96.85% targeted regions were covered by at least 20 folds, the accuracy of variants detection was 99.994%. In 4 of the 68 samples previously tested by Sanger sequencing, mutations of other diseases not consisting with the clinical diagnosis were detected by next-generation sequencing (NGS) not Sanger. Among the 99 RP patients, 64 (64.6%) were detected with pathogenic mutations, while in 3 patients, it was inconsistent between molecular diagnosis and their initial clinical diagnosis. After revisiting, one patient’s clinical diagnosis was reclassified. In addition, 3 patients were found carrying large deletions. Conclusions We have systematically evaluated our method and compared it with Sanger sequencing, and have identified a large number of novel mutations in a cohort of 99 RP patients. The results showed a sufficient accuracy of our method and suggested the importance of molecular diagnosis in clinical diagnosis. PMID:29641573

Single-Center Experience with a Targeted Next Generation Sequencing Assay for Assessment of Relevant Somatic Alterations in Solid Tumors.

PubMed

Paasinen-Sohns, Aino; Koelzer, Viktor H; Frank, Angela; Schafroth, Julian; Gisler, Aline; Sachs, Melanie; Graber, Anne; Rothschild, Sacha I; Wicki, Andreas; Cathomas, Gieri; Mertz, Kirsten D

2017-03-01

Companion diagnostics rely on genomic testing of molecular alterations to enable effective cancer treatment. Here we report the clinical application and validation of the Oncomine Focus Assay (OFA), an integrated, commercially available next-generation sequencing (NGS) assay for the rapid and simultaneous detection of single nucleotide variants, short insertions and deletions, copy number variations, and gene rearrangements in 52 cancer genes with therapeutic relevance. Two independent patient cohorts were investigated to define the workflow, turnaround times, feasibility, and reliability of OFA targeted sequencing in clinical application and using archival material. Cohort I consisted of 59 diagnostic clinical samples from the daily routine submitted for molecular testing over a 4-month time period. Cohort II consisted of 39 archival melanoma samples that were up to 15years old. Libraries were prepared from isolated nucleic acids and sequenced on the Ion Torrent PGM sequencer. Sequencing datasets were analyzed using the Ion Reporter software. Genomic alterations were identified and validated by orthogonal conventional assays including pyrosequencing and immunohistochemistry. Sequencing results of both cohorts, including archival formalin-fixed, paraffin-embedded material stored up to 15years, were consistent with published variant frequencies. A concordance of 100% between established assays and OFA targeted NGS was observed. The OFA workflow enabled a turnaround of 3½ days. Taken together, OFA was found to be a convenient tool for fast, reliable, broadly applicable and cost-effective targeted NGS of tumor samples in routine diagnostics. Thus, OFA has strong potential to become an important asset for precision oncology. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Cost-Effectiveness of Treatment Sequences of Chemotherapies and Targeted Biologics for Elderly Metastatic Colorectal Cancer Patients.

PubMed

Parikh, Rohan C; Du, Xianglin L; Robert, Morgan O; Lairson, David R

2017-01-01

Treatment patterns for metastatic colorectal cancer (mCRC) patients have changed considerably over the last decade with the introduction of new chemotherapies and targeted biologics. These treatments are often administered in various sequences with limited evidence regarding their cost-effectiveness. To conduct a pharmacoeconomic evaluation of commonly administered treatment sequences among elderly mCRC patients. A probabilistic discrete event simulation model assuming Weibull distribution was developed to evaluate the cost-effectiveness of the following common treatment sequences: (a) first-line oxaliplatin/irinotecan followed by second-line oxaliplatin/irinotecan + bevacizumab (OI-OIB); (b) first-line oxaliplatin/irinotecan + bevacizumab followed by second-line oxaliplatin/irinotecan + bevacizumab (OIB-OIB); (c) OI-OIB followed by a third-line targeted biologic (OI-OIB-TB); and (d) OIB-OIB followed by a third-line targeted biologic (OIB-OIB-TB). Input parameters for the model were primarily obtained from the Surveillance, Epidemiology, and End Results-Medicare linked dataset for incident mCRC patients aged 65 years and older diagnosed from January 2004 through December 2009. A probabilistic sensitivity analysis was performed to account for parameter uncertainty. Costs (2014 U.S. dollars) and effectiveness were discounted at an annual rate of 3%. In the base case analyses, at the willingness-to-pay (WTP) threshold of $100,000/quality-adjusted life-year (QALY) gained, the treatment sequence OIB-OIB (vs. OI-OIB) was not cost-effective with an incremental cost-effectiveness ratio (ICER) per patient of $119,007/QALY; OI-OIB-TB (vs. OIB-OIB) was dominated; and OIB-OIB-TB (vs. OIB-OIB) was not cost-effective with an ICER of $405,857/QALY. Results similar to the base case analysis were obtained assuming log-normal distribution. Cost-effectiveness acceptability curves derived from a probabilistic sensitivity analysis showed that at a WTP of $100,000/QALY gained, sequence

A Systematic Prediction of Drug-Target Interactions Using Molecular Fingerprints and Protein Sequences.

PubMed

Huang, Yu-An; You, Zhu-Hong; Chen, Xing

2018-01-01

Drug-Target Interactions (DTI) play a crucial role in discovering new drug candidates and finding new proteins to target for drug development. Although the number of detected DTI obtained by high-throughput techniques has been increasing, the number of known DTI is still limited. On the other hand, the experimental methods for detecting the interactions among drugs and proteins are costly and inefficient. Therefore, computational approaches for predicting DTI are drawing increasing attention in recent years. In this paper, we report a novel computational model for predicting the DTI using extremely randomized trees model and protein amino acids information. More specifically, the protein sequence is represented as a Pseudo Substitution Matrix Representation (Pseudo-SMR) descriptor in which the influence of biological evolutionary information is retained. For the representation of drug molecules, a novel fingerprint feature vector is utilized to describe its substructure information. Then the DTI pair is characterized by concatenating the two vector spaces of protein sequence and drug substructure. Finally, the proposed method is explored for predicting the DTI on four benchmark datasets: Enzyme, Ion Channel, GPCRs and Nuclear Receptor. The experimental results demonstrate that this method achieves promising prediction accuracies of 89.85%, 87.87%, 82.99% and 81.67%, respectively. For further evaluation, we compared the performance of Extremely Randomized Trees model with that of the state-of-the-art Support Vector Machine classifier. And we also compared the proposed model with existing computational models, and confirmed 15 potential drug-target interactions by looking for existing databases. The experiment results show that the proposed method is feasible and promising for predicting drug-target interactions for new drug candidate screening based on sizeable features. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

High throughput deep degradome sequencing reveals microRNAs and their targets in response to drought stress in mulberry (Morus alba).

PubMed

Li, Ruixue; Chen, Dandan; Wang, Taichu; Wan, Yizhen; Li, Rongfang; Fang, Rongjun; Wang, Yuting; Hu, Fei; Zhou, Hong; Li, Long; Zhao, Weiguo

2017-01-01

MicroRNAs (miRNAs) play important regulatory roles by targeting mRNAs for cleavage or translational repression. Identification of miRNA targets is essential to better understanding the roles of miRNAs. miRNA targets have not been well characterized in mulberry (Morus alba). To anatomize miRNA guided gene regulation under drought stress, transcriptome-wide high throughput degradome sequencing was used in this study to directly detect drought stress responsive miRNA targets in mulberry. A drought library (DL) and a contrast library (CL) were constructed to capture the cleaved mRNAs for sequencing. In CL, 409 target genes of 30 conserved miRNA families and 990 target genes of 199 novel miRNAs were identified. In DL, 373 target genes of 30 conserved miRNA families and 950 target genes of 195 novel miRNAs were identified. Of the conserved miRNA families in DL, mno-miR156, mno-miR172, and mno-miR396 had the highest number of targets with 54, 52 and 41 transcripts, respectively, indicating that these three miRNA families and their target genes might play important functions in response to drought stress in mulberry. Additionally, we found that many of the target genes were transcription factors. By analyzing the miRNA-target molecular network, we found that the DL independent networks consisted of 838 miRNA-mRNA pairs (63.34%). The expression patterns of 11 target genes and 12 correspondent miRNAs were detected using qRT-PCR. Six miRNA targets were further verified by RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-5' RACE). Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these target transcripts were implicated in a broad range of biological processes and various metabolic pathways. This is the first study to comprehensively characterize target genes and their associated miRNAs in response to drought stress by degradome sequencing in mulberry. This study provides a framework for understanding

Bamgineer: Introduction of simulated allele-specific copy number variants into exome and targeted sequence data sets.

PubMed

Samadian, Soroush; Bruce, Jeff P; Pugh, Trevor J

2018-03-01

Somatic copy number variations (CNVs) play a crucial role in development of many human cancers. The broad availability of next-generation sequencing data has enabled the development of algorithms to computationally infer CNV profiles from a variety of data types including exome and targeted sequence data; currently the most prevalent types of cancer genomics data. However, systemic evaluation and comparison of these tools remains challenging due to a lack of ground truth reference sets. To address this need, we have developed Bamgineer, a tool written in Python to introduce user-defined haplotype-phased allele-specific copy number events into an existing Binary Alignment Mapping (BAM) file, with a focus on targeted and exome sequencing experiments. As input, this tool requires a read alignment file (BAM format), lists of non-overlapping genome coordinates for introduction of gains and losses (bed file), and an optional file defining known haplotypes (vcf format). To improve runtime performance, Bamgineer introduces the desired CNVs in parallel using queuing and parallel processing on a local machine or on a high-performance computing cluster. As proof-of-principle, we applied Bamgineer to a single high-coverage (mean: 220X) exome sequence file from a blood sample to simulate copy number profiles of 3 exemplar tumors from each of 10 tumor types at 5 tumor cellularity levels (20-100%, 150 BAM files in total). To demonstrate feasibility beyond exome data, we introduced read alignments to a targeted 5-gene cell-free DNA sequencing library to simulate EGFR amplifications at frequencies consistent with circulating tumor DNA (10, 1, 0.1 and 0.01%) while retaining the multimodal insert size distribution of the original data. We expect Bamgineer to be of use for development and systematic benchmarking of CNV calling algorithms by users using locally-generated data for a variety of applications. The source code is freely available at http://github.com/pughlab/bamgineer.

Influence of quasi-specific sites on kinetics of target DNA search by a sequence-specific DNA-binding protein.

PubMed

Kemme, Catherine A; Esadze, Alexandre; Iwahara, Junji

2015-11-10

Functions of transcription factors require formation of specific complexes at particular sites in cis-regulatory elements of genes. However, chromosomal DNA contains numerous sites that are similar to the target sequences recognized by transcription factors. The influence of such "quasi-specific" sites on functions of the transcription factors is not well understood at present by experimental means. In this work, using fluorescence methods, we have investigated the influence of quasi-specific DNA sites on the efficiency of target location by the zinc finger DNA-binding domain of the inducible transcription factor Egr-1, which recognizes a 9 bp sequence. By stopped-flow assays, we measured the kinetics of Egr-1's association with a target site on 143 bp DNA in the presence of various competitor DNAs, including nonspecific and quasi-specific sites. The presence of quasi-specific sites on competitor DNA significantly decelerated the target association by the Egr-1 protein. The impact of the quasi-specific sites depended strongly on their affinity, their concentration, and the degree of their binding to the protein. To quantitatively describe the kinetic impact of the quasi-specific sites, we derived an analytical form of the apparent kinetic rate constant for the target association and used it for fitting to the experimental data. Our kinetic data with calf thymus DNA as a competitor suggested that there are millions of high-affinity quasi-specific sites for Egr-1 among the 3 billion bp of genomic DNA. This study quantitatively demonstrates that naturally abundant quasi-specific sites on DNA can considerably impede the target search processes of sequence-specific DNA-binding proteins.

Influence of Quasi-Specific Sites on Kinetics of Target DNA Search by a Sequence-Specific DNA-Binding Protein

PubMed Central

2015-01-01

Functions of transcription factors require formation of specific complexes at particular sites in cis-regulatory elements of genes. However, chromosomal DNA contains numerous sites that are similar to the target sequences recognized by transcription factors. The influence of such “quasi-specific” sites on functions of the transcription factors is not well understood at present by experimental means. In this work, using fluorescence methods, we have investigated the influence of quasi-specific DNA sites on the efficiency of target location by the zinc finger DNA-binding domain of the inducible transcription factor Egr-1, which recognizes a 9 bp sequence. By stopped-flow assays, we measured the kinetics of Egr-1’s association with a target site on 143 bp DNA in the presence of various competitor DNAs, including nonspecific and quasi-specific sites. The presence of quasi-specific sites on competitor DNA significantly decelerated the target association by the Egr-1 protein. The impact of the quasi-specific sites depended strongly on their affinity, their concentration, and the degree of their binding to the protein. To quantitatively describe the kinetic impact of the quasi-specific sites, we derived an analytical form of the apparent kinetic rate constant for the target association and used it for fitting to the experimental data. Our kinetic data with calf thymus DNA as a competitor suggested that there are millions of high-affinity quasi-specific sites for Egr-1 among the 3 billion bp of genomic DNA. This study quantitatively demonstrates that naturally abundant quasi-specific sites on DNA can considerably impede the target search processes of sequence-specific DNA-binding proteins. PMID:26502071

Vacuolar ATPase in Phagosome-Lysosome Fusion

PubMed Central

Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S.; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

2015-01-01

The vacuolar H+-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. PMID:25903133

Vacuolar ATPase in phagosome-lysosome fusion.

PubMed

Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

2015-05-29

The vacuolar H(+)-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The emerging structure of vacuolar ATPases.

PubMed

Drory, Omri; Nelson, Nathan

2006-10-01

Bioenergetics and physiology of primary pumps have been revitalized by new insights into the mechanism of energizing biomembranes. Structural information is becoming available, and the three-dimensional structure of F-ATPase is being resolved. The growing understanding of the fundamental mechanism of energy coupling may revolutionize our view of biological processes. The F- and V-ATPases (vacuolar-type ATPase) exhibit a common mechanical design in which nucleotide-binding on the catalytic sector, through a cycle of conformation changes, drives the transmembrane passage of protons by turning a membrane-embedded rotor. This motor can run in forward or reverse directions, hydrolyzing ATP as it pumps protons uphill or creating ATP as protons flow downhill. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as an ATP-dependent proton pump. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. V- and F-ATPases have similar structure and mechanism of action, and several of their subunits evolved from common ancestors. Electron microscopy studies of V-ATPase revealed its general structure at low resolution. Recently, several structures of V-ATPase subunits, solved by X-ray crystallography with atomic resolution, were published. This, together with electron microscopy low-resolution maps of the whole complex, and biochemistry cross-linking experiments, allows construction of a structural model for a part of the complex that may be used as a working hypothesis for future research.

Hi-Plex for Simple, Accurate, and Cost-Effective Amplicon-based Targeted DNA Sequencing.

PubMed

Pope, Bernard J; Hammet, Fleur; Nguyen-Dumont, Tu; Park, Daniel J

2018-01-01

Hi-Plex is a suite of methods to enable simple, accurate, and cost-effective highly multiplex PCR-based targeted sequencing (Nguyen-Dumont et al., Biotechniques 58:33-36, 2015). At its core is the principle of using gene-specific primers (GSPs) to "seed" (or target) the reaction and universal primers to "drive" the majority of the reaction. In this manner, effects on amplification efficiencies across the target amplicons can, to a large extent, be restricted to early seeding cycles. Product sizes are defined within a relatively narrow range to enable high-specificity size selection, replication uniformity across target sites (including in the context of fragmented input DNA such as that derived from fixed tumor specimens (Nguyen-Dumont et al., Biotechniques 55:69-74, 2013; Nguyen-Dumont et al., Anal Biochem 470:48-51, 2015), and application of high-specificity genetic variant calling algorithms (Pope et al., Source Code Biol Med 9:3, 2014; Park et al., BMC Bioinformatics 17:165, 2016). Hi-Plex offers a streamlined workflow that is suitable for testing large numbers of specimens without the need for automation.

CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection.

PubMed

Kim, Heon Seok; Lee, Kyungjin; Bae, Sangsu; Park, Jeongbin; Lee, Chong-Kyo; Kim, Meehyein; Kim, Eunji; Kim, Minju; Kim, Seokjoong; Kim, Chonsaeng; Kim, Jin-Soo

2017-06-23

Several groups have used genome-wide libraries of lentiviruses encoding small guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids and target gene identification via whole-genome sequencing (WGS) confirmation of authentic mutations rather than statistical estimation through targeted amplicon sequencing. We used 30,840 pairs of individually synthesized oligonucleotides to construct the genome-scale sgRNA library, collectively targeting 10,280 human genes ( i.e. three sgRNAs per gene). These plasmid libraries were co-transfected with a Cas9-expression plasmid into human cells, which were then treated with cytotoxic drugs or viruses. Only cells lacking key factors essential for cytotoxic drug metabolism or viral infection were able to survive. Genomic DNA isolated from cells that survived these challenges was subjected to WGS to directly identify CRISPR/Cas9-mediated causal mutations essential for cell survival. With this approach, we were able to identify known and novel genes essential for viral infection in human cells. We propose that genome-wide sgRNA screens based on plasmids coupled with WGS are powerful tools for forward genetics studies and drug target discovery. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The Loss of Vacuolar Protein Sorting 11 (vps11) Causes Retinal Pathogenesis in a Vertebrate Model of Syndromic Albinism

PubMed Central

Thomas, Jennifer L.; Vihtelic, Thomas S.; denDekker, Aaron D.; Willer, Gregory; Luo, Xixia; Murphy, Taylor R.; Gregg, Ronald G.; Hyde, David R.

2011-01-01

Purpose. To establish the zebrafish platinum mutant as a model for studying vision defects caused by syndromic albinism diseases such as Chediak-Higashi syndrome, Griscelli syndrome, and Hermansky-Pudlak syndrome (HPS). Methods. Bulked segregant analysis and candidate gene sequencing revealed that the zebrafish platinum mutation is a single-nucleotide insertion in the vps11 (vacuolar protein sorting 11) gene. Expression of vps11 was determined by RT-PCR and in situ hybridization. Mutants were analyzed for pigmentation defects and retinal disease by histology, immunohistochemistry, and transmission electron microscopy. Results. Phenocopy and rescue experiments determined that a loss of Vps11 results in the platinum phenotype. Expression of vps11 appeared ubiquitous during zebrafish development, with stronger expression in the developing retina and retinal pigmented epithelium (RPE). Zebrafish platinum mutants exhibited reduced pigmentation in the body and RPE; however, melanophore development, migration, and dispersion occurred normally. RPE, photoreceptors, and inner retinal neurons formed normally in zebrafish platinum mutants. However, a gradual loss of RPE, an absence of mature melanosomes, and the subsequent degradation of RPE/photoreceptor interdigitation was observed. Conclusions. These data show that Vps11 is not necessary for normal retinal development or initiation of melanin biosynthesis, but is essential for melanosome maturation and healthy maintenance of the RPE and photoreceptors. PMID:21330665

vph6 mutants of Saccharomyces cerevisiae require calcineurin for growth and are defective in vacuolar H(+)-ATPase assembly.

PubMed

Hemenway, C S; Dolinski, K; Cardenas, M E; Hiller, M A; Jones, E W; Heitman, J

1995-11-01

We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.

Evaluation of targeted exome sequencing for 28 protein-based blood group systems, including the homologous gene systems, for blood group genotyping.

PubMed

Schoeman, Elizna M; Lopez, Genghis H; McGowan, Eunike C; Millard, Glenda M; O'Brien, Helen; Roulis, Eileen V; Liew, Yew-Wah; Martin, Jacqueline R; McGrath, Kelli A; Powley, Tanya; Flower, Robert L; Hyland, Catherine A

2017-04-01

Blood group single nucleotide polymorphism genotyping probes for a limited range of polymorphisms. This study investigated whether massively parallel sequencing (also known as next-generation sequencing), with a targeted exome strategy, provides an extended blood group genotype and the extent to which massively parallel sequencing correctly genotypes in homologous gene systems, such as RH and MNS. Donor samples (n = 28) that were extensively phenotyped and genotyped using single nucleotide polymorphism typing, were analyzed using the TruSight One Sequencing Panel and MiSeq platform. Genes for 28 protein-based blood group systems, GATA1, and KLF1 were analyzed. Copy number variation analysis was used to characterize complex structural variants in the GYPC and RH systems. The average sequencing depth per target region was 66.2 ± 39.8. Each sample harbored on average 43 ± 9 variants, of which 10 ± 3 were used for genotyping. For the 28 samples, massively parallel sequencing variant sequences correctly matched expected sequences based on single nucleotide polymorphism genotyping data. Copy number variation analysis defined the Rh C/c alleles and complex RHD hybrids. Hybrid RHD*D-CE-D variants were correctly identified, but copy number variation analysis did not confidently distinguish between D and CE exon deletion versus rearrangement. The targeted exome sequencing strategy employed extended the range of blood group genotypes detected compared with single nucleotide polymorphism typing. This single-test format included detection of complex MNS hybrid cases and, with copy number variation analysis, defined RH hybrid genes along with the RHCE*C allele hitherto difficult to resolve by variant detection. The approach is economical compared with whole-genome sequencing and is suitable for a red blood cell reference laboratory setting. © 2017 AABB.

Comparative Analysis of Predicted Plastid-Targeted Proteomes of Sequenced Higher Plant Genomes

PubMed Central

Schaeffer, Scott; Harper, Artemus; Raja, Rajani; Jaiswal, Pankaj; Dhingra, Amit

2014-01-01

Plastids are actively involved in numerous plant processes critical to growth, development and adaptation. They play a primary role in photosynthesis, pigment and monoterpene synthesis, gravity sensing, starch and fatty acid synthesis, as well as oil, and protein storage. We applied two complementary methods to analyze the recently published apple genome (Malus × domestica) to identify putative plastid-targeted proteins, the first using TargetP and the second using a custom workflow utilizing a set of predictive programs. Apple shares roughly 40% of its 10,492 putative plastid-targeted proteins with that of the Arabidopsis (Arabidopsis thaliana) plastid-targeted proteome as identified by the Chloroplast 2010 project and ∼57% of its entire proteome with Arabidopsis. This suggests that the plastid-targeted proteomes between apple and Arabidopsis are different, and interestingly alludes to the presence of differential targeting of homologs between the two species. Co-expression analysis of 2,224 genes encoding putative plastid-targeted apple proteins suggests that they play a role in plant developmental and intermediary metabolism. Further, an inter-specific comparison of Arabidopsis, Prunus persica (Peach), Malus × domestica (Apple), Populus trichocarpa (Black cottonwood), Fragaria vesca (Woodland Strawberry), Solanum lycopersicum (Tomato) and Vitis vinifera (Grapevine) also identified a large number of novel species-specific plastid-targeted proteins. This analysis also revealed the presence of alternatively targeted homologs across species. Two separate analyses revealed that a small subset of proteins, one representing 289 protein clusters and the other 737 unique protein sequences, are conserved between seven plastid-targeted angiosperm proteomes. Majority of the novel proteins were annotated to play roles in stress response, transport, catabolic processes, and cellular component organization. Our results suggest that the current state of knowledge regarding

Targeted Sequencing of Venom Genes from Cone Snail Genomes Improves Understanding of Conotoxin Molecular Evolution

PubMed Central

Mahardika, Gusti N

2018-01-01

Abstract To expand our capacity to discover venom sequences from the genomes of venomous organisms, we applied targeted sequencing techniques to selectively recover venom gene superfamilies and nontoxin loci from the genomes of 32 cone snail species (family, Conidae), a diverse group of marine gastropods that capture their prey using a cocktail of neurotoxic peptides (conotoxins). We were able to successfully recover conotoxin gene superfamilies across all species with high confidence (> 100× coverage) and used these data to provide new insights into conotoxin evolution. First, we found that conotoxin gene superfamilies are composed of one to six exons and are typically short in length (mean = ∼85 bp). Second, we expanded our understanding of the following genetic features of conotoxin evolution: 1) positive selection, where exons coding the mature toxin region were often three times more divergent than their adjacent noncoding regions, 2) expression regulation, with comparisons to transcriptome data showing that cone snails only express a fraction of the genes available in their genome (24–63%), and 3) extensive gene turnover, where Conidae species varied from 120 to 859 conotoxin gene copies. Finally, using comparative phylogenetic methods, we found that while diet specificity did not predict patterns of conotoxin evolution, dietary breadth was positively correlated with total conotoxin gene diversity. Overall, the targeted sequencing technique demonstrated here has the potential to radically increase the pace at which venom gene families are sequenced and studied, reshaping our ability to understand the impact of genetic changes on ecologically relevant phenotypes and subsequent diversification. PMID:29514313

Sequence- and Interactome-Based Prediction of Viral Protein Hotspots Targeting Host Proteins: A Case Study for HIV Nef

PubMed Central

Sarmady, Mahdi; Dampier, William; Tozeren, Aydin

2011-01-01

Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk. PMID:21738584

Epizootiologic studies of avian vacuolar myelinopathy in waterbirds

USGS Publications Warehouse

Rocke, Tonie E.; Thomas, N.J.; Augspurger, T.; Miller, Kimberli J.

2002-01-01

Epizootic avian vacuolar myelinopathy (AVM) was first recognized as a neurologic disease in bald eagles (Haliaeetus leucocephalus) and American coots (Fulica americana) in Arkansas, USA in 1994 and 1996, respectively, but attempts to identify the etiology of the disease have been unsuccessful to date. Between 1998 and 2001, wing clipped sentinel birds (wild American coots and game farm mallards [Anas platyrhynchos]) were released at Lake Surf, North Carolina, a lake with recurrent outbreaks of AVM, in order to gain a better understanding of the epizootiology of the disease. As early as 5-7 days post-release, sentinel coots and mallards showed neurologic signs of disease and were confirmed with AVM upon histologic examination of their brains. Serial releases of sentinel mallards during the summer, fall, and winter of 2000-01 demonstrated that exposure to the causative agent at a threshold sufficient to manifest disease was seasonal and occurred over about a 2 mo period, during November and December. Our findings that disease onset can be very rapid (5-7 days) and that exposure to the causative agent of AVM is site-specific, seasonal (late fall to early winter), and occurs over a relatively short duration (several months) supports the hypothesis that the disease is caused by a chemical substance, most likely of natural origin.

Evaluation of Targeted Next-Generation Sequencing for Detection of Bovine Pathogens in Clinical Samples.

PubMed

Anis, Eman; Hawkins, Ian K; Ilha, Marcia R S; Woldemeskel, Moges W; Saliki, Jeremiah T; Wilkes, Rebecca P

2018-07-01

The laboratory diagnosis of infectious diseases, especially those caused by mixed infections, is challenging. Routinely, it requires submission of multiple samples to separate laboratories. Advances in next-generation sequencing (NGS) have provided the opportunity for development of a comprehensive method to identify infectious agents. This study describes the use of target-specific primers for PCR-mediated amplification with the NGS technology in which pathogen genomic regions of interest are enriched and selectively sequenced from clinical samples. In the study, 198 primers were designed to target 43 common bovine and small-ruminant bacterial, fungal, viral, and parasitic pathogens, and a bioinformatics tool was specifically constructed for the detection of targeted pathogens. The primers were confirmed to detect the intended pathogens by testing reference strains and isolates. The method was then validated using 60 clinical samples (including tissues, feces, and milk) that were also tested with other routine diagnostic techniques. The detection limits of the targeted NGS method were evaluated using 10 representative pathogens that were also tested by quantitative PCR (qPCR), and the NGS method was able to detect the organisms from samples with qPCR threshold cycle ( C T ) values in the 30s. The method was successful for the detection of multiple pathogens in the clinical samples, including some additional pathogens missed by the routine techniques because the specific tests needed for the particular organisms were not performed. The results demonstrate the feasibility of the approach and indicate that it is possible to incorporate NGS as a diagnostic tool in a cost-effective manner into a veterinary diagnostic laboratory. Copyright © 2018 Anis et al.

Comparative Analysis of Fruit Ripening-Related miRNAs and Their Targets in Blueberry Using Small RNA and Degradome Sequencing

PubMed Central

Hou, Yanming; Zhai, Lulu; Li, Xuyan; Xue, Yu; Wang, Jingjing; Yang, Pengjie; Cao, Chunmei; Li, Hongxue; Cui, Yuhai; Bian, Shaomin

2017-01-01

MicroRNAs (miRNAs) play vital roles in the regulation of fruit development and ripening. Blueberry is an important small berry fruit crop with economical and nutritional value. However, nothing is known about the miRNAs and their targets involved in blueberry fruit ripening. In this study, using high-throughput sequencing of small RNAs, 84 known miRNAs belonging to 28 families and 16 novel miRNAs were identified in white fruit (WF) and blue fruit (BF) libraries, which represent fruit ripening onset and in progress, respectively. Among them, 41 miRNAs were shown to be differentially expressed during fruit maturation, and 16 miRNAs representing 16 families were further chosen to validate the sRNA sequencing data by stem-loop qRT-PCR. Meanwhile, 178 targets were identified for 41 known and 7 novel miRNAs in WF and BF libraries using degradome sequencing, and targets of miR160 were validated using RLM-RACE (RNA Ligase-Mediated (RLM)-Rapid Amplification of cDNA Ends) approach. Moreover, the expression patterns of 6 miRNAs and their targets were examined during fruit development and ripening. Finally, integrative analysis of miRNAs and their targets revealed a complex miRNA-mRNA regulatory network involving a wide variety of biological processes. The findings will facilitate future investigations of the miRNA-mediated mechanisms that regulate fruit development and ripening in blueberry. PMID:29257112

Activity of the C-terminal-dependent vacuolar sorting signal of horseradish peroxidase C1a is enhanced by its secondary structure.

PubMed

Matsui, Takeshi; Tabayashi, Ayako; Iwano, Megumi; Shinmyo, Atsuhiko; Kato, Ko; Nakayama, Hideki

2011-02-01

Plant class III peroxidase (PRX) catalyzes the oxidation and oxidative polymerization of a variety of phenolic compounds while reducing hydrogen peroxide. PRX proteins are classified into apoplast type and vacuole type based on the absence or the presence of C-terminal propeptides, which probably function as vacuolar sorting signals (VSSs). In this study, in order to improve our understanding of vacuole-type PRX, we analyzed regulatory mechanisms of vacuolar sorting of a model vacuole-type PRX, the C1a isozyme of horseradish (Armoracia rusticana) (HRP C1a). Using cultured transgenic tobacco cells and protoplasts derived from horseradish leaves, we characterized HRP C1a's VSS, which is a 15 amino acid C-terminal propeptide (C15). We found that the C-terminal hexapeptide of C15 (C6), which is well conserved among vacuole-type PRX proteins, forms the core of the C-terminal-dependent VSS. We also found that the function of C6 is enhanced by the remaining N-terminal part of C15 which probably folds into an amphiphilic α-helix.

Insights into Deep-Sea Sediment Fungal Communities from the East Indian Ocean Using Targeted Environmental Sequencing Combined with Traditional Cultivation

PubMed Central

Zhang, Xiao-yong; Tang, Gui-ling; Xu, Xin-ya; Nong, Xu-hua; Qi, Shu-Hua

2014-01-01

The fungal diversity in deep-sea environments has recently gained an increasing amount attention. Our knowledge and understanding of the true fungal diversity and the role it plays in deep-sea environments, however, is still limited. We investigated the fungal community structure in five sediments from a depth of ∼4000 m in the East India Ocean using a combination of targeted environmental sequencing and traditional cultivation. This approach resulted in the recovery of a total of 45 fungal operational taxonomic units (OTUs) and 20 culturable fungal phylotypes. This finding indicates that there is a great amount of fungal diversity in the deep-sea sediments collected in the East Indian Ocean. Three fungal OTUs and one culturable phylotype demonstrated high divergence (89%–97%) from the existing sequences in the GenBank. Moreover, 44.4% fungal OTUs and 30% culturable fungal phylotypes are new reports for deep-sea sediments. These results suggest that the deep-sea sediments from the East India Ocean can serve as habitats for new fungal communities compared with other deep-sea environments. In addition, different fungal community could be detected when using targeted environmental sequencing compared with traditional cultivation in this study, which suggests that a combination of targeted environmental sequencing and traditional cultivation will generate a more diverse fungal community in deep-sea environments than using either targeted environmental sequencing or traditional cultivation alone. This study is the first to report new insights into the fungal communities in deep-sea sediments from the East Indian Ocean, which increases our knowledge and understanding of the fungal diversity in deep-sea environments. PMID:25272044

Vacuolar SPX-MFS transporters are essential for phosphate adaptation in plants.

PubMed

Liu, Jinlong; Fu, Shaomin; Yang, Lei; Luan, Mingda; Zhao, Fugeng; Luan, Sheng; Lan, Wenzhi

2016-08-02

To survive in most soils in which inorganic phosphate (Pi) levels are limited and constantly changing, plants universally use the vacuoles as cellular Pi "sink" and "source" to maintain Pi homeostasis. However, the transporters that mediate Pi sequestration into the vacuoles remain unknown. Recently, we and other 2 groups independently identified the members of SPS-MSF family as the candidates for tonoplast Pi transporters in Arabidopsis thaliana and Oryza sativa. We and Liu et al. demonstrated that one of SPS-MSF member, VPT1 (Vacuolar Phosphate Transporter 1), also named as PHT5;1 (Phosphate Transporter 5;1), plays a predominant role in Pi sequestration of vacuoles in Arabidopsis. Here we show that vpt1 mutants and VPT1-GFP overexpressing lines displayed sensitive to Pi stress under the hydroponic system containing the medium with low iron, supporting that VPT1 is essential for Arabidopsis to adapt phosphate stress.

PACCMIT/PACCMIT-CDS: identifying microRNA targets in 3' UTRs and coding sequences.

PubMed

Šulc, Miroslav; Marín, Ray M; Robins, Harlan S; Vaníček, Jiří

2015-07-01

The purpose of the proposed web server, publicly available at http://paccmit.epfl.ch, is to provide a user-friendly interface to two algorithms for predicting messenger RNA (mRNA) molecules regulated by microRNAs: (i) PACCMIT (Prediction of ACcessible and/or Conserved MIcroRNA Targets), which identifies primarily mRNA transcripts targeted in their 3' untranslated regions (3' UTRs), and (ii) PACCMIT-CDS, designed to find mRNAs targeted within their coding sequences (CDSs). While PACCMIT belongs among the accurate algorithms for predicting conserved microRNA targets in the 3' UTRs, the main contribution of the web server is 2-fold: PACCMIT provides an accurate tool for predicting targets also of weakly conserved or non-conserved microRNAs, whereas PACCMIT-CDS addresses the lack of similar portals adapted specifically for targets in CDS. The web server asks the user for microRNAs and mRNAs to be analyzed, accesses the precomputed P-values for all microRNA-mRNA pairs from a database for all mRNAs and microRNAs in a given species, ranks the predicted microRNA-mRNA pairs, evaluates their significance according to the false discovery rate and finally displays the predictions in a tabular form. The results are also available for download in several standard formats. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

External Guide Sequences Targeting the aac(6′)-Ib mRNA Induce Inhibition of Amikacin Resistance▿

PubMed Central

Bistué, Alfonso J. C. Soler; Ha, Hongphuc; Sarno, Renee; Don, Michelle; Zorreguieta, Angeles; Tolmasky, Marcelo E.

2007-01-01

The dissemination of AAC(6′)-I-type acetyltransferases have rendered amikacin and other aminoglycosides all but useless in some parts of the world. Antisense technologies could be an alternative to extend the life of these antibiotics. External guide sequences are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. Thirteen-nucleotide external guide sequences complementary to locations within five regions accessible for interaction with antisense oligonucleotides in the mRNA that encodes AAC(6′)-Ib were analyzed. While small variations in the location targeted by different external guide sequences resulted in big changes in efficiency of binding to native aac(6′)-Ib mRNA, most of them induced high levels of RNase P-mediated cleavage in vitro. Recombinant plasmids coding for selected external guide sequences were introduced into Escherichia coli harboring aac(6′)-Ib, and the transformant strains were tested to determine their resistance to amikacin. The two external guide sequences that showed the strongest binding efficiency to the mRNA in vitro, EGSC3 and EGSA2, interfered with expression of the resistance phenotype at different degrees. Growth curve experiments showed that E. coli cells harboring a plasmid coding for EGSC3, the external guide sequence with the highest mRNA binding affinity in vitro, did not grow for at least 300 min in the presence of 15 μg of amikacin/ml. EGSA2, which had a lower mRNA-binding affinity in vitro than EGSC3, inhibited the expression of amikacin resistance at a lesser level; growth of E. coli harboring a plasmid coding for EGSA2, in the presence of 15 μg of amikacin/ml was undetectable for 200 min but reached an optical density at 600 nm of 0.5 after 5 h of incubation. Our results indicate that the use of external guide sequences could be a viable strategy to preserve the efficacy of amikacin. PMID:17387154

Identification of a novel LMF1 nonsense mutation responsible for severe hypertriglyceridemia by targeted next-generation sequencing.

PubMed

Cefalù, Angelo B; Spina, Rossella; Noto, Davide; Ingrassia, Valeria; Valenti, Vincenza; Giammanco, Antonina; Fayer, Francesca; Misiano, Gabriella; Cocorullo, Gianfranco; Scrimali, Chiara; Palesano, Ornella; Altieri, Grazia I; Ganci, Antonina; Barbagallo, Carlo M; Averna, Maurizio R

Severe hypertriglyceridemia (HTG) may result from mutations in genes affecting the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. The aim of this study was to develop a targeted next-generation sequencing panel for the molecular diagnosis of disorders characterized by severe HTG. We developed a targeted customized panel for next-generation sequencing Ion Torrent Personal Genome Machine to capture the coding exons and intron/exon boundaries of 18 genes affecting the main pathways of TG synthesis and metabolism. We sequenced 11 samples of patients with severe HTG (TG>885 mg/dL-10 mmol/L): 4 positive controls in whom pathogenic mutations had previously been identified by Sanger sequencing and 7 patients in whom the molecular defect was still unknown. The customized panel was accurate, and it allowed to confirm genetic variants previously identified in all positive controls with primary severe HTG. Only 1 patient of 7 with HTG was found to be carrier of a homozygous pathogenic mutation of the third novel mutation of LMF1 gene (c.1380C>G-p.Y460X). The clinical and molecular familial cascade screening allowed the identification of 2 additional affected siblings and 7 heterozygous carriers of the mutation. We showed that our targeted resequencing approach for genetic diagnosis of severe HTG appears to be accurate, less time consuming, and more economical compared with traditional Sanger resequencing. The identification of pathogenic mutations in candidate genes remains challenging and clinical resequencing should mainly intended for patients with strong clinical criteria for monogenic severe HTG. Copyright © 2017 National Lipid Association. Published by Elsevier Inc. All rights reserved.

Kilo-sequencing: an ordered strategy for rapid DNA sequence data acquisition.

PubMed Central

Barnes, W M; Bevan, M

1983-01-01

A strategy for rapid DNA sequence acquisition in an ordered, nonrandom manner, while retaining all of the conveniences of the dideoxy method with M13 transducing phage DNA template, is described. Target DNA 3 to 14 kb in size can be stably carried by our M13 vectors. Suitable targets are stretches of DNA which lack an enzyme recognition site which is unique on our cloning vectors and adjacent to the sequencing primer; current sites that are so useful when lacking are Pst, Xba, HindIII, BglII, EcoRI. By an in vitro procedure, we cut RF DNA once randomly and once specifically, to create thousands of deletions which start at the unique restriction site adjacent to the dideoxy sequencing primer and extend various distances across the target DNA. Phage carrying a desired size of deletions, whose DNA as template will give rise to DNA sequence data in a desired location along the target DNA, may be purified by electrophoresis alive on agarose gels. Phage running in the same location on the agarose gel thus conveniently give rise to nucleotide sequence data from the same kilobase of target DNA. Images PMID:6298723

The GARP Complex Is Involved in Intracellular Cholesterol Transport via Targeting NPC2 to Lysosomes.

PubMed

Wei, Jian; Zhang, Ying-Yu; Luo, Jie; Wang, Ju-Qiong; Zhou, Yu-Xia; Miao, Hong-Hua; Shi, Xiong-Jie; Qu, Yu-Xiu; Xu, Jie; Li, Bo-Liang; Song, Bao-Liang

2017-06-27

Proper intracellular cholesterol trafficking is critical for cellular function. Two lysosome-resident proteins, NPC1 and NPC2, mediate the egress of low-density lipoprotein-derived cholesterol from lysosomes. However, other proteins involved in this process remain largely unknown. Through amphotericin B-based selection, we isolated two cholesterol transport-defective cell lines. Subsequent whole-transcriptome-sequencing analysis revealed two cell lines bearing the same mutation in the vacuolar protein sorting 53 (Vps53) gene. Depletion of VPS53 or other subunits of the Golgi-associated retrograde protein (GARP) complex impaired NPC2 sorting to lysosomes and caused cholesterol accumulation. GARP deficiency blocked the retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network. Further, Vps54 mutant mice displayed reduced cellular NPC2 protein levels and increased cholesterol accumulation, underscoring the physiological role of the GARP complex in cholesterol transport. We conclude that the GARP complex contributes to intracellular cholesterol transport by targeting NPC2 to lysosomes in a CI-MPR-dependent manner. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Targeted or whole genome sequencing of formalin fixed tissue samples: potential applications in cancer genomics.

PubMed

Munchel, Sarah; Hoang, Yen; Zhao, Yue; Cottrell, Joseph; Klotzle, Brandy; Godwin, Andrew K; Koestler, Devin; Beyerlein, Peter; Fan, Jian-Bing; Bibikova, Marina; Chien, Jeremy

2015-09-22

Current genomic studies are limited by the poor availability of fresh-frozen tissue samples. Although formalin-fixed diagnostic samples are in abundance, they are seldom used in current genomic studies because of the concern of formalin-fixation artifacts. Better characterization of these artifacts will allow the use of archived clinical specimens in translational and clinical research studies. To provide a systematic analysis of formalin-fixation artifacts on Illumina sequencing, we generated 26 DNA sequencing data sets from 13 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissue samples. The results indicate high rate of concordant calls between matched FF/FFPE pairs at reference and variant positions in three commonly used sequencing approaches (whole genome, whole exome, and targeted exon sequencing). Global mismatch rates and C · G > T · A substitutions were comparable between matched FF/FFPE samples, and discordant rates were low (<0.26%) in all samples. Finally, low-pass whole genome sequencing produces similar pattern of copy number alterations between FF/FFPE pairs. The results from our studies suggest the potential use of diagnostic FFPE samples for cancer genomic studies to characterize and catalog variations in cancer genomes.

Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test

PubMed Central

Lionel, Anath C; Costain, Gregory; Monfared, Nasim; Walker, Susan; Reuter, Miriam S; Hosseini, S Mohsen; Thiruvahindrapuram, Bhooma; Merico, Daniele; Jobling, Rebekah; Nalpathamkalam, Thomas; Pellecchia, Giovanna; Sung, Wilson W L; Wang, Zhuozhi; Bikangaga, Peter; Boelman, Cyrus; Carter, Melissa T; Cordeiro, Dawn; Cytrynbaum, Cheryl; Dell, Sharon D; Dhir, Priya; Dowling, James J; Heon, Elise; Hewson, Stacy; Hiraki, Linda; Inbar-Feigenberg, Michal; Klatt, Regan; Kronick, Jonathan; Laxer, Ronald M; Licht, Christoph; MacDonald, Heather; Mercimek-Andrews, Saadet; Mendoza-Londono, Roberto; Piscione, Tino; Schneider, Rayfel; Schulze, Andreas; Silverman, Earl; Siriwardena, Komudi; Snead, O Carter; Sondheimer, Neal; Sutherland, Joanne; Vincent, Ajoy; Wasserman, Jonathan D; Weksberg, Rosanna; Shuman, Cheryl; Carew, Chris; Szego, Michael J; Hayeems, Robin Z; Basran, Raveen; Stavropoulos, Dimitri J; Ray, Peter N; Bowdin, Sarah; Meyn, M Stephen; Cohn, Ronald D; Scherer, Stephen W; Marshall, Christian R

2018-01-01

Purpose Genetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use. Methods We prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing. Results WGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24% P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A. Conclusion WGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort. PMID:28771251

Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test.

PubMed

Lionel, Anath C; Costain, Gregory; Monfared, Nasim; Walker, Susan; Reuter, Miriam S; Hosseini, S Mohsen; Thiruvahindrapuram, Bhooma; Merico, Daniele; Jobling, Rebekah; Nalpathamkalam, Thomas; Pellecchia, Giovanna; Sung, Wilson W L; Wang, Zhuozhi; Bikangaga, Peter; Boelman, Cyrus; Carter, Melissa T; Cordeiro, Dawn; Cytrynbaum, Cheryl; Dell, Sharon D; Dhir, Priya; Dowling, James J; Heon, Elise; Hewson, Stacy; Hiraki, Linda; Inbar-Feigenberg, Michal; Klatt, Regan; Kronick, Jonathan; Laxer, Ronald M; Licht, Christoph; MacDonald, Heather; Mercimek-Andrews, Saadet; Mendoza-Londono, Roberto; Piscione, Tino; Schneider, Rayfel; Schulze, Andreas; Silverman, Earl; Siriwardena, Komudi; Snead, O Carter; Sondheimer, Neal; Sutherland, Joanne; Vincent, Ajoy; Wasserman, Jonathan D; Weksberg, Rosanna; Shuman, Cheryl; Carew, Chris; Szego, Michael J; Hayeems, Robin Z; Basran, Raveen; Stavropoulos, Dimitri J; Ray, Peter N; Bowdin, Sarah; Meyn, M Stephen; Cohn, Ronald D; Scherer, Stephen W; Marshall, Christian R

2018-04-01

PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use.MethodsWe prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing.ResultsWGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24%; P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A.ConclusionWGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort.

Failure to transmit avian vacuolar myelinopathy to mallard ducks

USGS Publications Warehouse

Larsen, R.S.; Nutter, F.B.; Augspurger, T.; Rocke, T.E.; Thomas, N.J.; Stoskopf, M.K.

2003-01-01

Avian vacuolar myelinopathy (AVM) is a neurologic disease that has been diagnosed in free-ranging birds in the southeastern United States. Bald eagles (Haliaeetus leuocephalus), American coots (Fulica americana), and mallards (Anas platyrhynchos) have been affected. Previous investigations have not determined the etiology of this disease. In November and December 2002, we attempted to induce AVM in game-farmed mallards through four, 7-day exposure trials. Mallards were housed in six groups of eight, with two of these groups serving as controls. One group was housed with AVM-affected coots; one group was tube fed daily with water from the lake where affected coots were captured; one group was tube fed daily with aquatic vegetation (Hydrilla verticillata) from the same lake; and another group was tube fed daily with sediment from the lake. No ducks exhibited clinical neurologic abnormalities consistent with AVM and no evidence of AVM was present at histopathologic examination of brain tissue. Although limitations in sample size, quantity of individual doses, frequency of dose administration, duration of exposure, and timing of these trials restrict the interpretation of the findings, AVM was not readily transmitted by direct contact, water, hydrilla, or sediment in this investigation.

A Multidimensional Strategy to Detect Polypharmacological Targets in the Absence of Structural and Sequence Homology

PubMed Central

Durrant, Jacob D.; Amaro, Rommie E.; Xie, Lei; Urbaniak, Michael D.; Ferguson, Michael A. J.; Haapalainen, Antti; Chen, Zhijun; Di Guilmi, Anne Marie; Wunder, Frank; Bourne, Philip E.; McCammon, J. Andrew

2010-01-01

Conventional drug design embraces the “one gene, one drug, one disease” philosophy. Polypharmacology, which focuses on multi-target drugs, has emerged as a new paradigm in drug discovery. The rational design of drugs that act via polypharmacological mechanisms can produce compounds that exhibit increased therapeutic potency and against which resistance is less likely to develop. Additionally, identifying multiple protein targets is also critical for side-effect prediction. One third of potential therapeutic compounds fail in clinical trials or are later removed from the market due to unacceptable side effects often caused by off-target binding. In the current work, we introduce a multidimensional strategy for the identification of secondary targets of known small-molecule inhibitors in the absence of global structural and sequence homology with the primary target protein. To demonstrate the utility of the strategy, we identify several targets of 4,5-dihydroxy-3-(1-naphthyldiazenyl)-2,7-naphthalenedisulfonic acid, a known micromolar inhibitor of Trypanosoma brucei RNA editing ligase 1. As it is capable of identifying potential secondary targets, the strategy described here may play a useful role in future efforts to reduce drug side effects and/or to increase polypharmacology. PMID:20098496

A multidimensional strategy to detect polypharmacological targets in the absence of structural and sequence homology.

PubMed

Durrant, Jacob D; Amaro, Rommie E; Xie, Lei; Urbaniak, Michael D; Ferguson, Michael A J; Haapalainen, Antti; Chen, Zhijun; Di Guilmi, Anne Marie; Wunder, Frank; Bourne, Philip E; McCammon, J Andrew

2010-01-22

Conventional drug design embraces the "one gene, one drug, one disease" philosophy. Polypharmacology, which focuses on multi-target drugs, has emerged as a new paradigm in drug discovery. The rational design of drugs that act via polypharmacological mechanisms can produce compounds that exhibit increased therapeutic potency and against which resistance is less likely to develop. Additionally, identifying multiple protein targets is also critical for side-effect prediction. One third of potential therapeutic compounds fail in clinical trials or are later removed from the market due to unacceptable side effects often caused by off-target binding. In the current work, we introduce a multidimensional strategy for the identification of secondary targets of known small-molecule inhibitors in the absence of global structural and sequence homology with the primary target protein. To demonstrate the utility of the strategy, we identify several targets of 4,5-dihydroxy-3-(1-naphthyldiazenyl)-2,7-naphthalenedisulfonic acid, a known micromolar inhibitor of Trypanosoma brucei RNA editing ligase 1. As it is capable of identifying potential secondary targets, the strategy described here may play a useful role in future efforts to reduce drug side effects and/or to increase polypharmacology.

The chaperonin-60 universal target is a barcode for bacteria that enables de novo assembly of metagenomic sequence data.

PubMed

Links, Matthew G; Dumonceaux, Tim J; Hemmingsen, Sean M; Hill, Janet E

2012-01-01

Barcoding with molecular sequences is widely used to catalogue eukaryotic biodiversity. Studies investigating the community dynamics of microbes have relied heavily on gene-centric metagenomic profiling using two genes (16S rRNA and cpn60) to identify and track Bacteria. While there have been criteria formalized for barcoding of eukaryotes, these criteria have not been used to evaluate gene targets for other domains of life. Using the framework of the International Barcode of Life we evaluated DNA barcodes for Bacteria. Candidates from the 16S rRNA gene and the protein coding cpn60 gene were evaluated. Within complete bacterial genomes in the public domain representing 983 species from 21 phyla, the largest difference between median pairwise inter- and intra-specific distances ("barcode gap") was found from cpn60. Distribution of sequence diversity along the ∼555 bp cpn60 target region was remarkably uniform. The barcode gap of the cpn60 universal target facilitated the faithful de novo assembly of full-length operational taxonomic units from pyrosequencing data from a synthetic microbial community. Analysis supported the recognition of both 16S rRNA and cpn60 as DNA barcodes for Bacteria. The cpn60 universal target was found to have a much larger barcode gap than 16S rRNA suggesting cpn60 as a preferred barcode for Bacteria. A large barcode gap for cpn60 provided a robust target for species-level characterization of data. The assembly of consensus sequences for barcodes was shown to be a reliable method for the identification and tracking of novel microbes in metagenomic studies.

Involvement of Vacuolar Sequestration and Active Transport in Tolerance of Saccharomyces cerevisiae to Hop Iso-α-Acids▿ † ¶

PubMed Central

Hazelwood, Lucie A.; Walsh, Michael C.; Pronk, Jack T.; Daran, Jean-Marc

2010-01-01

The hop plant, Humulus lupulus L., has an exceptionally high content of secondary metabolites, the hop α-acids, which possess a range of beneficial properties, including antiseptic action. Studies performed on the mode of action of hop iso-α-acids have hitherto been restricted to lactic acid bacteria. The present study investigated molecular mechanisms of hop iso-α-acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso-α-acids that were an order of magnitude higher than those found with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso-α-acid detoxification and tolerance. This screening and further analysis of deletion mutants confirmed that yeast tolerance to hop iso-α-acids involves three major processes, active proton pumping into the vacuole by the vacuolar-type ATPase to enable vacuolar sequestration of iso-α-acids and alteration of cell wall structure and, to a lesser extent, active export of iso-α-acids across the plasma membrane. Furthermore, iso-α-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelators. PMID:19915041

Genome and transcriptome sequencing identifies breeding targets in the orphan crop tef (Eragrostis tef).

PubMed

Cannarozzi, Gina; Plaza-Wüthrich, Sonia; Esfeld, Korinna; Larti, Stéphanie; Wilson, Yi Song; Girma, Dejene; de Castro, Edouard; Chanyalew, Solomon; Blösch, Regula; Farinelli, Laurent; Lyons, Eric; Schneider, Michel; Falquet, Laurent; Kuhlemeier, Cris; Assefa, Kebebew; Tadele, Zerihun

2014-07-09

Tef (Eragrostis tef), an indigenous cereal critical to food security in the Horn of Africa, is rich in minerals and protein, resistant to many biotic and abiotic stresses and safe for diabetics as well as sufferers of immune reactions to wheat gluten. We present the genome of tef, the first species in the grass subfamily Chloridoideae and the first allotetraploid assembled de novo. We sequenced the tef genome for marker-assisted breeding, to shed light on the molecular mechanisms conferring tef's desirable nutritional and agronomic properties, and to make its genome publicly available as a community resource. The draft genome contains 672 Mbp representing 87% of the genome size estimated from flow cytometry. We also sequenced two transcriptomes, one from a normalized RNA library and another from unnormalized RNASeq data. The normalized RNA library revealed around 38000 transcripts that were then annotated by the SwissProt group. The CoGe comparative genomics platform was used to compare the tef genome to other genomes, notably sorghum. Scaffolds comprising approximately half of the genome size were ordered by syntenic alignment to sorghum producing tef pseudo-chromosomes, which were sorted into A and B genomes as well as compared to the genetic map of tef. The draft genome was used to identify novel SSR markers, investigate target genes for abiotic stress resistance studies, and understand the evolution of the prolamin family of proteins that are responsible for the immune response to gluten. It is highly plausible that breeding targets previously identified in other cereal crops will also be valuable breeding targets in tef. The draft genome and transcriptome will be of great use for identifying these targets for genetic improvement of this orphan crop that is vital for feeding 50 million people in the Horn of Africa.

Program Synthesizes UML Sequence Diagrams

NASA Technical Reports Server (NTRS)

Barry, Matthew R.; Osborne, Richard N.

2006-01-01

A computer program called "Rational Sequence" generates Universal Modeling Language (UML) sequence diagrams of a target Java program running on a Java virtual machine (JVM). Rational Sequence thereby performs a reverse engineering function that aids in the design documentation of the target Java program. Whereas previously, the construction of sequence diagrams was a tedious manual process, Rational Sequence generates UML sequence diagrams automatically from the running Java code.

Germline TRAV5D-4 T-Cell Receptor Sequence Targets a Primary Insulin Peptide of NOD Mice

PubMed Central

Nakayama, Maki; Castoe, Todd; Sosinowski, Tomasz; He, XiangLing; Johnson, Kelly; Haskins, Kathryn; Vignali, Dario A.A.; Gapin, Laurent; Pollock, David; Eisenbarth, George S.

2012-01-01

There is accumulating evidence that autoimmunity to insulin B chain peptide, amino acids 9–23 (insulin B:9–23), is central to development of autoimmune diabetes of the NOD mouse model. We hypothesized that enhanced susceptibility to autoimmune diabetes is the result of targeting of insulin by a T-cell receptor (TCR) sequence commonly encoded in the germline. In this study, we aimed to demonstrate that a particular Vα gene TRAV5D-4 with multiple junction sequences is sufficient to induce anti-islet autoimmunity by studying retrogenic mouse lines expressing α-chains with different Vα TRAV genes. Retrogenic NOD strains expressing Vα TRAV5D-4 α-chains with many different complementarity determining region (CDR) 3 sequences, even those derived from TCRs recognizing islet-irrelevant molecules, developed anti-insulin autoimmunity. Induction of insulin autoantibodies by TRAV5D-4 α-chains was abrogated by the mutation of insulin peptide B:9–23 or that of two amino acid residues in CDR1 and 2 of the TRAV5D-4. TRAV13–1, the human ortholog of murine TRAV5D-4, was also capable of inducing in vivo anti-insulin autoimmunity when combined with different murine CDR3 sequences. Targeting primary autoantigenic peptides by simple germline-encoded TCR motifs may underlie enhanced susceptibility to the development of autoimmune diabetes. PMID:22315318

Increased Activity of the Vacuolar Monosaccharide Transporter TMT1 Alters Cellular Sugar Partitioning, Sugar Signaling, and Seed Yield in Arabidopsis1[OA

PubMed Central

Wingenter, Karina; Schulz, Alexander; Wormit, Alexandra; Wic, Stefan; Trentmann, Oliver; Hoermiller, Imke I.; Heyer, Arnd G.; Marten, Irene; Hedrich, Rainer; Neuhaus, H. Ekkehard

2010-01-01

The extent to which vacuolar sugar transport activity affects molecular, cellular, and developmental processes in Arabidopsis (Arabidopsis thaliana) is unknown. Electrophysiological analysis revealed that overexpression of the tonoplast monosaccharide transporter TMT1 in a tmt1-2::tDNA mutant led to increased proton-coupled monosaccharide import into isolated mesophyll vacuoles in comparison with wild-type vacuoles. TMT1 overexpressor mutants grew faster than wild-type plants on soil and in high-glucose (Glc)-containing liquid medium. These effects were correlated with increased vacuolar monosaccharide compartmentation, as revealed by nonaqueous fractionation and by chlorophyllab-binding protein1 and nitrate reductase1 gene expression studies. Soil-grown TMT1 overexpressor plants respired less Glc than wild-type plants and only about half the amount of Glc respired by tmt1-2::tDNA mutants. In sum, these data show that TMT activity in wild-type plants limits vacuolar monosaccharide loading. Remarkably, TMT1 overexpressor mutants produced larger seeds and greater total seed yield, which was associated with increased lipid and protein content. These changes in seed properties were correlated with slightly decreased nocturnal CO2 release and increased sugar export rates from detached source leaves. The SUC2 gene, which codes for a sucrose transporter that may be critical for phloem loading in leaves, has been identified as Glc repressed. Thus, the observation that SUC2 mRNA increased slightly in TMT1 overexpressor leaves, characterized by lowered cytosolic Glc levels than wild-type leaves, provided further evidence of a stimulated source capacity. In summary, increased TMT activity in Arabidopsis induced modified subcellular sugar compartmentation, altered cellular sugar sensing, affected assimilate allocation, increased the biomass of Arabidopsis seeds, and accelerated early plant development. PMID:20709831

Non-selective cation channels in plasma and vacuolar membranes and their contribution to K+ transport.

PubMed

Pottosin, Igor; Dobrovinskaya, Oxana

2014-05-15

Both in vacuolar and plasma membranes, in addition to truly K(+)-selective channels there is a variety of non-selective channels, which conduct K(+) and other ions with little preference. Many non-selective channels in the plasma membrane are active at depolarized potentials, thus, contributing to K(+) efflux rather than to K(+) uptake. They may play important roles in xylem loading or contribute to a K(+) leak, induced by salt or oxidative stress. Here, three currents, expressed in root cells, are considered: voltage-insensitive cation current, non-selective outwardly rectifying current, and low-selective conductance, activated by reactive oxygen species. The latter two do not only poorly discriminate between different cations (like K(+)vs Na(+)), but also conduct anions. Such solute channels may mediate massive electroneutral transport of salts and might be involved in osmotic adjustment or volume decrease, associated with cell death. In the tonoplast two major currents are mediated by SV (slow) and FV (fast) vacuolar channels, respectively, which are virtually impermeable for anions. SV channels conduct mono- and divalent cations indiscriminately and are activated by high cytosolic Ca(2+) and depolarized voltages. FV channels are inhibited by micromolar cytosolic Ca(2+), Mg(2+), and polyamines, and conduct a variety of monovalent cations, including K(+). Strikingly, both SV and FV channels sense the K(+) content of vacuoles, which modulates their voltage dependence, and in case of SV, also alleviates channel's inhibition by luminal Ca(2+). Therefore, SV and FV channels may operate as K(+)-sensing valves, controlling K(+) distribution between the vacuole and the cytosol. Copyright © 2014 Elsevier GmbH. All rights reserved.

Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space.

PubMed

Coppens, Isabelle; Dunn, Joe Dan; Romano, Julia D; Pypaert, Marc; Zhang, Hui; Boothroyd, John C; Joiner, Keith A

2006-04-21

The intracellular compartment harboring Toxoplasma gondii satisfies the parasite's nutritional needs for rapid growth in mammalian cells. We demonstrate that the parasitophorous vacuole (PV) of T. gondii accumulates material coming from the host mammalian cell via the exploitation of the host endo-lysosomal system. The parasite actively recruits host microtubules, resulting in selective attraction of endo-lysosomes to the PV. Microtubule-based invaginations of the PV membrane serve as conduits for the delivery of host endo-lysosomes within the PV. These tubular conduits are decorated by a parasite coat, including the tubulogenic protein GRA7, which acts like a garrote that sequesters host endocytic organelles in the vacuolar space. These data define an unanticipated process allowing the parasite intimate and concentrated access to a diverse range of low molecular weight components produced by the endo-lysosomal system. More generally, they identify a unique mechanism for unidirectional transport and sequestration of host organelles.

Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity

PubMed Central

Farsi, Zohreh; Rammner, Burkhard; Woehler, Andrew; Lafer, Eileen M; Mim, Carsten; Jahn, Reinhard

2018-01-01

Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling. PMID:29652249

A vacuolar iron-transporter homologue acts as a detoxifier in Plasmodium

PubMed Central

Slavic, Ksenija; Krishna, Sanjeev; Lahree, Aparajita; Bouyer, Guillaume; Hanson, Kirsten K.; Vera, Iset; Pittman, Jon K.; Staines, Henry M.; Mota, Maria M.

2016-01-01

Iron is an essential micronutrient but is also highly toxic. In yeast and plant cells, a key detoxifying mechanism involves iron sequestration into intracellular storage compartments, mediated by members of the vacuolar iron-transporter (VIT) family of proteins. Here we study the VIT homologue from the malaria parasites Plasmodium falciparum (PfVIT) and Plasmodium berghei (PbVIT). PfVIT-mediated iron transport in a yeast heterologous expression system is saturable (Km∼14.7 μM), and selective for Fe2+ over other divalent cations. PbVIT-deficient P. berghei lines (Pbvit−) show a reduction in parasite load in both liver and blood stages of infection in mice. Moreover, Pbvit− parasites have higher levels of labile iron in blood stages and are more sensitive to increased iron levels in liver stages, when compared with wild-type parasites. Our data are consistent with Plasmodium VITs playing a major role in iron detoxification and, thus, normal development of malaria parasites in their mammalian host. PMID:26786069

A vacuolar iron-transporter homologue acts as a detoxifier in Plasmodium.

PubMed

Slavic, Ksenija; Krishna, Sanjeev; Lahree, Aparajita; Bouyer, Guillaume; Hanson, Kirsten K; Vera, Iset; Pittman, Jon K; Staines, Henry M; Mota, Maria M

2016-01-20

Iron is an essential micronutrient but is also highly toxic. In yeast and plant cells, a key detoxifying mechanism involves iron sequestration into intracellular storage compartments, mediated by members of the vacuolar iron-transporter (VIT) family of proteins. Here we study the VIT homologue from the malaria parasites Plasmodium falciparum (PfVIT) and Plasmodium berghei (PbVIT). PfVIT-mediated iron transport in a yeast heterologous expression system is saturable (Km ∼ 14.7 μM), and selective for Fe(2+) over other divalent cations. PbVIT-deficient P. berghei lines (Pbvit(-)) show a reduction in parasite load in both liver and blood stages of infection in mice. Moreover, Pbvit(-) parasites have higher levels of labile iron in blood stages and are more sensitive to increased iron levels in liver stages, when compared with wild-type parasites. Our data are consistent with Plasmodium VITs playing a major role in iron detoxification and, thus, normal development of malaria parasites in their mammalian host.

Detecting very low allele fraction variants using targeted DNA sequencing and a novel molecular barcode-aware variant caller.

PubMed

Xu, Chang; Nezami Ranjbar, Mohammad R; Wu, Zhong; DiCarlo, John; Wang, Yexun

2017-01-03

Detection of DNA mutations at very low allele fractions with high accuracy will significantly improve the effectiveness of precision medicine for cancer patients. To achieve this goal through next generation sequencing, researchers need a detection method that 1) captures rare mutation-containing DNA fragments efficiently in the mix of abundant wild-type DNA; 2) sequences the DNA library extensively to deep coverage; and 3) distinguishes low level true variants from amplification and sequencing errors with high accuracy. Targeted enrichment using PCR primers provides researchers with a convenient way to achieve deep sequencing for a small, yet most relevant region using benchtop sequencers. Molecular barcoding (or indexing) provides a unique solution for reducing sequencing artifacts analytically. Although different molecular barcoding schemes have been reported in recent literature, most variant calling has been done on limited targets, using simple custom scripts. The analytical performance of barcode-aware variant calling can be significantly improved by incorporating advanced statistical models. We present here a highly efficient, simple and scalable enrichment protocol that integrates molecular barcodes in multiplex PCR amplification. In addition, we developed smCounter, an open source, generic, barcode-aware variant caller based on a Bayesian probabilistic model. smCounter was optimized and benchmarked on two independent read sets with SNVs and indels at 5 and 1% allele fractions. Variants were called with very good sensitivity and specificity within coding regions. We demonstrated that we can accurately detect somatic mutations with allele fractions as low as 1% in coding regions using our enrichment protocol and variant caller.

A Phylogenomic Approach Based on PCR Target Enrichment and High Throughput Sequencing: Resolving the Diversity within the South American Species of Bartsia L. (Orobanchaceae)

PubMed Central

Tank, David C.

2016-01-01

Advances in high-throughput sequencing (HTS) have allowed researchers to obtain large amounts of biological sequence information at speeds and costs unimaginable only a decade ago. Phylogenetics, and the study of evolution in general, is quickly migrating towards using HTS to generate larger and more complex molecular datasets. In this paper, we present a method that utilizes microfluidic PCR and HTS to generate large amounts of sequence data suitable for phylogenetic analyses. The approach uses the Fluidigm Access Array System (Fluidigm, San Francisco, CA, USA) and two sets of PCR primers to simultaneously amplify 48 target regions across 48 samples, incorporating sample-specific barcodes and HTS adapters (2,304 unique amplicons per Access Array). The final product is a pooled set of amplicons ready to be sequenced, and thus, there is no need to construct separate, costly genomic libraries for each sample. Further, we present a bioinformatics pipeline to process the raw HTS reads to either generate consensus sequences (with or without ambiguities) for every locus in every sample or—more importantly—recover the separate alleles from heterozygous target regions in each sample. This is important because it adds allelic information that is well suited for coalescent-based phylogenetic analyses that are becoming very common in conservation and evolutionary biology. To test our approach and bioinformatics pipeline, we sequenced 576 samples across 96 target regions belonging to the South American clade of the genus Bartsia L. in the plant family Orobanchaceae. After sequencing cleanup and alignment, the experiment resulted in ~25,300bp across 486 samples for a set of 48 primer pairs targeting the plastome, and ~13,500bp for 363 samples for a set of primers targeting regions in the nuclear genome. Finally, we constructed a combined concatenated matrix from all 96 primer combinations, resulting in a combined aligned length of ~40,500bp for 349 samples. PMID:26828929

Solid phase sequencing of biopolymers

DOEpatents

Cantor, Charles; Koster, Hubert

2010-09-28

This invention relates to methods for detecting and sequencing target nucleic acid sequences, to mass modified nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probes comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include DNA or RNA in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated molecular weight analysis and identification of the target sequence.

A fluorescent reporter protein containing AtRMR1 domains is targeted to the storage and central vacuoles in Arabidopsis thaliana and tobacco leaf cells.

PubMed

Scabone, Camila María; Frigerio, Lorenzo; Petruccelli, Silvana

2011-10-01

To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles.

Rational Design of a Transferrin-Binding Peptide Sequence Tailored to Targeted Nanoparticle Internalization.

PubMed

Santi, Melissa; Maccari, Giuseppe; Mereghetti, Paolo; Voliani, Valerio; Rocchiccioli, Silvia; Ucciferri, Nadia; Luin, Stefano; Signore, Giovanni

2017-02-15

The transferrin receptor (TfR) is a promising target in cancer therapy owing to its overexpression in most solid tumors and on the blood-brain barrier. Nanostructures chemically derivatized with transferrin are employed in TfR targeting but often lose their functionality upon injection in the bloodstream. As an alternative strategy, we rationally designed a peptide coating able to bind transferrin on suitable pockets not involved in binding to TfR or iron by using an iterative multiscale-modeling approach coupled with quantitative structure-activity and relationship (QSAR) analysis and evolutionary algorithms. We tested that selected sequences have low aspecific protein adsorption and high binding energy toward transferrin, and one of them is efficiently internalized in cells with a transferrin-dependent pathway. Furthermore, it promotes transferrin-mediated endocytosis of gold nanoparticles by modifying their protein corona and promoting oriented adsorption of transferrin. This strategy leads to highly effective nanostructures, potentially useful in diagnostic and therapeutic applications, which exploit (and do not suffer) the protein solvation for achieving a better targeting.

Discovery and Annotation of Plant Endogenous Target Mimicry Sequences from Public Transcriptome Libraries: A Case Study of Prunus persica.

PubMed

Karakülah, Gökhan

2017-06-28

Novel transcript discovery through RNA sequencing has substantially improved our understanding of the transcriptome dynamics of biological systems. Endogenous target mimicry (eTM) transcripts, a novel class of regulatory molecules, bind to their target microRNAs (miRNAs) by base pairing and block their biological activity. The objective of this study was to provide a computational analysis framework for the prediction of putative eTM sequences in plants, and as an example, to discover previously un-annotated eTMs in Prunus persica (peach) transcriptome. Therefore, two public peach transcriptome libraries downloaded from Sequence Read Archive (SRA) and a previously published set of long non-coding RNAs (lncRNAs) were investigated with multi-step analysis pipeline, and 44 putative eTMs were found. Additionally, an eTM-miRNA-mRNA regulatory network module associated with peach fruit organ development was built via integration of the miRNA target information and predicted eTM-miRNA interactions. My findings suggest that one of the most widely expressed miRNA families among diverse plant species, miR156, might be potentially sponged by seven putative eTMs. Besides, the study indicates eTMs potentially play roles in the regulation of development processes in peach fruit via targeting specific miRNAs. In conclusion, by following the step-by step instructions provided in this study, novel eTMs can be identified and annotated effectively in public plant transcriptome libraries.

Evaluation of cysteine proteases of Plasmodium vivax as antimalarial drug targets: sequence analysis and sensitivity to cysteine protease inhibitors.

PubMed

Na, Byoung-Kuk; Kim, Tong-Soo; Rosenthal, Philip J; Lee, Jong-Koo; Kong, Yoon

2004-10-01

Cysteine proteases perform critical roles in the life cycles of malaria parasites. In Plasmodium falciparum, treatment of cysteine protease inhibitors inhibits hemoglobin hydrolysis and blocks the parasite development in vitro and in vivo, suggesting that plasmodial cysteine proteases may be interesting targets for new chemotherapeutics. To determine whether sequence diversity may limit chemotherapy against Plasmodium vivax, we analyzed sequence variations in the genes encoding three cysteine proteases, vivapain-1, -2 and -3, in 22 wild isolates of P. vivax. The sequences were highly conserved among wild isolates. A small number of substitutions leading to amino acid changes were found, while they did not modify essential residues for the function or structure of the enzymes. The substrate specificities and sensitivities to synthetic cysteine protease inhibitors of vivapain-2 and -3 from wild isolates were also very similar. These results support the suggestion that cysteine proteases of P. vivax are promising antimalarial chemotherapeutic targets.

BAC sequencing using pooled methods.

PubMed

Saski, Christopher A; Feltus, F Alex; Parida, Laxmi; Haiminen, Niina

2015-01-01

Shotgun sequencing and assembly of a large, complex genome can be both expensive and challenging to accurately reconstruct the true genome sequence. Repetitive DNA arrays, paralogous sequences, polyploidy, and heterozygosity are main factors that plague de novo genome sequencing projects that typically result in highly fragmented assemblies and are difficult to extract biological meaning. Targeted, sub-genomic sequencing offers complexity reduction by removing distal segments of the genome and a systematic mechanism for exploring prioritized genomic content through BAC sequencing. If one isolates and sequences the genome fraction that encodes the relevant biological information, then it is possible to reduce overall sequencing costs and efforts that target a genomic segment. This chapter describes the sub-genome assembly protocol for an organism based upon a BAC tiling path derived from a genome-scale physical map or from fine mapping using BACs to target sub-genomic regions. Methods that are described include BAC isolation and mapping, DNA sequencing, and sequence assembly.

Sequencing of a new target genome: the Pediculus humanus humanus (Phthiraptera: Pediculidae) genome project.

PubMed

Pittendrigh, B R; Clark, J M; Johnston, J S; Lee, S H; Romero-Severson, J; Dasch, G A

2006-11-01

The human body louse, Pediculus humanus humanus (L.), and the human head louse, Pediculus humanus capitis, belong to the hemimetabolous order Phthiraptera. The body louse is the primary vector that transmits the bacterial agents of louse-borne relapsing fever, trench fever, and epidemic typhus. The genomes of the bacterial causative agents of several of these aforementioned diseases have been sequenced. Thus, determining the body louse genome will enhance studies of host-vector-pathogen interactions. Although not important as a major disease vector, head lice are of major social concern. Resistance to traditional pesticides used to control head and body lice have developed. It is imperative that new molecular targets be discovered for the development of novel compounds to control these insects. No complete genome sequence exists for a hemimetabolous insect species primarily because hemimetabolous insects often have large (2000 Mb) to very large (up to 16,300 Mb) genomes. Fortuitously, we determined that the human body louse has one of the smallest genome sizes known in insects, suggesting it may be a suitable choice as a minimal hemimetabolous genome in which many genes have been eliminated during its adaptation to human parasitism. Because many louse species infest birds and mammals, the body louse genome-sequencing project will facilitate studies of their comparative genomics. A 6-8X coverage of the body louse genome, plus sequenced expressed sequence tags, should provide the entomological, evolutionary biology, medical, and public health communities with useful genetic information.

Targeted next-generation sequencing in chronic lymphocytic leukemia: a high-throughput yet tailored approach will facilitate implementation in a clinical setting.

PubMed

Sutton, Lesley-Ann; Ljungström, Viktor; Mansouri, Larry; Young, Emma; Cortese, Diego; Navrkalova, Veronika; Malcikova, Jitka; Muggen, Alice F; Trbusek, Martin; Panagiotidis, Panagiotis; Davi, Frederic; Belessi, Chrysoula; Langerak, Anton W; Ghia, Paolo; Pospisilova, Sarka; Stamatopoulos, Kostas; Rosenquist, Richard

2015-03-01

Next-generation sequencing has revealed novel recurrent mutations in chronic lymphocytic leukemia, particularly in patients with aggressive disease. Here, we explored targeted re-sequencing as a novel strategy to assess the mutation status of genes with prognostic potential. To this end, we utilized HaloPlex targeted enrichment technology and designed a panel including nine genes: ATM, BIRC3, MYD88, NOTCH1, SF3B1 and TP53, which have been linked to the prognosis of chronic lymphocytic leukemia, and KLHL6, POT1 and XPO1, which are less characterized but were found to be recurrently mutated in various sequencing studies. A total of 188 chronic lymphocytic leukemia patients with poor prognostic features (unmutated IGHV, n=137; IGHV3-21 subset #2, n=51) were sequenced on the HiSeq 2000 and data were analyzed using well-established bioinformatics tools. Using a conservative cutoff of 10% for the mutant allele, we found that 114/180 (63%) patients carried at least one mutation, with mutations in ATM, BIRC3, NOTCH1, SF3B1 and TP53 accounting for 149/177 (84%) of all mutations. We selected 155 mutations for Sanger validation (variant allele frequency, 10-99%) and 93% (144/155) of mutations were confirmed; notably, all 11 discordant variants had a variant allele frequency between 11-27%, hence at the detection limit of conventional Sanger sequencing. Technical precision was assessed by repeating the entire HaloPlex procedure for 63 patients; concordance was found for 77/82 (94%) mutations. In summary, this study demonstrates that targeted next-generation sequencing is an accurate and reproducible technique potentially suitable for routine screening, eventually as a stand-alone test without the need for confirmation by Sanger sequencing. Copyright© Ferrata Storti Foundation.

Simultaneous human platelet antigen genotyping and detection of novel single nucleotide polymorphisms by targeted next-generation sequencing.

PubMed

Davey, Sue; Navarrete, Cristina; Brown, Colin

2017-06-01

Twenty-nine human platelet antigen systems have been described to date, but the majority of current genotyping methods are restricted to the identification of those most commonly associated with alloantibody production in a clinical context. This can result in a protracted investigation if causative human platelet antigens are rare or novel. A targeted next-generation sequencing approach was designed to detect all known human platelet antigens with the additional capability of identifying novel mutations in the encoding genes. A targeted enrichment, high-sensitivity HaloPlex assay was designed to sequence all exons and flanking regions of the six genes known to encode human platelet antigens. Indexed DNA libraries were prepared from 47 previously human platelet antigen-genotyped samples and subsequently combined into one of three pools for sequencing on an Illumina MiSeq platform. The generated FASTQ files were aligned and scrutinized for each human platelet antigen polymorphism using SureCall data analysis software. Forty-six samples were successfully genotyped for human platelet antigens 1 through 29bw, with an average per base coverage depth of 1144. Concordance with historical human platelet antigen genotypes was 100%. A putative novel mutation in Exon 10 of the integrin β-3 (ITGB3) gene from an unsolved case of fetal neonatal alloimmune thrombocytopenia was also detected. A next-generation sequencing-based method that can accurately define all known human platelet antigen polymorphisms was developed. With the ability to sequence up to 96 samples simultaneously, our HaloPlex design could be used for high-throughput human platelet antigen genotyping. This method is also applicable for investigating fetal neonatal alloimmune thrombocytopenia when rare or novel human platelet antigens are suspected. © 2017 AABB.

PACCMIT/PACCMIT-CDS: identifying microRNA targets in 3′ UTRs and coding sequences

PubMed Central

Šulc, Miroslav; Marín, Ray M.; Robins, Harlan S.; Vaníček, Jiří

2015-01-01

The purpose of the proposed web server, publicly available at http://paccmit.epfl.ch, is to provide a user-friendly interface to two algorithms for predicting messenger RNA (mRNA) molecules regulated by microRNAs: (i) PACCMIT (Prediction of ACcessible and/or Conserved MIcroRNA Targets), which identifies primarily mRNA transcripts targeted in their 3′ untranslated regions (3′ UTRs), and (ii) PACCMIT-CDS, designed to find mRNAs targeted within their coding sequences (CDSs). While PACCMIT belongs among the accurate algorithms for predicting conserved microRNA targets in the 3′ UTRs, the main contribution of the web server is 2-fold: PACCMIT provides an accurate tool for predicting targets also of weakly conserved or non-conserved microRNAs, whereas PACCMIT-CDS addresses the lack of similar portals adapted specifically for targets in CDS. The web server asks the user for microRNAs and mRNAs to be analyzed, accesses the precomputed P-values for all microRNA–mRNA pairs from a database for all mRNAs and microRNAs in a given species, ranks the predicted microRNA–mRNA pairs, evaluates their significance according to the false discovery rate and finally displays the predictions in a tabular form. The results are also available for download in several standard formats. PMID:25948580

Development and Validation of Targeted Next-Generation Sequencing Panels for Detection of Germline Variants in Inherited Diseases.

PubMed

Santani, Avni; Murrell, Jill; Funke, Birgit; Yu, Zhenming; Hegde, Madhuri; Mao, Rong; Ferreira-Gonzalez, Andrea; Voelkerding, Karl V; Weck, Karen E

2017-06-01

- The number of targeted next-generation sequencing (NGS) panels for genetic diseases offered by clinical laboratories is rapidly increasing. Before an NGS-based test is implemented in a clinical laboratory, appropriate validation studies are needed to determine the performance characteristics of the test. - To provide examples of assay design and validation of targeted NGS gene panels for the detection of germline variants associated with inherited disorders. - The approaches used by 2 clinical laboratories for the development and validation of targeted NGS gene panels are described. Important design and validation considerations are examined. - Clinical laboratories must validate performance specifications of each test prior to implementation. Test design specifications and validation data are provided, outlining important steps in validation of targeted NGS panels by clinical diagnostic laboratories.

Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence.

PubMed Central

Ridley, R G; Patel, H V; Gerber, G E; Morton, R C; Freeman, K B

1986-01-01

A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein. Images PMID:3012461

Structure-Function Analysis of Rgs1 in Magnaporthe oryzae: Role of DEP Domains in Subcellular Targeting

PubMed Central

Ramanujam, Ravikrishna; Yishi, Xu; Liu, Hao; Naqvi, Naweed I.

2012-01-01

Background Rgs1, a prototypical Regulator of G protein Signaling, negatively modulates the cyclic AMP pathway thereby influencing various aspects of asexual development and pathogenesis in the rice-blast fungus Magnaporthe oryzae. Rgs1 possesses tandem DEP motifs (termed DEP-A and DEP-B; for Dishevelled, Egl-10, Pleckstrin) at the N-terminus, and a Gα-GTP interacting RGS catalytic core domain at the C-terminus. In this study, we focused on gaining further insights into the mechanisms of Rgs1 regulation and subcellular localization by characterizing the role(s) of the individual domains and the full-length protein during asexual development and pathogenesis in Magnaporthe. Methodology/Principal Findings Utilizing western blot analysis and specific antisera against the N- and C-terminal halves of Rgs1, we identify and report the in vivo endoproteolytic processing/cleavage of full-length Rgs1 that yields an N-terminal DEP and a RGS core domain. Independent expression of the resultant DEP-DEP half (N-Rgs1) or RGS core (C-Rgs1) fragments, failed to complement the rgs1Δ defects in colony morphology, aerial hyphal growth, surface hydrophobicity, conidiation, appressorium formation and infection. Interestingly, the full-length Rgs1-mCherry, as well as the tagged N-terminal DEP domains (individually or in conjunction) localized to distinct punctate vesicular structures in the cytosol, while the catalytic RGS core motif was predominantly vacuolar. Conclusions/Significance Based on our data from sequence alignments, immuno-blot and microscopic analysis, we propose that the post-translational proteolytic processing of Rgs1 and the vacuolar sequestration of the catalytic RGS domain represents an important means of down regulating Rgs1 function and thus forming an additional and alternative means of regulating G protein signaling in Magnaporthe. We further hypothesize the prevalence of analogous mechanisms functioning in other filamentous fungi. Furthermore, we conclusively

Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple).

PubMed

Antony, Edna; Taybi, Tahar; Courbot, Mikaël; Mugford, Sam T; Smith, J Andrew C; Borland, Anne M

2008-01-01

In photosynthetic tissues of the CAM plant pineapple (Ananas comosus), storage of soluble sugars in the central vacuole during the daytime and their remobilization at night is required to provide carbon skeletons for nocturnal CO(2) fixation. However, soluble sugars produced photosynthetically must also be exported to support growth processes in heterotrophic tissues. To begin to address how vacuolar sugar storage and assimilate partitioning are regulated in A. comosus, degenerate PCR and cDNA library screening were used to clone three candidate sugar transporters from the leaves of this species. Subcellular localization of the three transporters was investigated via expression of YFP-fusion proteins in tobacco epidermal cells and their co-localization with subcellular markers by confocal microscopy. Using this strategy, a putative hexose transporter (AcMST1) and a putative inositol transporter (AcINT1) were identified that both localized to the tonoplast, whereas a putative sucrose transporter (AcSUT1) was found to localize to prevacuolar compartments. A cDNA (AcMST2) with high similarity to a recently characterized tonoplast hexose transporter in Arabidopsis was also identified from an A. comosus fruit EST database. Analyses of transcript abundance indicated that AcMST1 was more highly expressed in fruits compared to leaves of A. comosus, whilst transcripts of AcINT1, AcSUT1, and AcMST2 were more abundant in leaves. Transcript abundance of AcINT1, the putative inositol transporter, showed day-night changes comparable to those of other CAM-related transcripts described in Mesembryanthemum crystallinum. The results are discussed in terms of the role of vacuolar sugar transporters in regulating carbon flow during the diel cycle in CAM plants.

High expression in leaves of the zinc hyperaccumulator Arabidopsis halleri of AhMHX, a homolog of an Arabidopsis thaliana vacuolar metal/proton exchanger.

PubMed

Elbaz, Benayahu; Shoshani-Knaani, Noa; David-Assael, Ora; Mizrachy-Dagri, Talya; Mizrahi, Keren; Saul, Helen; Brook, Emil; Berezin, Irina; Shaul, Orit

2006-06-01

Zn hyperaccumulator plants sequester Zn into their shoot vacuoles. To date, the only transporters implicated in Zn sequestration into the vacuoles of hyperaccumulator plants are cation diffusion facilitators (CDFs). We investigated the expression in Arabidopsis halleri of a homolog of AtMHX, an A. thaliana tonoplast transporter that exchanges protons with Mg, Zn and Fe ions. A. halleri has a single copy of a homologous gene, encoding a protein that shares 98% sequence identity with AtMHX. Western blot analysis with vacuolar-enriched membrane fractions suggests localization of AhMHX in the tonoplast. The levels of MHX proteins are much higher in leaves of A. halleri than in leaves of the non-accumulator plant A. thaliana. At the same time, the levels of MHX transcripts are similar in leaves of the two species. This suggests that the difference in MHX levels is regulated at the post-transcriptional level. In vitro translation studies indicated that the difference between AhMHX and AtMHX expression is not likely to result from the variations in the sequence of their 5' untranslated regions (5'UTRs). The high expression of AhMHX in A. halleri leaves is constitutive and not significantly affected by the metal status of the plants. In both species, MHX transcript levels are higher in leaves than in roots, but the difference is higher in A. halleri. Metal sequestration into root vacuoles was suggested to inhibit hyperaccumulation in the shoot. Our data implicate AhMHX as a candidate gene in metal accumulation or tolerance in A. halleri.

Sequence-Specific Targeting of Dosage Compensation in Drosophila Favors an Active Chromatin Context

PubMed Central

Gelbart, Marnie; Tolstorukov, Michael Y.; Plachetka, Annette; Kharchenko, Peter V.; Jung, Youngsook L.; Gorchakov, Andrey A.; Larschan, Erica; Gu, Tingting; Minoda, Aki; Riddle, Nicole C.; Schwartz, Yuri B.; Elgin, Sarah C. R.; Karpen, Gary H.; Pirrotta, Vincenzo; Kuroda, Mitzi I.; Park, Peter J.

2012-01-01

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at “entry sites” that contain a consensus sequence motif (“MSL recognition element” or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome. PMID:22570616

Unlocking hidden genomic sequence

PubMed Central

Keith, Jonathan M.; Cochran, Duncan A. E.; Lala, Gita H.; Adams, Peter; Bryant, Darryn; Mitchelson, Keith R.

2004-01-01

Despite the success of conventional Sanger sequencing, significant regions of many genomes still present major obstacles to sequencing. Here we propose a novel approach with the potential to alleviate a wide range of sequencing difficulties. The technique involves extracting target DNA sequence from variants generated by introduction of random mutations. The introduction of mutations does not destroy original sequence information, but distributes it amongst multiple variants. Some of these variants lack problematic features of the target and are more amenable to conventional sequencing. The technique has been successfully demonstrated with mutation levels up to an average 18% base substitution and has been used to read previously intractable poly(A), AT-rich and GC-rich motifs. PMID:14973330

Inorganic polyphosphate in the yeast Saccharomyces cerevisiae with a mutation disturbing the function of vacuolar ATPase.

PubMed

Tomaschevsky, A A; Ryasanova, L P; Kulakovskaya, T V; Kulaev, I S

2010-08-01

A mutation in the vma2 gene disturbing V-ATPase function in the yeast Saccharomyces cerevisiae results in a five- and threefold decrease in inorganic polyphosphate content in the stationary and active phases of growth on glucose, respectively. The average polyphosphate chain length in the mutant cells is decreased. The mutation does not prevent polyphosphate utilization during cultivation in a phosphate-deficient medium and recovery of its level on reinoculation in complete medium after phosphate deficiency. The content of short chain acid-soluble polyphosphates is recovered first. It is supposed that these polyphosphates are less dependent on the electrochemical gradient on the vacuolar membrane.

DNA sequencing using polymerase substrate-binding kinetics

PubMed Central

Previte, Michael John Robert; Zhou, Chunhong; Kellinger, Matthew; Pantoja, Rigo; Chen, Cheng-Yao; Shi, Jin; Wang, BeiBei; Kia, Amirali; Etchin, Sergey; Vieceli, John; Nikoomanzar, Ali; Bomati, Erin; Gloeckner, Christian; Ronaghi, Mostafa; He, Molly Min

2015-01-01

Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications. PMID:25612848

Identification of miRNAs and their targets in wild tomato at moderately and acutely elevated temperatures by high-throughput sequencing and degradome analysis

PubMed Central

Zhou, Rong; Wang, Qian; Jiang, Fangling; Cao, Xue; Sun, Mintao; Liu, Min; Wu, Zhen

2016-01-01

MicroRNAs (miRNAs) are 19–24 nucleotide (nt) noncoding RNAs that play important roles in abiotic stress responses in plants. High temperatures have been the subject of considerable attention due to their negative effects on plant growth and development. Heat-responsive miRNAs have been identified in some plants. However, there have been no reports on the global identification of miRNAs and their targets in tomato at high temperatures, especially at different elevated temperatures. Here, three small-RNA libraries and three degradome libraries were constructed from the leaves of the heat-tolerant tomato at normal, moderately and acutely elevated temperatures (26/18 °C, 33/33 °C and 40/40 °C, respectively). Following high-throughput sequencing, 662 conserved and 97 novel miRNAs were identified in total with 469 conserved and 91 novel miRNAs shared in the three small-RNA libraries. Of these miRNAs, 96 and 150 miRNAs were responsive to the moderately and acutely elevated temperature, respectively. Following degradome sequencing, 349 sequences were identified as targets of 138 conserved miRNAs, and 13 sequences were identified as targets of eight novel miRNAs. The expression levels of seven miRNAs and six target genes obtained by quantitative real-time PCR (qRT-PCR) were largely consistent with the sequencing results. This study enriches the number of heat-responsive miRNAs and lays a foundation for the elucidation of the miRNA-mediated regulatory mechanism in tomatoes at elevated temperatures. PMID:27653374

Identification of rare genetic variants in Italian patients with dementia by targeted gene sequencing.

PubMed

Bartoletti-Stella, Anna; Baiardi, Simone; Stanzani-Maserati, Michelangelo; Piras, Silvia; Caffarra, Paolo; Raggi, Alberto; Pantieri, Roberta; Baldassari, Sara; Caporali, Leonardo; Abu-Rumeileh, Samir; Linarello, Simona; Liguori, Rocco; Parchi, Piero; Capellari, Sabina

2018-06-01

Genetics is intricately involved in the etiology of neurodegenerative dementias. The incidence of monogenic dementia among all neurodegenerative forms is unknown due to the lack of systematic studies and of patient/clinician access to extensive diagnostic procedures. In this study, we conducted targeted sequencing in 246 clinically heterogeneous patients, mainly with early-onset and/or familial neurodegenerative dementia, using a custom-designed next-generation sequencing panel covering 27 genes known to harbor mutations that can cause different types of dementia, in addition to the detection of C9orf72 repeat expansions. Forty-nine patients (19.9%) carried known pathogenic or novel, likely pathogenic, variants, involving both common (presenilin 1, presenilin 2, C9orf72, and granulin) and rare (optineurin, serpin family I member 1 and protein kinase cyclic adenosine monophosphate (cAMP)-dependent type I regulatory subunit beta) dementia-associated genes. Our results support the use of an extended next-generation sequencing panels as a quick, accurate, and cost-effective method for diagnosis in clinical practice. This approach could have a significant impact on the proportion of tested patients, especially among those with an early disease onset. Copyright © 2018 Elsevier Inc. All rights reserved.

Structure of the vacuolar H+-ATPase rotary motor reveals new mechanistic insights.

PubMed

Rawson, Shaun; Phillips, Clair; Huss, Markus; Tiburcy, Felix; Wieczorek, Helmut; Trinick, John; Harrison, Michael A; Muench, Stephen P

2015-03-03

Vacuolar H(+)-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼ 1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Prediction of Drug-Target Interaction Networks from the Integration of Protein Sequences and Drug Chemical Structures.

PubMed

Meng, Fan-Rong; You, Zhu-Hong; Chen, Xing; Zhou, Yong; An, Ji-Yong

2017-07-05

Knowledge of drug-target interaction (DTI) plays an important role in discovering new drug candidates. Unfortunately, there are unavoidable shortcomings; including the time-consuming and expensive nature of the experimental method to predict DTI. Therefore, it motivates us to develop an effective computational method to predict DTI based on protein sequence. In the paper, we proposed a novel computational approach based on protein sequence, namely PDTPS (Predicting Drug Targets with Protein Sequence) to predict DTI. The PDTPS method combines Bi-gram probabilities (BIGP), Position Specific Scoring Matrix (PSSM), and Principal Component Analysis (PCA) with Relevance Vector Machine (RVM). In order to evaluate the prediction capacity of the PDTPS, the experiment was carried out on enzyme, ion channel, GPCR, and nuclear receptor datasets by using five-fold cross-validation tests. The proposed PDTPS method achieved average accuracy of 97.73%, 93.12%, 86.78%, and 87.78% on enzyme, ion channel, GPCR and nuclear receptor datasets, respectively. The experimental results showed that our method has good prediction performance. Furthermore, in order to further evaluate the prediction performance of the proposed PDTPS method, we compared it with the state-of-the-art support vector machine (SVM) classifier on enzyme and ion channel datasets, and other exiting methods on four datasets. The promising comparison results further demonstrate that the efficiency and robust of the proposed PDTPS method. This makes it a useful tool and suitable for predicting DTI, as well as other bioinformatics tasks.

Clinical Validation of Targeted Next Generation Sequencing for Colon and Lung Cancers

PubMed Central

D’Haene, Nicky; Le Mercier, Marie; De Nève, Nancy; Blanchard, Oriane; Delaunoy, Mélanie; El Housni, Hakim; Dessars, Barbara; Heimann, Pierre; Remmelink, Myriam; Demetter, Pieter; Tejpar, Sabine; Salmon, Isabelle

2015-01-01

Objective Recently, Next Generation Sequencing (NGS) has begun to supplant other technologies for gene mutation testing that is now required for targeted therapies. However, transfer of NGS technology to clinical daily practice requires validation. Methods We validated the Ion Torrent AmpliSeq Colon and Lung cancer panel interrogating 1850 hotspots in 22 genes using the Ion Torrent Personal Genome Machine. First, we used commercial reference standards that carry mutations at defined allelic frequency (AF). Then, 51 colorectal adenocarcinomas (CRC) and 39 non small cell lung carcinomas (NSCLC) were retrospectively analyzed. Results Sensitivity and accuracy for detecting variants at an AF >4% was 100% for commercial reference standards. Among the 90 cases, 89 (98.9%) were successfully sequenced. Among the 86 samples for which NGS and the reference test were both informative, 83 showed concordant results between NGS and the reference test; i.e. KRAS and BRAF for CRC and EGFR for NSCLC, with the 3 discordant cases each characterized by an AF <10%. Conclusions Overall, the AmpliSeq colon/lung cancer panel was specific and sensitive for mutation analysis of gene panels and can be incorporated into clinical daily practice. PMID:26366557

The vacuolar ATPase from Entamoeba histolytica: molecular cloning of the gene encoding for the B subunit and subcellular localization of the protein.

PubMed

Meléndez-Hernández, Mayra Gisela; Barrios, María Luisa Labra; Orozco, Esther; Luna-Arias, Juan Pedro

2008-12-23

Entamoeba histolytica is a professional phagocytic cell where the vacuolar ATPase plays a key role. This enzyme is a multisubunit complex that regulates pH in many subcellular compartments, even in those that are not measurably acidic. It participates in a wide variety of cellular processes such as endocytosis, intracellular transport and membrane fusion. The presence of a vacuolar type H+-ATPase in E. histolytica trophozoites has been inferred previously from inhibition assays of its activity, the isolation of the Ehvma1 and Ehvma3 genes, and by proteomic analysis of purified phagosomes. We report the isolation and characterization of the Ehvma2 gene, which encodes for the subunit B of the vacuolar ATPase. This polypeptide is a 55.3 kDa highly conserved protein with 34 to 80% identity to orthologous proteins from other species. Particularly, in silico studies showed that EhV-ATPase subunit B displays 78% identity and 90% similarity to its Dictyostelium ortholog. A 462 bp DNA fragment of the Ehvma2 gene was expressed in bacteria and recombinant polypeptide was used to raise mouse polyclonal antibodies. EhV-ATPase subunit B antibodies detected a 55 kDa band in whole cell extracts and in an enriched fraction of DNA-containing organelles named EhkOs. The V-ATPase subunit B was located by immunofluorescence and confocal microscopy in many vesicles, in phagosomes, plasma membrane and in EhkOs. We also identified the genes encoding for the majority of the V-ATPase subunits in the E. histolytica genome, and proposed a putative model for this proton pump. We have isolated the Ehvma2 gene which encodes for the V-ATPase subunit B from the E. histolytica clone A. This gene has a 154 bp intron and encodes for a highly conserved polypeptide. Specific antibodies localized EhV-ATPase subunit B in many vesicles, phagosomes, plasma membrane and in EhkOs. Most of the orthologous genes encoding for the EhV-ATPase subunits were found in the E. histolytica genome, indicating the

Is sequence awareness mandatory for perceptual sequence learning: An assessment using a pure perceptual sequence learning design.

PubMed

Deroost, Natacha; Coomans, Daphné

2018-02-01

We examined the role of sequence awareness in a pure perceptual sequence learning design. Participants had to react to the target's colour that changed according to a perceptual sequence. By varying the mapping of the target's colour onto the response keys, motor responses changed randomly. The effect of sequence awareness on perceptual sequence learning was determined by manipulating the learning instructions (explicit versus implicit) and assessing the amount of sequence awareness after the experiment. In the explicit instruction condition (n = 15), participants were instructed to intentionally search for the colour sequence, whereas in the implicit instruction condition (n = 15), they were left uninformed about the sequenced nature of the task. Sequence awareness after the sequence learning task was tested by means of a questionnaire and the process-dissociation-procedure. The results showed that the instruction manipulation had no effect on the amount of perceptual sequence learning. Based on their report to have actively applied their sequence knowledge during the experiment, participants were subsequently regrouped in a sequence strategy group (n = 14, of which 4 participants from the implicit instruction condition and 10 participants from the explicit instruction condition) and a no-sequence strategy group (n = 16, of which 11 participants from the implicit instruction condition and 5 participants from the explicit instruction condition). Only participants of the sequence strategy group showed reliable perceptual sequence learning and sequence awareness. These results indicate that perceptual sequence learning depends upon the continuous employment of strategic cognitive control processes on sequence knowledge. Sequence awareness is suggested to be a necessary but not sufficient condition for perceptual learning to take place. Copyright © 2018 Elsevier B.V. All rights reserved.

Targeting of Repeated Sequences Unique to a Gene Results in Significant Increases in Antisense Oligonucleotide Potency

PubMed Central

Vickers, Timothy A.; Freier, Susan M.; Bui, Huynh-Hoa; Watt, Andrew; Crooke, Stanley T.

2014-01-01

A new strategy for identifying potent RNase H-dependent antisense oligonucleotides (ASOs) is presented. Our analysis of the human transcriptome revealed that a significant proportion of genes contain unique repeated sequences of 16 or more nucleotides in length. Activities of ASOs targeting these repeated sites in several representative genes were compared to those of ASOs targeting unique single sites in the same transcript. Antisense activity at repeated sites was also evaluated in a highly controlled minigene system. Targeting both native and minigene repeat sites resulted in significant increases in potency as compared to targeting of non-repeated sites. The increased potency at these sites is a result of increased frequency of ASO/RNA interactions which, in turn, increases the probability of a productive interaction between the ASO/RNA heteroduplex and human RNase H1 in the cell. These results suggest a new, highly efficient strategy for rapid identification of highly potent ASOs. PMID:25334092

Targeted next-generation sequencing helps to decipher the genetic and phenotypic heterogeneity of hypertrophic cardiomyopathy

PubMed Central

Cecconi, Massimiliano; Parodi, Maria I.; Formisano, Francesco; Spirito, Paolo; Autore, Camillo; Musumeci, Maria B.; Favale, Stefano; Forleo, Cinzia; Rapezzi, Claudio; Biagini, Elena; Davì, Sabrina; Canepa, Elisabetta; Pennese, Loredana; Castagnetta, Mauro; Degiorgio, Dario; Coviello, Domenico A.

2016-01-01

Hypertrophic cardiomyopathy (HCM) is mainly associated with myosin, heavy chain 7 (MYH7) and myosin binding protein C, cardiac (MYBPC3) mutations. In order to better explain the clinical and genetic heterogeneity in HCM patients, in this study, we implemented a target-next generation sequencing (NGS) assay. An Ion AmpliSeq™ Custom Panel for the enrichment of 19 genes, of which 9 of these did not encode thick/intermediate and thin myofilament (TTm) proteins and, among them, 3 responsible of HCM phenocopy, was created. Ninety-two DNA samples were analyzed by the Ion Personal Genome Machine: 73 DNA samples (training set), previously genotyped in some of the genes by Sanger sequencing, were used to optimize the NGS strategy, whereas 19 DNA samples (discovery set) allowed the evaluation of NGS performance. In the training set, we identified 72 out of 73 expected mutations and 15 additional mutations: the molecular diagnosis was achieved in one patient with a previously wild-type status and the pre-excitation syndrome was explained in another. In the discovery set, we identified 20 mutations, 5 of which were in genes encoding non-TTm proteins, increasing the diagnostic yield by approximately 20%: a single mutation in genes encoding non-TTm proteins was identified in 2 out of 3 borderline HCM patients, whereas co-occuring mutations in genes encoding TTm and galactosidase alpha (GLA) altered proteins were characterized in a male with HCM and multiorgan dysfunction. Our combined targeted NGS-Sanger sequencing-based strategy allowed the molecular diagnosis of HCM with greater efficiency than using the conventional (Sanger) sequencing alone. Mutant alleles encoding non-TTm proteins may aid in the complete understanding of the genetic and phenotypic heterogeneity of HCM: co-occuring mutations of genes encoding TTm and non-TTm proteins could explain the wide variability of the HCM phenotype, whereas mutations in genes encoding only the non-TTm proteins are identifiable in

High-throughput sequencing of retrotransposon integration provides a saturated profile of target activity in Schizosaccharomyces pombe.

PubMed

Guo, Yabin; Levin, Henry L

2010-02-01

The biological impact of transposons on the physiology of the host depends greatly on the frequency and position of integration. Previous studies of Tf1, a long terminal repeat retrotransposon in Schizosaccharomyces pombe, showed that integration occurs at the promoters of RNA polymerase II (Pol II) transcribed genes. To determine whether specific promoters are preferred targets of integration, we sequenced large numbers of insertions using high-throughput pyrosequencing. In four independent experiments we identified a total of 73,125 independent integration events. These data provided strong support for the conclusion that Pol II promoters are the targets of Tf1 integration. The size and number of the integration experiments resulted in reproducible measures of integration for each intergenic region and ORF in the S. pombe genome. The reproducibility of the integration activity from experiment to experiment demonstrates that we have saturated the full set of insertion sites that are actively targeted by Tf1. We found Tf1 integration was highly biased in favor of a specific set of Pol II promoters. The overwhelming majority (76%) of the insertions were distributed in intergenic sequences that contained 31% of the promoters of S. pombe. Interestingly, there was no correlation between the amount of integration at these promoters and their level of transcription. Instead, we found Tf1 had a strong preference for promoters that are induced by conditions of stress. This targeting of stress response genes coupled with the ability of Tf1 to regulate the expression of adjacent genes suggests Tf1 may improve the survival of S. pombe when cells are exposed to environmental stress.

High-throughput sequencing of retrotransposon integration provides a saturated profile of target activity in Schizosaccharomyces pombe

PubMed Central

Guo, Yabin; Levin, Henry L.

2010-01-01

The biological impact of transposons on the physiology of the host depends greatly on the frequency and position of integration. Previous studies of Tf1, a long terminal repeat retrotransposon in Schizosaccharomyces pombe, showed that integration occurs at the promoters of RNA polymerase II (Pol II) transcribed genes. To determine whether specific promoters are preferred targets of integration, we sequenced large numbers of insertions using high-throughput pyrosequencing. In four independent experiments we identified a total of 73,125 independent integration events. These data provided strong support for the conclusion that Pol II promoters are the targets of Tf1 integration. The size and number of the integration experiments resulted in reproducible measures of integration for each intergenic region and ORF in the S. pombe genome. The reproducibility of the integration activity from experiment to experiment demonstrates that we have saturated the full set of insertion sites that are actively targeted by Tf1. We found Tf1 integration was highly biased in favor of a specific set of Pol II promoters. The overwhelming majority (76%) of the insertions were distributed in intergenic sequences that contained 31% of the promoters of S. pombe. Interestingly, there was no correlation between the amount of integration at these promoters and their level of transcription. Instead, we found Tf1 had a strong preference for promoters that are induced by conditions of stress. This targeting of stress response genes coupled with the ability of Tf1 to regulate the expression of adjacent genes suggests Tf1 may improve the survival of S. pombe when cells are exposed to environmental stress. PMID:20040583

Generic detection of poleroviruses using an RT-PCR assay targeting the RdRp coding sequence.

PubMed

Lotos, Leonidas; Efthimiou, Konstantinos; Maliogka, Varvara I; Katis, Nikolaos I

2014-03-01

In this study a two-step RT-PCR assay was developed for the generic detection of poleroviruses. The RdRp coding region was selected as the primers' target, since it differs significantly from that of other members in the family Luteoviridae and its sequence can be more informative than other regions in the viral genome. Species specific RT-PCR assays targeting the same region were also developed for the detection of the six most widespread poleroviral species (Beet mild yellowing virus, Beet western yellows virus, Cucurbit aphid-borne virus, Carrot red leaf virus, Potato leafroll virus and Turnip yellows virus) in Greece and the collection of isolates. These isolates along with other characterized ones were used for the evaluation of the generic PCR's detection range. The developed assay efficiently amplified a 593bp RdRp fragment from 46 isolates of 10 different Polerovirus species. Phylogenetic analysis using the generic PCR's amplicon sequence showed that although it cannot accurately infer evolutionary relationships within the genus it can differentiate poleroviruses at the species level. Overall, the described generic assay could be applied for the reliable detection of Polerovirus infections and, in combination with the specific PCRs, for the identification of new and uncharacterized species in the genus. Copyright © 2013 Elsevier B.V. All rights reserved.

Properties of the intracellular transient receptor potential (TRP) channel in yeast, Yvc1.

PubMed

Chang, Yiming; Schlenstedt, Gabriel; Flockerzi, Veit; Beck, Andreas

2010-05-17

Transient receptor potential (TRP) channels are found among mammals, flies, worms, ciliates, Chlamydomonas, and yeast but are absent in plants. These channels are believed to be tetramers of proteins containing six transmembrane domains (TMs). Their primary structures are diverse with sequence similarities only in some short amino acid sequence motifs mainly within sequences covering TM5, TM6, and adjacent domains. In the yeast genome, there is one gene encoding a TRP-like sequence. This protein forms an ion channel in the vacuolar membrane and is therefore called Yvc1 for yeast vacuolar conductance 1. In the following we summarize its prominent features. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Combined mutation and copy-number variation detection by targeted next-generation sequencing in uveal melanoma.

PubMed

Smit, Kyra N; van Poppelen, Natasha M; Vaarwater, Jolanda; Verdijk, Robert; van Marion, Ronald; Kalirai, Helen; Coupland, Sarah E; Thornton, Sophie; Farquhar, Neil; Dubbink, Hendrikus-Jan; Paridaens, Dion; de Klein, Annelies; Kiliç, Emine

2018-05-01

Uveal melanoma is a highly aggressive cancer of the eye, in which nearly 50% of the patients die from metastasis. It is the most common type of primary eye cancer in adults. Chromosome and mutation status have been shown to correlate with the disease-free survival. Loss of chromosome 3 and inactivating mutations in BAP1, which is located on chromosome 3, are strongly associated with 'high-risk' tumors that metastasize early. Other genes often involved in uveal melanoma are SF3B1 and EIF1AX, which are found to be mutated in intermediate- and low-risk tumors, respectively. To obtain genetic information of all genes in one test, we developed a targeted sequencing method that can detect mutations in uveal melanoma genes and chromosomal anomalies in chromosome 1, 3, and 8. With as little as 10 ng DNA, we obtained enough coverage on all genes to detect mutations, such as substitutions, deletions, and insertions. These results were validated with Sanger sequencing in 28 samples. In >90% of the cases, the BAP1 mutation status corresponded to the BAP1 immunohistochemistry. The results obtained in the Ion Torrent single-nucleotide polymorphism assay were confirmed with several other techniques, such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, and Illumina SNP array. By validating our assay in 27 formalin-fixed paraffin-embedded and 43 fresh uveal melanomas, we show that mutations and chromosome status can reliably be obtained using targeted next-generation sequencing. Implementing this technique as a diagnostic pathology application for uveal melanoma will allow prediction of the patients' metastatic risk and potentially assess eligibility for new therapies.

Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy.

PubMed

Huang, Xiao-Yan; Zhuang, Hong; Wu, Ji-Hong; Li, Jian-Kang; Hu, Fang-Yuan; Zheng, Yu; Tellier, Laurent Christian Asker M; Zhang, Sheng-Hai; Gao, Feng-Juan; Zhang, Jian-Guo; Xu, Ge-Zhi

2017-01-01

Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [ LRP5 , c.4053 DelC (p.Ile1351IlefsX88); TSPAN12 , EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. We identified two novel heterozygous deletion mutations [ LRP5 , c.4053 DelC (p.Ile1351IlefsX88); TSPAN12 , EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype-phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling.

Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy

PubMed Central

Huang, Xiao-Yan; Zhuang, Hong; Wu, Ji-Hong; Li, Jian-Kang; Hu, Fang-Yuan; Zheng, Yu; Tellier, Laurent Christian Asker M.; Zhang, Sheng-Hai; Gao, Feng-Juan; Zhang, Jian-Guo

2017-01-01

Purpose Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. Methods To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). Results Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. Conclusions We identified two novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype–phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling. PMID:28867931

Targeted next-generation sequencing at copy-number breakpoints for personalized analysis of rearranged ends in solid tumors.

PubMed

Kim, Hyun-Kyoung; Park, Won Cheol; Lee, Kwang Man; Hwang, Hai-Li; Park, Seong-Yeol; Sorn, Sungbin; Chandra, Vishal; Kim, Kwang Gi; Yoon, Woong-Bae; Bae, Joon Seol; Shin, Hyoung Doo; Shin, Jong-Yeon; Seoh, Ju-Young; Kim, Jong-Il; Hong, Kyeong-Man

2014-01-01

The concept of the utilization of rearranged ends for development of personalized biomarkers has attracted much attention owing to its clinical applicability. Although targeted next-generation sequencing (NGS) for recurrent rearrangements has been successful in hematologic malignancies, its application to solid tumors is problematic due to the paucity of recurrent translocations. However, copy-number breakpoints (CNBs), which are abundant in solid tumors, can be utilized for identification of rearranged ends. As a proof of concept, we performed targeted next-generation sequencing at copy-number breakpoints (TNGS-CNB) in nine colon cancer cases including seven primary cancers and two cell lines, COLO205 and SW620. For deduction of CNBs, we developed a novel competitive single-nucleotide polymorphism (cSNP) microarray method entailing CNB-region refinement by competitor DNA. Using TNGS-CNB, 19 specific rearrangements out of 91 CNBs (20.9%) were identified, and two polymerase chain reaction (PCR)-amplifiable rearrangements were obtained in six cases (66.7%). And significantly, TNGS-CNB, with its high positive identification rate (82.6%) of PCR-amplifiable rearrangements at candidate sites (19/23), just from filtering of aligned sequences, requires little effort for validation. Our results indicate that TNGS-CNB, with its utility for identification of rearrangements in solid tumors, can be successfully applied in the clinical laboratory for cancer-relapse and therapy-response monitoring.

Targeted gene panel sequencing in children with very early onset inflammatory bowel disease--evaluation and prospective analysis.

PubMed

Kammermeier, Jochen; Drury, Suzanne; James, Chela T; Dziubak, Robert; Ocaka, Louise; Elawad, Mamoun; Beales, Philip; Lench, Nicholas; Uhlig, Holm H; Bacchelli, Chiara; Shah, Neil

2014-11-01

Multiple monogenetic conditions with partially overlapping phenotypes can present with inflammatory bowel disease (IBD)-like intestinal inflammation. With novel genotype-specific therapies emerging, establishing a molecular diagnosis is becoming increasingly important. We have introduced targeted next-generation sequencing (NGS) technology as a prospective screening tool in children with very early onset IBD (VEOIBD). We evaluated the coverage of 40 VEOIBD genes in two separate cohorts undergoing targeted gene panel sequencing (TGPS) (n=25) and whole exome sequencing (WES) (n=20). TGPS revealed causative mutations in four genes (IL10RA, EPCAM, TTC37 and SKIV2L) discovered unexpected phenotypes and directly influenced clinical decision making by supporting as well as avoiding haematopoietic stem cell transplantation. TGPS resulted in significantly higher median coverage when compared with WES, fewer coverage deficiencies and improved variant detection across established VEOIBD genes. Excluding or confirming known VEOIBD genotypes should be considered early in the disease course in all cases of therapy-refractory VEOIBD, as it can have a direct impact on patient management. To combine both described NGS technologies would compensate for the limitations of WES for disease-specific application while offering the opportunity for novel gene discovery in the research setting. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L.

PubMed Central

Barkla, B. J.; Zingarelli, L.; Blumwald, E.; Smith, JAC.

1995-01-01

Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast. PMID:12228611

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L.

PubMed

Barkla, B. J.; Zingarelli, L.; Blumwald, E.; Smith, JAC.

1995-10-01

Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast.

The impact of targeting repetitive BamHI-W sequences on the sensitivity and precision of EBV DNA quantification.

PubMed

Sanosyan, Armen; Fayd'herbe de Maudave, Alexis; Bollore, Karine; Zimmermann, Valérie; Foulongne, Vincent; Van de Perre, Philippe; Tuaillon, Edouard

2017-01-01

Viral load monitoring and early Epstein-Barr virus (EBV) DNA detection are essential in routine laboratory testing, especially in preemptive management of Post-transplant Lymphoproliferative Disorder. Targeting the repetitive BamHI-W sequence was shown to increase the sensitivity of EBV DNA quantification, but the variability of BamHI-W reiterations was suggested to be a source of quantification bias. We aimed to assess the extent of variability associated with BamHI-W PCR and its impact on the sensitivity of EBV DNA quantification using the 1st WHO international standard, EBV strains and clinical samples. Repetitive BamHI-W- and LMP2 single- sequences were amplified by in-house qPCRs and BXLF-1 sequence by a commercial assay (EBV R-gene™, BioMerieux). Linearity and limits of detection of in-house methods were assessed. The impact of repeated versus single target sequences on EBV DNA quantification precision was tested on B95.8 and Raji cell lines, possessing 11 and 7 copies of the BamHI-W sequence, respectively, and on clinical samples. BamHI-W qPCR demonstrated a lower limit of detection compared to LMP2 qPCR (2.33 log10 versus 3.08 log10 IU/mL; P = 0.0002). BamHI-W qPCR underestimated the EBV DNA load on Raji strain which contained fewer BamHI-W copies than the WHO standard derived from the B95.8 EBV strain (mean bias: - 0.21 log10; 95% CI, -0.54 to 0.12). Comparison of BamHI-W qPCR versus LMP2 and BXLF-1 qPCR showed an acceptable variability between EBV DNA levels in clinical samples with the mean bias being within 0.5 log10 IU/mL EBV DNA, whereas a better quantitative concordance was observed between LMP2 and BXLF-1 assays. Targeting BamHI-W resulted to a higher sensitivity compared to LMP2 but the variable reiterations of BamHI-W segment are associated with higher quantification variability. BamHI-W can be considered for clinical and therapeutic monitoring to detect an early EBV DNA and a dynamic change in viral load.

The impact of targeting repetitive BamHI-W sequences on the sensitivity and precision of EBV DNA quantification

PubMed Central

Fayd’herbe de Maudave, Alexis; Bollore, Karine; Zimmermann, Valérie; Foulongne, Vincent; Van de Perre, Philippe; Tuaillon, Edouard

2017-01-01

Background Viral load monitoring and early Epstein-Barr virus (EBV) DNA detection are essential in routine laboratory testing, especially in preemptive management of Post-transplant Lymphoproliferative Disorder. Targeting the repetitive BamHI-W sequence was shown to increase the sensitivity of EBV DNA quantification, but the variability of BamHI-W reiterations was suggested to be a source of quantification bias. We aimed to assess the extent of variability associated with BamHI-W PCR and its impact on the sensitivity of EBV DNA quantification using the 1st WHO international standard, EBV strains and clinical samples. Methods Repetitive BamHI-W- and LMP2 single- sequences were amplified by in-house qPCRs and BXLF-1 sequence by a commercial assay (EBV R-gene™, BioMerieux). Linearity and limits of detection of in-house methods were assessed. The impact of repeated versus single target sequences on EBV DNA quantification precision was tested on B95.8 and Raji cell lines, possessing 11 and 7 copies of the BamHI-W sequence, respectively, and on clinical samples. Results BamHI-W qPCR demonstrated a lower limit of detection compared to LMP2 qPCR (2.33 log10 versus 3.08 log10 IU/mL; P = 0.0002). BamHI-W qPCR underestimated the EBV DNA load on Raji strain which contained fewer BamHI-W copies than the WHO standard derived from the B95.8 EBV strain (mean bias: - 0.21 log10; 95% CI, -0.54 to 0.12). Comparison of BamHI-W qPCR versus LMP2 and BXLF-1 qPCR showed an acceptable variability between EBV DNA levels in clinical samples with the mean bias being within 0.5 log10 IU/mL EBV DNA, whereas a better quantitative concordance was observed between LMP2 and BXLF-1 assays. Conclusions Targeting BamHI-W resulted to a higher sensitivity compared to LMP2 but the variable reiterations of BamHI-W segment are associated with higher quantification variability. BamHI-W can be considered for clinical and therapeutic monitoring to detect an early EBV DNA and a dynamic change in viral load

Niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects.

PubMed

Jurgeit, Andreas; McDowell, Robert; Moese, Stefan; Meldrum, Eric; Schwendener, Reto; Greber, Urs F

2012-01-01

Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV) and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H(+)-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals.

Electron tomographic characterization of a vacuolar reticulum and of six vesicle types that occupy different cytoplasmic domains in the apex of tip-growing Chara rhizoids.

PubMed

Limbach, Christoph; Staehelin, L Andrew; Sievers, Andreas; Braun, Markus

2008-04-01

We provide a 3D ultrastructural analysis of the membrane systems involved in tip growth of rhizoids of the green alga Chara. Electron tomography of cells preserved by high-pressure freeze fixation has enabled us to distinguish six different types of vesicles in the apical cytoplasm where the tip growth machinery is accommodated. The vesicle types are: dark and light secretory vesicles, plasma membrane-associated clathrin-coated vesicles (PM-CCVs), Spitzenkoerper-associated clathrin-coated vesicles (Sp-CCVs) and coated vesicles (Sp-CVs), and microvesicles. Each of these vesicle types exhibits a distinct distribution pattern, which provides insights into their possible function for tip growth. The PM-CCVs are confined to the cytoplasm adjacent to the apical plasma membrane. Within this space they are arranged in clusters often surrounding tubular plasma membrane invaginations from which CCVs bud. This suggests that endocytosis and membrane recycling are locally confined to specialized apical endocytosis sites. In contrast, exocytosis of secretory vesicles occurs over the entire membrane area of the apical dome. The Sp-CCVs and the Sp-CVs are associated with the aggregate of endoplasmic reticulum membranes in the center of the growth-organizing Spitzenkoerper complex. Here, Sp-CCVs are seen to bud from undefined tubular membranes. The subapical region of rhizoids contains a vacuolar reticulum that extends along the longitudinal cell axis and consists of large, vesicle-like segments interconnected by thin tubular domains. The tubular domains are encompassed by thin filamentous structures resembling dynamin spirals which could drive peristaltic movements of the vacuolar reticulum similar to those observed in fungal hyphae. The vacuolar reticulum appears to serve as a lytic compartment into which multivesicular bodies deliver their internal vesicles for molecular recycling and degradation.

Composition for nucleic acid sequencing

DOEpatents

Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

2008-08-26

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Genomic Characterization of Non–Small-Cell Lung Cancer in African Americans by Targeted Massively Parallel Sequencing

PubMed Central

Araujo, Luiz H.; Timmers, Cynthia; Bell, Erica Hlavin; Shilo, Konstantin; Lammers, Philip E.; Zhao, Weiqiang; Natarajan, Thanemozhi G.; Miller, Clinton J.; Zhang, Jianying; Yilmaz, Ayse S.; Liu, Tom; Coombes, Kevin; Amann, Joseph; Carbone, David P.

2015-01-01

Purpose Technologic advances have enabled the comprehensive analysis of genetic perturbations in non–small-cell lung cancer (NSCLC); however, African Americans have often been underrepresented in these studies. This ethnic group has higher lung cancer incidence and mortality rates, and some studies have suggested a lower incidence of epidermal growth factor receptor mutations. Herein, we report the most in-depth molecular profile of NSCLC in African Americans to date. Methods A custom panel was designed to cover the coding regions of 81 NSCLC-related genes and 40 ancestry-informative markers. Clinical samples were sequenced on a massively parallel sequencing instrument, and anaplastic lymphoma kinase translocation was evaluated by fluorescent in situ hybridization. Results The study cohort included 99 patients (61% males, 94% smokers) comprising 31 squamous and 68 nonsquamous cell carcinomas. We detected 227 nonsilent variants in the coding sequence, including 24 samples with nonoverlapping, classic driver alterations. The frequency of driver mutations was not significantly different from that of whites, and no association was found between genetic ancestry and the presence of somatic mutations. Copy number alteration analysis disclosed distinguishable amplifications in the 3q chromosome arm in squamous cell carcinomas and pointed toward a handful of targetable alterations. We also found frequent SMARCA4 mutations and protein loss, mostly in driver-negative tumors. Conclusion Our data suggest that African American ancestry may not be significantly different from European/white background for the presence of somatic driver mutations in NSCLC. Furthermore, we demonstrated that using a comprehensive genotyping approach could identify numerous targetable alterations, with potential impact on therapeutic decisions. PMID:25918285

UniDrug-target: a computational tool to identify unique drug targets in pathogenic bacteria.

PubMed

Chanumolu, Sree Krishna; Rout, Chittaranjan; Chauhan, Rajinder S

2012-01-01

Targeting conserved proteins of bacteria through antibacterial medications has resulted in both the development of resistant strains and changes to human health by destroying beneficial microbes which eventually become breeding grounds for the evolution of resistances. Despite the availability of more than 800 genomes sequences, 430 pathways, 4743 enzymes, 9257 metabolic reactions and protein (three-dimensional) 3D structures in bacteria, no pathogen-specific computational drug target identification tool has been developed. A web server, UniDrug-Target, which combines bacterial biological information and computational methods to stringently identify pathogen-specific proteins as drug targets, has been designed. Besides predicting pathogen-specific proteins essentiality, chokepoint property, etc., three new algorithms were developed and implemented by using protein sequences, domains, structures, and metabolic reactions for construction of partial metabolic networks (PMNs), determination of conservation in critical residues, and variation analysis of residues forming similar cavities in proteins sequences. First, PMNs are constructed to determine the extent of disturbances in metabolite production by targeting a protein as drug target. Conservation of pathogen-specific protein's critical residues involved in cavity formation and biological function determined at domain-level with low-matching sequences. Last, variation analysis of residues forming similar cavities in proteins sequences from pathogenic versus non-pathogenic bacteria and humans is performed. The server is capable of predicting drug targets for any sequenced pathogenic bacteria having fasta sequences and annotated information. The utility of UniDrug-Target server was demonstrated for Mycobacterium tuberculosis (H37Rv). The UniDrug-Target identified 265 mycobacteria pathogen-specific proteins, including 17 essential proteins which can be potential drug targets. UniDrug-Target is expected to accelerate

Spectrum of mutations in leiomyosarcomas identified by clinical targeted next-generation sequencing.

PubMed

Lee, Paul J; Yoo, Naomi S; Hagemann, Ian S; Pfeifer, John D; Cottrell, Catherine E; Abel, Haley J; Duncavage, Eric J

2017-02-01

Recurrent genomic mutations in uterine and non-uterine leiomyosarcomas have not been well established. Using a next generation sequencing (NGS) panel of common cancer-associated genes, 25 leiomyosarcomas arising from multiple sites were examined to explore genetic alterations, including single nucleotide variants (SNV), small insertions/deletions (indels), and copy number alterations (CNA). Sequencing showed 86 non-synonymous, coding region somatic variants within 151 gene targets in 21 cases, with a mean of 4.1 variants per case; 4 cases had no putative mutations in the panel of genes assayed. The most frequently altered genes were TP53 (36%), ATM and ATRX (16%), and EGFR and RB1 (12%). CNA were identified in 85% of cases, with the most frequent copy number losses observed in chromosomes 10 and 13 including PTEN and RB1; the most frequent gains were seen in chromosomes 7 and 17. Our data show that deletions in canonical cancer-related genes are common in leiomyosarcomas. Further, the spectrum of gene mutations observed shows that defects in DNA repair and chromosomal maintenance are central to the biology of leiomyosarcomas, and that activating mutations observed in other common cancer types are rare in leiomyosarcomas. Copyright © 2017 Elsevier Inc. All rights reserved.

Efficient strategy for the molecular diagnosis of intellectual disability using targeted high-throughput sequencing

PubMed Central

Redin, Claire; Gérard, Bénédicte; Lauer, Julia; Herenger, Yvan; Muller, Jean; Quartier, Angélique; Masurel-Paulet, Alice; Willems, Marjolaine; Lesca, Gaétan; El-Chehadeh, Salima; Le Gras, Stéphanie; Vicaire, Serge; Philipps, Muriel; Dumas, Michaël; Geoffroy, Véronique; Feger, Claire; Haumesser, Nicolas; Alembik, Yves; Barth, Magalie; Bonneau, Dominique; Colin, Estelle; Dollfus, Hélène; Doray, Bérénice; Delrue, Marie-Ange; Drouin-Garraud, Valérie; Flori, Elisabeth; Fradin, Mélanie; Francannet, Christine; Goldenberg, Alice; Lumbroso, Serge; Mathieu-Dramard, Michèle; Martin-Coignard, Dominique; Lacombe, Didier; Morin, Gilles; Polge, Anne; Sukno, Sylvie; Thauvin-Robinet, Christel; Thevenon, Julien; Doco-Fenzy, Martine; Genevieve, David; Sarda, Pierre; Edery, Patrick; Isidor, Bertrand; Jost, Bernard; Olivier-Faivre, Laurence; Mandel, Jean-Louis; Piton, Amélie

2014-01-01

Background Intellectual disability (ID) is characterised by an extreme genetic heterogeneity. Several hundred genes have been associated to monogenic forms of ID, considerably complicating molecular diagnostics. Trio-exome sequencing was recently proposed as a diagnostic approach, yet remains costly for a general implementation. Methods We report the alternative strategy of targeted high-throughput sequencing of 217 genes in which mutations had been reported in patients with ID or autism as the major clinical concern. We analysed 106 patients with ID of unknown aetiology following array-CGH analysis and other genetic investigations. Ninety per cent of these patients were males, and 75% sporadic cases. Results We identified 26 causative mutations: 16 in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA, MECP2, SLC9A6, SLC16A2, PHF8) and 10 de novo in autosomal-dominant genes (DYRK1A, GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We also detected four possibly causative mutations (eg, in NLGN3) requiring further investigations. We present detailed reasoning for assigning causality for each mutation, and associated patients’ clinical information. Some genes were hit more than once in our cohort, suggesting they correspond to more frequent ID-associated conditions (KDM5C, MECP2, DYRK1A, TCF4). We highlight some unexpected genotype to phenotype correlations, with causative mutations being identified in genes associated to defined syndromes in patients deviating from the classic phenotype (DMD, TCF4, MECP2). We also bring additional supportive (HCFC1, MED13L) or unsupportive (SHROOM4, SRPX2) evidences for the implication of previous candidate genes or mutations in cognitive disorders. Conclusions With a diagnostic yield of 25% targeted sequencing appears relevant as a first intention test for the diagnosis of ID, but importantly will also contribute to a better understanding regarding the specific contribution of the many genes

Measuring the diversity of the human microbiota with targeted next-generation sequencing.

PubMed

Finotello, Francesca; Mastrorilli, Eleonora; Di Camillo, Barbara

2016-12-26

The human microbiota is a complex ecological community of commensal, symbiotic and pathogenic microorganisms harboured by the human body. Next-generation sequencing (NGS) technologies, in particular targeted amplicon sequencing of the 16S ribosomal RNA gene (16S-seq), are enabling the identification and quantification of human-resident microorganisms at unprecedented resolution, providing novel insights into the role of the microbiota in health and disease. Once microbial abundances are quantified through NGS data analysis, diversity indices provide valuable mathematical tools to describe the ecological complexity of a single sample or to detect species differences between samples. However, diversity is not a determined physical quantity for which a consensus definition and unit of measure have been established, and several diversity indices are currently available. Furthermore, they were originally developed for macroecology and their robustness to the possible bias introduced by sequencing has not been characterized so far. To assist the reader with the selection and interpretation of diversity measures, we review a panel of broadly used indices, describing their mathematical formulations, purposes and properties, and characterize their behaviour and criticalities in dependence of the data features using simulated data as ground truth. In addition, we make available an R package, DiversitySeq, which implements in a unified framework the full panel of diversity indices and a simulator of 16S-seq data, and thus represents a valuable resource for the analysis of diversity from NGS count data and for the benchmarking of computational methods for 16S-seq. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Analysis of Genes Involved in Body Weight Regulation by Targeted Re-Sequencing.

PubMed

Volckmar, Anna-Lena; Han, Chung Ting; Pütter, Carolin; Haas, Stefan; Vogel, Carla I G; Knoll, Nadja; Struve, Christoph; Göbel, Maria; Haas, Katharina; Herrfurth, Nikolas; Jarick, Ivonne; Grallert, Harald; Schürmann, Annette; Al-Hasani, Hadi; Hebebrand, Johannes; Sauer, Sascha; Hinney, Anke

2016-01-01

Genes involved in body weight regulation that were previously investigated in genome-wide association studies (GWAS) and in animal models were target-enriched followed by massive parallel next generation sequencing. We enriched and re-sequenced continuous genomic regions comprising FTO, MC4R, TMEM18, SDCCAG8, TKNS, MSRA and TBC1D1 in a screening sample of 196 extremely obese children and adolescents with age and sex specific body mass index (BMI) ≥ 99th percentile and 176 lean adults (BMI ≤ 15th percentile). 22 variants were confirmed by Sanger sequencing. Genotyping was performed in up to 705 independent obesity trios (extremely obese child and both parents), 243 extremely obese cases and 261 lean adults. We detected 20 different non-synonymous variants, one frame shift and one nonsense mutation in the 7 continuous genomic regions in study groups of different weight extremes. For SNP Arg695Cys (rs58983546) in TBC1D1 we detected nominal association with obesity (pTDT = 0.03 in 705 trios). Eleven of the variants were rare, thus were only detected heterozygously in up to ten individual(s) of the complete screening sample of 372 individuals. Two of them (in FTO and MSRA) were found in lean individuals, nine in extremely obese. In silico analyses of the 11 variants did not reveal functional implications for the mutations. Concordant with our hypothesis we detected a rare variant that potentially leads to loss of FTO function in a lean individual. For TBC1D1, in contrary to our hypothesis, the loss of function variant (Arg443Stop) was found in an obese individual. Functional in vitro studies are warranted.

Fluorescence self-quenching assay for the detection of target collagen sequences using a short probe peptide.

PubMed

Nian, Linge; Hu, Yue; Fu, Caihong; Song, Chen; Wang, Jie; Xiao, Jianxi

2018-01-01

The development of novel assays to detect collagen fragments is of utmost importance for diagnostic, prognostic and therapeutic decisions in various collagen-related diseases, and one essential question is to discover probe peptides that can specifically recognize target collagen sequences. Herein we have developed the fluorescence self-quenching assay as a convenient tool to screen the capability of a series of fluorescent probe peptides of variable lengths to bind with target collagen peptides. We have revealed that the targeting ability of probe peptides is length-dependent, and have discovered a relatively short probe peptide FAM-G(POG) 8 capable to identify the target peptide. We have further demonstrated that fluorescence self-quenching assay together with this short probe peptide can be applied to specifically detect the desired collagen fragment in complex biological media. Fluorescence self-quenching assay provides a powerful new tool to discover effective peptides for the recognition of collagen biomarkers, and it may have great potential to identify probe peptides for various protein biomarkers involved in pathological conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

Targeted next generation sequencing of well-differentiated/dedifferentiated liposarcoma reveals novel gene amplifications and mutations.

PubMed

Somaiah, Neeta; Beird, Hannah C; Barbo, Andrea; Song, Juhee; Mills Shaw, Kenna R; Wang, Wei-Lien; Eterovic, Karina; Chen, Ken; Lazar, Alexander; Conley, Anthony P; Ravi, Vinod; Hwu, Patrick; Futreal, Andrew; Simon, George; Meric-Bernstam, Funda; Hong, David

2018-04-13

Well-differentiated/dedifferentiated liposarcoma is a common soft tissue sarcoma with approximately 1500 new cases per year. Surgery is the mainstay of treatment but recurrences are frequent and systemic options are limited. 'Tumor genotyping' is becoming more common in clinical practice as it offers the hope of personalized targeted therapy. We wanted to evaluate the results and the clinical utility of available next-generation sequencing panels in WD/DD liposarcoma. Patients who had their tumor sequenced by either FoundationOne ( n = 13) or the institutional T200/T200.1 panels ( n = 7) were included in this study. Significant copy number alterations were identified, but mutations were infrequent. Out of the 27 mutations detected in 7 samples, 8 ( CTNNB1, MECOM, ZNF536, EGFR, EML4, CSMD3, PBRM1, PPP1R3A ) were identified as deleterious (on Condel, PolyPhen and SIFT) and a truncating mutation was found in NF2 . Of these, EGFR and NF2 are potential driver mutations and have not been reported previously in liposarcoma. MDM2 and CDK4 amplification was universally present in all the tested samples and multiple other recurrent genes with high amplification or high deletion were detected. Many of these targets are potentially actionable. Eight patients went on to receive an MDM2 inhibitor with a median time to progression of 23 months (95% CI: 10-83 months).

Modified Cross-Linking, Ligation, and Sequencing of Hybrids (qCLASH) Identifies Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Targets in Endothelial Cells.

PubMed

Gay, Lauren A; Sethuraman, Sunantha; Thomas, Merin; Turner, Peter C; Renne, Rolf

2018-04-15

Kaposi's sarcoma (KS) tumors are derived from endothelial cells and express Kaposi's sarcoma-associated herpesvirus (KSHV) microRNAs (miRNAs). Although miRNA targets have been identified in B cell lymphoma-derived cells and epithelial cells, little has been done to characterize the KSHV miRNA targetome in endothelial cells. A recent innovation in the identification of miRNA targetomes, cross-linking, ligation, and sequencing of hybrids (CLASH), unambiguously identifies miRNAs and their targets by ligating the two species while both species are still bound within the RNA-induced silencing complex (RISC). We developed a streamlined quick CLASH (qCLASH) protocol that requires a lower cell input than the original method and therefore has the potential to be used on patient biopsy samples. Additionally, we developed a fast-growing, KSHV-negative endothelial cell line derived from telomerase-immortalized vein endothelial long-term culture (TIVE-LTC) cells. qCLASH was performed on uninfected cells and cells infected with either wild-type KSHV or a mutant virus lacking miR-K12-11/11*. More than 1,400 cellular targets of KSHV miRNAs were identified. Many of the targets identified by qCLASH lacked a canonical seed sequence match. Additionally, most target regions in mRNAs originated from the coding DNA sequence (CDS) rather than the 3' untranslated region (UTR). This set of genes includes some that were previously identified in B cells and some new genes that warrant further study. Pathway analysis of endothelial cell targets showed enrichment in cell cycle control, apoptosis, and glycolysis pathways, among others. Characterization of these new targets and the functional consequences of their repression will be important in furthering our understanding of the role of KSHV miRNAs in oncogenesis. IMPORTANCE KS lesions consist of endothelial cells latently infected with KSHV. Cells that make up these lesions express KSHV miRNAs. Identification of the targets of KSHV miRNAs will

Targeted Next Generation Sequencing in Patients with Inborn Errors of Metabolism

PubMed Central

Yubero, Dèlia; Brandi, Núria; Ormazabal, Aida; Garcia-Cazorla, Àngels; Pérez-Dueñas, Belén; Campistol, Jaime; Ribes, Antonia; Palau, Francesc

2016-01-01

Background Next-generation sequencing (NGS) technology has allowed the promotion of genetic diagnosis and are becoming increasingly inexpensive and faster. To evaluate the utility of NGS in the clinical field, a targeted genetic panel approach was designed for the diagnosis of a set of inborn errors of metabolism (IEM). The final aim of the study was to compare the findings for the diagnostic yield of NGS in patients who presented with consistent clinical and biochemical suspicion of IEM with those obtained for patients who did not have specific biomarkers. Methods The subjects studied (n = 146) were classified into two categories: Group 1 (n = 81), which consisted of patients with clinical and biochemical suspicion of IEM, and Group 2 (n = 65), which consisted of IEM cases with clinical suspicion and unspecific biomarkers. A total of 171 genes were analyzed using a custom targeted panel of genes followed by Sanger validation. Results Genetic diagnosis was achieved in 50% of patients (73/146). In addition, the diagnostic yield obtained for Group 1 was 78% (63/81), and this rate decreased to 15.4% (10/65) in Group 2 (X2 = 76.171; p < 0.0001). Conclusions A rapid and effective genetic diagnosis was achieved in our cohort, particularly the group that had both clinical and biochemical indications for the diagnosis. PMID:27243974

Molecular Diagnosis of Infantile Mitochondrial Disease with Targeted Next-Generation Sequencing

PubMed Central

Calvo, Sarah E.; Compton, Alison G.; Hershman, Steven G.; Lim, Sze Chern; Lieber, Daniel S.; Tucker, Elena J.; Laskowski, Adrienne; Garone, Caterina; Liu, Shangtao; Jaffe, David B.; Christodoulou, John; Fletcher, Janice M.; Bruno, Damien L; Goldblatt, Jack; DiMauro, Salvatore; Thorburn, David R.; Mootha, Vamsi K.

2012-01-01

Advances in next-generation sequencing (NGS) promise to facilitate diagnosis of inherited disorders. While in research settings NGS has pinpointed causal alleles using segregation in large families, the key challenge for clinical diagnosis is application to single individuals. To explore its diagnostic utility, we performed targeted NGS in 42 unrelated infants with clinical and biochemical evidence of mitochondrial oxidative phosphorylation disease, who were refractory to traditional molecular diagnosis. These devastating mitochondrial disorders are characterized by phenotypic and genetic heterogeneity, with over 100 causal genes identified to date. We performed “MitoExome” sequencing of the mitochondrial DNA (mtDNA) and exons of ~1000 nuclear genes encoding mitochondrial proteins and prioritized rare mutations predicted to disrupt function. Since patients and controls harbored a comparable number of such heterozygous alleles, we could not prioritize dominant acting genes. However, patients showed a five-fold enrichment of genes with two such mutations that could underlie recessive disease. In total, 23/42 (55%) patients harbored such recessive genes or pathogenic mtDNA variants. Firm diagnoses were enabled in 10 patients (24%) who had mutations in genes previously linked to disease. 13 patients (31%) had mutations in nuclear genes never linked to disease. The pathogenicity of two such genes, NDUFB3 and AGK, was supported by cDNA complementation and evidence from multiple patients, respectively. The results underscore the immediate potential and challenges of deploying NGS in clinical settings. PMID:22277967

PhytoCRISP-Ex: a web-based and stand-alone application to find specific target sequences for CRISPR/CAS editing.

PubMed

Rastogi, Achal; Murik, Omer; Bowler, Chris; Tirichine, Leila

2016-07-01

With the emerging interest in phytoplankton research, the need to establish genetic tools for the functional characterization of genes is indispensable. The CRISPR/Cas9 system is now well recognized as an efficient and accurate reverse genetic tool for genome editing. Several computational tools have been published allowing researchers to find candidate target sequences for the engineering of the CRISPR vectors, while searching possible off-targets for the predicted candidates. These tools provide built-in genome databases of common model organisms that are used for CRISPR target prediction. Although their predictions are highly sensitive, the applicability to non-model genomes, most notably protists, makes their design inadequate. This motivated us to design a new CRISPR target finding tool, PhytoCRISP-Ex. Our software offers CRIPSR target predictions using an extended list of phytoplankton genomes and also delivers a user-friendly standalone application that can be used for any genome. The software attempts to integrate, for the first time, most available phytoplankton genomes information and provide a web-based platform for Cas9 target prediction within them with high sensitivity. By offering a standalone version, PhytoCRISP-Ex maintains an independence to be used with any organism and widens its applicability in high throughput pipelines. PhytoCRISP-Ex out pars all the existing tools by computing the availability of restriction sites over the most probable Cas9 cleavage sites, which can be ideal for mutant screens. PhytoCRISP-Ex is a simple, fast and accurate web interface with 13 pre-indexed and presently updating phytoplankton genomes. The software was also designed as a UNIX-based standalone application that allows the user to search for target sequences in the genomes of a variety of other species.

Investigating the Feasibility of Targeted Next-Generation Sequencing to Guide the Treatment of Head and Neck Squamous Cell Carcinoma.

PubMed

Lim, Sun Min; Cho, Sang Hee; Hwang, In Gyu; Choi, Jae Woo; Chang, Hyun; Ahn, Myung-Ju; Park, Keon Uk; Kim, Ji-Won; Ko, Yoon Ho; Ahn, Hee Kyung; Cho, Byoung Chul; Nam, Byung-Ho; Chun, Sang Hoon; Hong, Ji Hyung; Kwon, Jung Hye; Choi, Jong Gwon; Kang, Eun Joo; Yun, Tak; Lee, Keun-Wook; Kim, Joo-Hang; Kim, Jin Soo; Lee, Hyun Woo; Kim, Min Kyoung; Jung, Dongmin; Kim, Ji Eun; Keam, Bhumsuk; Yun, Hwan Jung; Kim, Sangwoo; Kim, Hye Ryun

2018-05-09

Head and neck squamous cell carcinoma (HNSCC) is a deadly disease in which precision medicine needs to be incorporated. We aimed to implement next-generation sequencing (NGS) in determining actionable targets to guide appropriate molecular targeted therapy in HNSCC patients. Ninety-three tumors and matched blood samples underwent targeted sequencing of 244 genes using the Illumina HiSeq 2500 platform with an average depth of coverage of greater than 1,000×. Clinicopathological data from patients were obtained from 17 centers in Korea, and were analyzed in correlation with NGS data. Ninety-two of the 93 tumors were amenable to data analysis. TP53 was the most common mutation, occurring in 47 (51%) patients, followed by CDKN2A (n=23, 25%), CCND1 (n=22, 24%) and PIK3CA (n=19, 21%). The total mutational burden was similar between human papillomavirus (HPV)-negative vs. positive tumors, although TP53, CDKN2A and CCND1 gene alterations occurred more frequently in HPV-negative tumors. HPV-positive tumors were significantly associated with immune signature-related genes compared to HPV-negative tumors. Mutations of NOTCH1 (p=0.027), CDKN2A (p<0.001) and TP53 (p=0.038) were significantly associated with poorer overall survival. FAT1 mutations were highly enriched in cisplatin responders, and potentially targetable alterations such as PIK3CA E545K and CDKN2A R58X were noted in 14 (15%) patients. We found several targetable genetic alterations, and our findings suggest that implementation of precision medicine in HNSCC is feasible. The predictive value of each targetable alteration should be assessed in a future umbrella trial using matched molecular targeted agents.

Megabase sequencing of human genome by ordered-shotgun-sequencing (OSS) strategy

NASA Astrophysics Data System (ADS)

Chen, Ellson Y.

1997-05-01

So far we have used OSS strategy to sequence over 2 megabases DNA in large-insert clones from regions of human X chromosomes with different characteristic levels of GC content. The method starts by randomly fragmenting a BAC, YAC or PAC to 8-12 kb pieces and subcloning those into lambda phage. Insert-ends of these clones are sequenced and overlapped to create a partial map. Complete sequencing is then done on a minimal tiling path of selected subclones, recursively focusing on those at the edges of contigs to facilitate mergers of clones across the entire target. To reduce manual labor, PCR processes have been adapted to prepare sequencing templates throughout the entire operation. The streamlined process can thus lend itself to further automation. The OSS approach is suitable for large- scale genomic sequencing, providing considerable flexibility in the choice of subclones or regions for more or less intensive sequencing. For example, subclones containing contaminating host cell DNA or cloning vector can be recognized and ignored with minimal sequencing effort; regions overlapping a neighboring clone already sequenced need not be redone; and segments containing tandem repeats or long repetitive sequences can be spotted early on and targeted for additional attention.

Vacuolar Localization of Endoproteinases EP(1) and EP(2) in Barley Mesophyll Cells.

PubMed

Thayer, S S; Huffaker, R C

1984-05-01

The localization of two previously characterized endoproteinases (EP(1) and EP(2)) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP(1) and partially purified EP(2). It is therefore concluded that EP(1) and EP(2) are localized in the vacuoles of mesophyll cells.

Molecular Basis of ADP Inhibition of Vacuolar (V)-type ATPase/Synthase*

PubMed Central

Kishikawa, Jun-ichi; Nakanishi, Atsuko; Furuike, Shou; Tamakoshi, Masatada; Yokoyama, Ken

2014-01-01

Reduction of ATP hydrolysis activity of vacuolar-type ATPase/synthase (V0V1) as a result of ADP inhibition occurs as part of the normal mechanism of V0V1 of Thermus thermophilus but not V0V1 of Enterococcus hirae or eukaryotes. To investigate the molecular basis for this difference, domain-swapped chimeric V1 consisting of both T. thermophilus and E. hirae enzymes were generated, and their function was analyzed. The data showed that the interaction between the nucleotide binding and C-terminal domains of the catalytic A subunit from E. hirae V1 is central to increasing binding affinity of the chimeric V1 for phosphate, resulting in reduction of the ADP inhibition. These findings together with a comparison of the crystal structures of T. thermophilus V1 with E. hirae V1 strongly suggest that the A subunit adopts a conformation in T. thermophilus V1 different from that in E. hirae V1. This key difference results in ADP inhibition of T. thermophilus V1 by abolishing the binding affinity for phosphate during ATP hydrolysis. PMID:24247239

The N-terminus of survivin is a mitochondrial-targeting sequence and Src regulator

PubMed Central

Dunajová, Lucia; Cash, Emily; Markus, Robert; Rochette, Sophie; Townley, Amelia R.

2016-01-01

ABSTRACT Survivin (also known as BIRC5) is a cancer-associated protein that exists in several locations in the cell. Its cytoplasmic residence in interphase cells is governed by CRM1 (also known as XPO1)-mediated nuclear exportation, and its localisation during mitosis to the centromeres and midzone microtubules is that of a canonical chromosomal passenger protein. In addition to these well-established locations, survivin is also a mitochondrial protein, but how it gets there and its function therein is presently unclear. Here, we show that the first ten amino acids at the N-terminus of survivin are sufficient to target GFP to the mitochondria in vivo, and ectopic expression of this decapeptide decreases cell adhesion and accelerates proliferation. The data support a signalling mechanism in which this decapeptide regulates the tyrosine kinase Src, leading to reduced focal adhesion plaques and disruption of F-actin organisation. This strongly suggests that the N-terminus of survivin is a mitochondrial-targeting sequence that regulates Src, and that survivin acts in concert with Src to promote tumorigenesis. PMID:27246243

Short communication: Validation of 4 candidate causative trait variants in 2 cattle breeds using targeted sequence imputation.

PubMed

Pausch, Hubert; Wurmser, Christine; Reinhardt, Friedrich; Emmerling, Reiner; Fries, Ruedi

2015-06-01

Most association studies for pinpointing trait-associated variants are performed within breed. The availability of sequence data from key ancestors of several cattle breeds now enables immediate assessment of the frequency of trait-associated variants in populations different from the mapping population and their imputation into large validation populations. The objective of this study was to validate the effects of 4 putatively causative variants on milk production traits, male fertility, and stature in German Fleckvieh and Holstein-Friesian animals using targeted sequence imputation. We used whole-genome sequence data of 456 animals to impute 4 missense mutations in DGAT1, GHR, PRLR, and PROP1 into 10,363 Fleckvieh and 8,812 Holstein animals. The accuracy of the imputed genotypes exceeded 95% for all variants. Association testing with imputed variants revealed consistent antagonistic effects of the DGAT1 p.A232K and GHR p.F279Y variants on milk yield and protein and fat contents, respectively, in both breeds. The allele frequency of both polymorphisms has changed considerably in the past 20 yr, indicating that they were targets of recent selection for milk production traits. The PRLR p.S18N variant was associated with yield traits in Fleckvieh but not in Holstein, suggesting that it may be in linkage disequilibrium with a mutation affecting yield traits rather than being causal. The reported effects of the PROP1 p.H173R variant on milk production, male fertility, and stature could not be confirmed. Our results demonstrate that population-wide imputation of candidate causal variants from sequence data is feasible, enabling their rapid validation in large independent populations. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Sequencing Needs for Viral Diagnostics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, S N; Lam, M; Mulakken, N J

2004-01-26

We built a system to guide decisions regarding the amount of genomic sequencing required to develop diagnostic DNA signatures, which are short sequences that are sufficient to uniquely identify a viral species. We used our existing DNA diagnostic signature prediction pipeline, which selects regions of a target species genome that are conserved among strains of the target (for reliability, to prevent false negatives) and unique relative to other species (for specificity, to avoid false positives). We performed simulations, based on existing sequence data, to assess the number of genome sequences of a target species and of close phylogenetic relatives (''nearmore » neighbors'') that are required to predict diagnostic signature regions that are conserved among strains of the target species and unique relative to other bacterial and viral species. For DNA viruses such as variola (smallpox), three target genomes provide sufficient guidance for selecting species-wide signatures. Three near neighbor genomes are critical for species specificity. In contrast, most RNA viruses require four target genomes and no near neighbor genomes, since lack of conservation among strains is more limiting than uniqueness. SARS and Ebola Zaire are exceptional, as additional target genomes currently do not improve predictions, but near neighbor sequences are urgently needed. Our results also indicate that double stranded DNA viruses are more conserved among strains than are RNA viruses, since in most cases there was at least one conserved signature candidate for the DNA viruses and zero conserved signature candidates for the RNA viruses.« less

Proton pump inhibitors as anti vacuolar-ATPases drugs: a novel anticancer strategy.

PubMed

Spugnini, Enrico P; Citro, Gennaro; Fais, Stefano

2010-05-08

The vacuolar ATPases are ATP-dependent proton pumps whose functions include the acidification of intracellular compartments and the extrusion of protons through the cell cytoplasmic membrane. These pumps play a pivotal role in the regulation of cell pH in normal cells and, to a much greater extent, in tumor cells. In fact, the glucose metabolism in hypoxic conditions by the neoplasms leads to an intercellular pH drift towards acidity. The acid microenvironment is modulated through the over-expression of H+ transporters that are also involved in tumor progression, invasiveness, distant spread and chemoresistance. Several strategies to block/downmodulate the efficiency of these transporters are currently being investigated. Among them, proton pump inhibitors have shown to successfully block the H+ transporters in vitro and in vivo, leading to apoptotic death. Furthermore, their action seems to synergize with conventional chemotherapy protocols, leading to chemosensitization and reversal of chemoresistance. Aim of this article is to critically revise the current knowledge of this cellular machinery and to summarize the therapeutic strategies developed to counter this mechanism.

Differential expression of vacuolar and defective cell wall invertase genes in roots and seeds of metalliferous and non-metalliferous populations of Rumex dentatus under copper stress.

PubMed

Xu, Zhong-Rui; Cai, Shen-Wen; Huang, Wu-Xing; Liu, Rong-Xiang; Xiong, Zhi-Ting

2018-01-01

Acid invertase activities in roots and young seeds of a metalliferous population (MP) of Rumex dentatus were previously observed to be significantly higher than those of a non-metalliferous population (NMP) under Cu stress. To date, no acid invertase gene has been cloned from R. dentatus. Here, we isolated four full-length cDNAs from the two populations of R. dentatus, presumably encoding cell wall (RdnCIN1 and RdmCIN1 from the NMP and MP, respectively) and vacuolar invertases (RdnVIN1 and RdmVIN1 from the NMP and MP, respectively). Unexpectedly, RdnCIN1 and RdmCIN1 most likely encode special defective invertases with highly attenuated sucrose-hydrolyzing capacity. The transcript levels of RdmCIN1 were significantly higher than those of RdnCIN1 in roots and young seeds under Cu stress, whereas under control conditions, the former was initially lower than the latter. Unexpected high correlations were observed between the transcript levels of RdnCIN1 and RdmCIN1 and the activity of cell wall invertase, even though RdnCIN1 and RdmCIN1 do not encode catalytically active invertases. Similarly, the transcript levels of RdmVIN1 in roots and young seeds were increased under Cu stress, whereas those of RdnVIN1 were decreased. The high correlations between the transcript levels of RdnVIN1 and RdmVIN1 and the activity of vacuolar invertase indicate that RdnVIN1 and RdmVIN1 might control distinct vacuolar invertase activities in the two populations. Moreover, a possible indirect role for acid invertases in Cu tolerance, mediated by generating a range of sugars used as nutrients and signaling molecules, is discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC

PubMed Central

Kong, Daochun; Coleman, Thomas R.; DePamphilis, Melvin L.

2003-01-01

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S.pombe. PMID:12840006

Method and apparatus for biological sequence comparison

DOEpatents

Marr, T.G.; Chang, W.I.

1997-12-23

A method and apparatus are disclosed for comparing biological sequences from a known source of sequences, with a subject (query) sequence. The apparatus takes as input a set of target similarity levels (such as evolutionary distances in units of PAM), and finds all fragments of known sequences that are similar to the subject sequence at each target similarity level, and are long enough to be statistically significant. The invention device filters out fragments from the known sequences that are too short, or have a lower average similarity to the subject sequence than is required by each target similarity level. The subject sequence is then compared only to the remaining known sequences to find the best matches. The filtering member divides the subject sequence into overlapping blocks, each block being sufficiently large to contain a minimum-length alignment from a known sequence. For each block, the filter member compares the block with every possible short fragment in the known sequences and determines a best match for each comparison. The determined set of short fragment best matches for the block provide an upper threshold on alignment values. Regions of a certain length from the known sequences that have a mean alignment value upper threshold greater than a target unit score are concatenated to form a union. The current block is compared to the union and provides an indication of best local alignment with the subject sequence. 5 figs.

Method and apparatus for biological sequence comparison

DOEpatents

Marr, Thomas G.; Chang, William I-Wei

1997-01-01

A method and apparatus for comparing biological sequences from a known source of sequences, with a subject (query) sequence. The apparatus takes as input a set of target similarity levels (such as evolutionary distances in units of PAM), and finds all fragments of known sequences that are similar to the subject sequence at each target similarity level, and are long enough to be statistically significant. The invention device filters out fragments from the known sequences that are too short, or have a lower average similarity to the subject sequence than is required by each target similarity level. The subject sequence is then compared only to the remaining known sequences to find the best matches. The filtering member divides the subject sequence into overlapping blocks, each block being sufficiently large to contain a minimum-length alignment from a known sequence. For each block, the filter member compares the block with every possible short fragment in the known sequences and determines a best match for each comparison. The determined set of short fragment best matches for the block provide an upper threshold on alignment values. Regions of a certain length from the known sequences that have a mean alignment value upper threshold greater than a target unit score are concatenated to form a union. The current block is compared to the union and provides an indication of best local alignment with the subject sequence.

Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing

PubMed Central

Brownstein, Zippora; Abu-Rayyan, Amal; Karfunkel-Doron, Daphne; Sirigu, Serena; Davidov, Bella; Shohat, Mordechai; Frydman, Moshe; Houdusse, Anne; Kanaan, Moien; Avraham, Karen B

2014-01-01

Hereditary hearing loss is genetically heterogeneous, with a large number of genes and mutations contributing to this sensory, often monogenic, disease. This number, as well as large size, precludes comprehensive genetic diagnosis of all known deafness genes. A combination of targeted genomic capture and massively parallel sequencing (MPS), also referred to as next-generation sequencing, was applied to determine the deafness-causing genes in hearing-impaired individuals from Israeli Jewish and Palestinian Arab families. Among the mutations detected, we identified nine novel mutations in the genes encoding myosin VI, myosin VIIA and myosin XVA, doubling the number of myosin mutations in the Middle East. Myosin VI mutations were identified in this population for the first time. Modeling of the mutations provided predicted mechanisms for the damage they inflict in the molecular motors, leading to impaired function and thus deafness. The myosin mutations span all regions of these molecular motors, leading to a wide range of hearing phenotypes, reinforcing the key role of this family of proteins in auditory function. This study demonstrates that multiple mutations responsible for hearing loss can be identified in a relatively straightforward manner by targeted-gene MPS technology and concludes that this is the optimal genetic diagnostic approach for identification of mutations responsible for hearing loss. PMID:24105371

Kit for detecting nucleic acid sequences using competitive hybridization probes

DOEpatents

Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

2001-01-01

A kit is provided for detecting a target nucleic acid sequence in a sample, the kit comprising: a first hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the first hybridization probe including a first complexing agent for forming a binding pair with a second complexing agent; and a second hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the first hybridization probe does not selectively hybridize, the second hybridization probe including a detectable marker; a third hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the third hybridization probe including the same detectable marker as the second hybridization probe; and a fourth hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the third hybridization probe does not selectively hybridize, the fourth hybridization probe including the first complexing agent for forming a binding pair with the second complexing agent; wherein the first and second hybridization probes are capable of simultaneously hybridizing to the target sequence and the third and fourth hybridization probes are capable of simultaneously hybridizing to the target sequence, the detectable marker is not present on the first or fourth hybridization probes and the first, second, third, and fourth hybridization probes each include a competitive nucleic acid sequence which is sufficiently complementary to a third portion of the target sequence that the competitive sequences of the first, second, third, and fourth hybridization probes compete with each other to hybridize to the third portion of the

Vacuolar zinc transporter Zrc1 is required for detoxification of excess intracellular zinc in the human fungal pathogen Cryptococcus neoformans.

PubMed

Cho, Minsu; Hu, Guanggan; Caza, Mélissa; Horianopoulos, Linda C; Kronstad, James W; Jung, Won Hee

2018-01-01

Zinc is an important transition metal in all living organisms and is required for numerous biological processes. However, excess zinc can also be toxic to cells and cause cellular stress. In the model fungus Saccharomyces cerevisiae, a vacuolar zinc transporter, Zrc1, plays important roles in the storage and detoxification of excess intracellular zinc to protect the cell. In this study, we identified an ortholog of the S. cerevisiae ZRC1 gene in the human fungal pathogen Cryptococcus neoformans. Zrc1 was localized in the vacuolar membrane in C. neoformans, and a mutant lacking ZRC1 showed significant growth defects under high-zinc conditions. These results suggested a role for Zrc1 in zinc detoxification. However, contrary to our expectation, the expression of Zrc1 was induced in cells grown in zinc-limited conditions and decreased upon the addition of zinc. These expression patterns were similar to those of Zip1, the high-affinity zinc transporter in the plasma membrane of C. neoformans. Furthermore, we used the zrc1 mutant in a murine model of cryptococcosis to examine whether a mammalian host could inhibit the survival of C. neoformans using zinc toxicity. We found that the mutant showed no difference in virulence compared with the wildtype strain. This result suggests that Zrc1-mediated zinc detoxification is not required for the virulence of C. neoformans, and imply that zinc toxicity may not be an important aspect of the host immune response to the fungus.

Effect of inherent location uncertainty on detection of stationary targets in noisy image sequences.

PubMed

Manjeshwar, R M; Wilson, D L

2001-01-01

The effect of inherent location uncertainty on the detection of stationary targets was determined in noisy image sequences. Targets were thick and thin projected cylinders mimicking arteries, catheters, and guide wires in medical imaging x-ray fluoroscopy. With the use of an adaptive forced-choice method, detection contrast sensitivity (the inverse of contrast) was measured both with and without marker cues that directed the attention of observers to the target location. With the probability correct clamped at 80%, contrast sensitivity increased an average of 77% when the marker was added to the thin-cylinder target. There was an insignificant effect on the thick cylinder. The large enhancement with the thin cylinder was obtained even though the target was located exactly in the center of a small panel, giving observers the impression that it was well localized. Psychometric functions consisting of d' plotted as a function of the square root of the signal-energy-to-noise-ratio gave a positive x intercept for the case of the thin cylinder without a marker. This x intercept, characteristic of uncertainty in other types of detection experiments, disappeared when the marker was added or when the thick cylinder was used. Inherent location uncertainty was further characterized by using four different markers with varying proximity to the target. Visual detection by human observers increased monotonically as the markers better localized the target. Human performance was modeled as a matched-filter detector with an uncertainty in the placement of the template. The removal of a location cue was modeled by introducing a location uncertainty of approximately equals 0.4 mm on the display device or only 7 microm on the retina, a size on the order of a single photoreceptor field. We conclude that detection is affected by target location uncertainty on the order of cellular dimensions, an observation with important implications for detection mechanisms in humans. In medical

Improving mapping and SNP-calling performance in multiplexed targeted next-generation sequencing

PubMed Central

2012-01-01

Background Compared to classical genotyping, targeted next-generation sequencing (tNGS) can be custom-designed to interrogate entire genomic regions of interest, in order to detect novel as well as known variants. To bring down the per-sample cost, one approach is to pool barcoded NGS libraries before sample enrichment. Still, we lack a complete understanding of how this multiplexed tNGS approach and the varying performance of the ever-evolving analytical tools can affect the quality of variant discovery. Therefore, we evaluated the impact of different software tools and analytical approaches on the discovery of single nucleotide polymorphisms (SNPs) in multiplexed tNGS data. To generate our own test model, we combined a sequence capture method with NGS in three experimental stages of increasing complexity (E. coli genes, multiplexed E. coli, and multiplexed HapMap BRCA1/2 regions). Results We successfully enriched barcoded NGS libraries instead of genomic DNA, achieving reproducible coverage profiles (Pearson correlation coefficients of up to 0.99) across multiplexed samples, with <10% strand bias. However, the SNP calling quality was substantially affected by the choice of tools and mapping strategy. With the aim of reducing computational requirements, we compared conventional whole-genome mapping and SNP-calling with a new faster approach: target-region mapping with subsequent ‘read-backmapping’ to the whole genome to reduce the false detection rate. Consequently, we developed a combined mapping pipeline, which includes standard tools (BWA, SAMtools, etc.), and tested it on public HiSeq2000 exome data from the 1000 Genomes Project. Our pipeline saved 12 hours of run time per Hiseq2000 exome sample and detected ~5% more SNPs than the conventional whole genome approach. This suggests that more potential novel SNPs may be discovered using both approaches than with just the conventional approach. Conclusions We recommend applying our general �

Identification of microRNAs and their targets in Finger millet by high throughput sequencing.

PubMed

Usha, S; Jyothi, M N; Sharadamma, N; Dixit, Rekha; Devaraj, V R; Nagesh Babu, R

2015-12-15

MicroRNAs are short non-coding RNAs which play an important role in regulating gene expression by mRNA cleavage or by translational repression. The majority of identified miRNAs were evolutionarily conserved; however, others expressed in a species-specific manner. Finger millet is an important cereal crop; nonetheless, no practical information is available on microRNAs to date. In this study, we have identified 95 conserved microRNAs belonging to 39 families and 3 novel microRNAs by high throughput sequencing. For the identified conserved and novel miRNAs a total of 507 targets were predicted. 11 miRNAs were validated and tissue specificity was determined by stem loop RT-qPCR, Northern blot. GO analyses revealed targets of miRNA were involved in wide range of regulatory functions. This study implies large number of known and novel miRNAs found in Finger millet which may play important role in growth and development. Copyright © 2015 Elsevier B.V. All rights reserved.

Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs)

PubMed Central

Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

2016-01-01

Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the ‘Vitis vinifera cv. Pinot Noir’ and ‘Vitis vinifera cv. Thompson Seedless’ varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs. PMID:27551866

Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs).

PubMed

Tang, Yujin; Wang, Ruipu; Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

2016-01-01

Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the 'Vitis vinifera cv. Pinot Noir' and 'Vitis vinifera cv. Thompson Seedless' varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs.

Clinical features of avian vacuolar myelinopathy in American coots

USGS Publications Warehouse

Larsen, R.S.; Nutter, F.B.; Augspurger, T.; Rocke, T.E.; Tomlinson, L.; Thomas, N.J.; Stoskopf, M.K.

2002-01-01

Objectivea??To characterize clinical features of avian vacuolar myelinopathy (AVM) in American coots. Designa??Case-control study. Animalsa??26 AVM-affected American coots and 12 unaffected coots. Proceduresa??Complete physical, neurologic, hematologic, and plasma biochemical evaluations were performed. Affected coots received supportive care. All coots died or were euthanatized, and AVM status was confirmed via histopathologic findings. Resultsa??3 severely affected coots were euthanatized immediately after examination. Seventeen affected coots were found dead within 7 days of admission, but 5 affected coots survived > 21 days and had signs of clinical recovery. Abnormal physical examination findings appeared to be related to general debilitation. Ataxia (88%), decreased withdrawal reflexes (88%), proprioceptive deficits (81%), decreased vent responses (69%), beak or tongue weakness (42%), and head tremors (31%), as well as absent pupillary light responses (46%), anisocoria (15%), apparent blindness (4%), nystagmus (4%), and strabismus (4%) were detected. Few gross abnormalities were detected at necropsy, but histologically, all AVM-affected coots had severe vacuolation of white matter of the brain. None of the control coots had vacuolation. Conclusions and Clinical Relevancea??Although there was considerable variability in form and severity of clinical neurologic abnormalities, clinical signs common in AVM-affected birds were identified. Clinical recovery of some AVM-affected coots can occur when supportive care is administered. Until the etiology is identified, caution should be exercised when rehabilitating and releasing coots thought to be affected by AVM.

Species identification in mixed tuna samples with next-generation sequencing targeting two short cytochrome b gene fragments.

PubMed

Kappel, Kristina; Haase, Ilka; Käppel, Christine; Sotelo, Carmen G; Schröder, Ute

2017-11-01

Conventional Sanger sequencing of PCR products is the gold standard for species authentication of seafood products. However, this method is inappropriate for the analysis of products that might contain mixtures of species, such as tinned tuna. The purpose of this study was to test whether next-generation sequencing (NGS) can be a solution for the authentication of mixed products. Nine tuna samples containing mixtures of up to four species were prepared and subjected to an NGS approach targeting two short cytochrome b gene (cytb) fragments on the Illumina MiSeq platform. Sequence recovery was precise and admixtures of as low as 1% could be identified, depending on the species composition of the mixtures. Duplicate samples as well as two individual NGS runs produced very similar results. A first test of three commercial tinned tuna samples indicated the presence of different species in the same tin, although this is forbidden by EU law. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Application of targeted capture technology and next generation sequencing in molecular diagnosis of inherited myopathy].

PubMed

Fu, Xiaona; Liu, Aijie; Yang, Haipo; Wei, Cuijie; Ding, Juan; Wang, Shuang; Wang, Jingmin; Yuan, Yun; Jiang, Yuwu; Xiong, Hui

2015-10-01

To elucidate the usefulness of next generation sequencing for diagnosis of inherited myopathy, and to analyze the relevance between clinical phenotype and genotype in inherited myopathy. Related genes were selected for SureSelect target enrichment system kit (Panel Version 1 and Panel Version 2). A total of 134 patients who were diagnosed as inherited myopathy clinically underwent next generation sequencing in Department of Pediatrics, Peking University First Hospital from January 2013 to June 2014. Clinical information and gene detection result of the patients were collected and analyzed. Seventy-seven of 134 patients (89 males and 45 females, visiting ages from 6-month-old to 26-year-old, average visiting age was 6 years and 1 month) underwent next generation sequencing by Panel Version 1 in 2013, and 57 patients underwent next generation sequencing by Panel Version 2 in 2014. The gene detection revealed that 74 patients had pathogenic gene mutations, and the positive rate of genetic diagnosis was 55.22%. One patient was diagnosed as metabolic myopathy. Five patients were diagnosed as congenital myopathy; 68 were diagnosed as muscular dystrophy, including 22 with congenital muscular dystrophy 1A (MDC1A), 11 with Ullrich congenital muscular dystrophy (UCMD), 6 with Bethlem myopathy (BM), 12 with Duchenne muscular dystrophy (DMD) caused by point mutations in DMD gene, 5 with LMNA-related congenital muscular dystrophy (L-CMD), 1 with Emery-Dreifuss muscular dystrophy (EDMD), 7 with alpha-dystroglycanopathy (α-DG) patients, and 4 with limb-girdle muscular dystrophy (LGMD) patients. Next generation sequencing plays an important role in diagnosis of inherited myopathy. Clinical and biological information analysis was essential for screening pathogenic gene of inherited myopathy.

Enhanced salt stress tolerance of rice plants expressing a vacuolar H+-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Löisel

USDA-ARS?s Scientific Manuscript database

The physiological role of a vacuolar ATPase subunit c1 (SaVHAc1) from a halophyte grass Spartina alterniflora was studied through its expression in rice. The SaVHAc1– expressing plants showed enhanced tolerance to salt stress than the wild-type plants, mainly through adjustments in early stage and p...

Identification of Direct Target Genes Using Joint Sequence and Expression Likelihood with Application to DAF-16

PubMed Central

Yu, Ron X.; Liu, Jie; True, Nick; Wang, Wei

2008-01-01

A major challenge in the post-genome era is to reconstruct regulatory networks from the biological knowledge accumulated up to date. The development of tools for identifying direct target genes of transcription factors (TFs) is critical to this endeavor. Given a set of microarray experiments, a probabilistic model called TRANSMODIS has been developed which can infer the direct targets of a TF by integrating sequence motif, gene expression and ChIP-chip data. The performance of TRANSMODIS was first validated on a set of transcription factor perturbation experiments (TFPEs) involving Pho4p, a well studied TF in Saccharomyces cerevisiae. TRANSMODIS removed elements of arbitrariness in manual target gene selection process and produced results that concur with one's intuition. TRANSMODIS was further validated on a genome-wide scale by comparing it with two other methods in Saccharomyces cerevisiae. The usefulness of TRANSMODIS was then demonstrated by applying it to the identification of direct targets of DAF-16, a critical TF regulating ageing in Caenorhabditis elegans. We found that 189 genes were tightly regulated by DAF-16. In addition, DAF-16 has differential preference for motifs when acting as an activator or repressor, which awaits experimental verification. TRANSMODIS is computationally efficient and robust, making it a useful probabilistic framework for finding immediate targets. PMID:18350157

Disruption of the vacuolar-type H+-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes.

PubMed

Kissing, Sandra; Rudnik, Sönke; Damme, Markus; Lüllmann-Rauch, Renate; Ichihara, Atsuhiro; Kornak, Uwe; Eskelinen, Eeva-Liisa; Jabs, Sabrina; Heeren, Jörg; De Brabander, Jef K; Haas, Albert; Saftig, Paul

2017-04-03

The vacuolar-type H + -translocating ATPase (v-H + -ATPase) has been implicated in the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H + -ATPase and MTORC1, we destablilized v-H + -ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H + -ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H + -ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H + -ATPase-mediated regulation of MTORC1.

Targeted next generation sequencing of well-differentiated/dedifferentiated liposarcoma reveals novel gene amplifications and mutations

PubMed Central

Somaiah, Neeta; Beird, Hannah C; Barbo, Andrea; Song, Juhee; Mills Shaw, Kenna R.; Wang, Wei-Lien; Eterovic, Karina; Chen, Ken; Lazar, Alexander; Conley, Anthony P.; Ravi, Vinod; Hwu, Patrick; Futreal, Andrew; Simon, George; Meric-Bernstam, Funda; Hong, David

2018-01-01

Well-differentiated/dedifferentiated liposarcoma is a common soft tissue sarcoma with approximately 1500 new cases per year. Surgery is the mainstay of treatment but recurrences are frequent and systemic options are limited. ‘Tumor genotyping’ is becoming more common in clinical practice as it offers the hope of personalized targeted therapy. We wanted to evaluate the results and the clinical utility of available next-generation sequencing panels in WD/DD liposarcoma. Patients who had their tumor sequenced by either FoundationOne (n = 13) or the institutional T200/T200.1 panels (n = 7) were included in this study. Significant copy number alterations were identified, but mutations were infrequent. Out of the 27 mutations detected in 7 samples, 8 (CTNNB1, MECOM, ZNF536, EGFR, EML4, CSMD3, PBRM1, PPP1R3A) were identified as deleterious (on Condel, PolyPhen and SIFT) and a truncating mutation was found in NF2. Of these, EGFR and NF2 are potential driver mutations and have not been reported previously in liposarcoma. MDM2 and CDK4 amplification was universally present in all the tested samples and multiple other recurrent genes with high amplification or high deletion were detected. Many of these targets are potentially actionable. Eight patients went on to receive an MDM2 inhibitor with a median time to progression of 23 months (95% CI: 10-83 months). PMID:29731991

Method for sequencing nucleic acid molecules

DOEpatents

Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

2006-06-06

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Method for sequencing nucleic acid molecules

DOEpatents

Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

2006-05-30

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Targeted next generation sequencing of parotid gland cancer uncovers genetic heterogeneity.

PubMed

Grünewald, Inga; Vollbrecht, Claudia; Meinrath, Jeannine; Meyer, Moritz F; Heukamp, Lukas C; Drebber, Uta; Quaas, Alexander; Beutner, Dirk; Hüttenbrink, Karl-Bernd; Wardelmann, Eva; Hartmann, Wolfgang; Büttner, Reinhard; Odenthal, Margarete; Stenner, Markus

2015-07-20

Salivary gland cancer represents a heterogeneous group of malignant tumors. Due to their low incidence and the existence of multiple morphologically defined subtypes, these tumors are still poorly understood with regard to their molecular pathogenesis and therapeutically relevant genetic alterations.Performing a systematic and comprehensive study covering 13 subtypes of salivary gland cancer, next generation sequencing was done on 84 tissue samples of parotid gland cancer using multiplex PCR for enrichment of cancer related gene loci covering hotspots of 46 cancer genes.Mutations were identified in 22 different genes. The most frequent alterations affected TP53, followed by RAS genes, PIK3CA, SMAD4 and members of the ERB family. HRAS mutations accounted for more than 90% of RAS mutations, occurring especially in epithelial-myoepithelial carcinomas and salivary duct carcinomas. Additional mutations in PIK3CA also affected particularly epithelial-myoepithelial carcinomas and salivary duct carcinomas, occurring simultaneously with HRAS mutations in almost all cases, pointing to an unknown and therapeutically relevant molecular constellation. Interestingly, 14% of tumors revealed mutations in surface growth factor receptor genes including ALK, HER2, ERBB4, FGFR, cMET and RET, which might prove to be targetable by new therapeutic agents. 6% of tumors revealed mutations in SMAD4.In summary, our data provide novel insight into the fundamental molecular heterogeneity of salivary gland cancer, relevant in terms of tumor classification and the establishment of targeted therapeutic concepts.

Solid phase sequencing of double-stranded nucleic acids

DOEpatents

Fu, Dong-Jing; Cantor, Charles R.; Koster, Hubert; Smith, Cassandra L.

2002-01-01

This invention relates to methods for detecting and sequencing of target double-stranded nucleic acid sequences, to nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probe comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include nucleic acids in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated determination of molecular weights and identification of the target sequence.

Combined Targeted DNA Sequencing in Non-Small Cell Lung Cancer (NSCLC) Using UNCseq and NGScopy, and RNA Sequencing Using UNCqeR for the Detection of Genetic Aberrations in NSCLC

PubMed Central

Walter, Vonn; Patel, Nirali M.; Eberhard, David A.; Hayward, Michele C.; Salazar, Ashley H.; Jo, Heejoon; Soloway, Matthew G.; Wilkerson, Matthew D.; Parker, Joel S.; Yin, Xiaoying; Zhang, Guosheng; Siegel, Marni B.; Rosson, Gary B.; Earp, H. Shelton; Sharpless, Norman E.; Gulley, Margaret L.; Weck, Karen E.

2015-01-01

The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07–0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11–1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion. PMID:26076459

Pure Perceptual-Based Sequence Learning: A Role for Visuospatial Attention

ERIC Educational Resources Information Center

Remillard, Gilbert

2009-01-01

Learning the structure of a sequence of target locations when target location is not the response dimension and the sequence of target locations is uncorrelated with the sequence of responses is called pure perceptual-based sequence learning. The paradigm introduced by G. Remillard (2003) was used to determine whether orienting of visuospatial…

Vacuolar transport of the glutathione conjugate of trans-cinnamic acid.

PubMed

Walczak, H A; Dean, J V

2000-02-01

Red beet (Beta vulgaris L.) tonoplast membrane vesicles and [14C]trans-cinnamic acid-glutatione were used to study the vacuolar transport of phynylpropanoid-glutathione conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [14C]trans-cinnamic acid-glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-conjugated [14C]trans-cinnamic acid. Uptake of the conjugate in the presence of MgATP was not dependent on a trans-tonoblast H+-electrochemical gradient, because uptake was not affected by the addition of NH4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 microM; 14% inhibition). Uptake of the conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4-dinitrophenyl) glutathione (500 microM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated Km and Vmax values for uptake were 142 microM amd 5.95 nmol mg(-1) min(-1), respectively. Based on these results, phenylpropanoid-glutation conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the glutathione S-conjugate pump(s) located in the tonoplast membrane.

Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian

2009-11-06

Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3more » in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.« less

Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

DOE PAGES

Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric; ...

2015-03-30

Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric

Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

Efficient strategy for the molecular diagnosis of intellectual disability using targeted high-throughput sequencing.

PubMed

Redin, Claire; Gérard, Bénédicte; Lauer, Julia; Herenger, Yvan; Muller, Jean; Quartier, Angélique; Masurel-Paulet, Alice; Willems, Marjolaine; Lesca, Gaétan; El-Chehadeh, Salima; Le Gras, Stéphanie; Vicaire, Serge; Philipps, Muriel; Dumas, Michaël; Geoffroy, Véronique; Feger, Claire; Haumesser, Nicolas; Alembik, Yves; Barth, Magalie; Bonneau, Dominique; Colin, Estelle; Dollfus, Hélène; Doray, Bérénice; Delrue, Marie-Ange; Drouin-Garraud, Valérie; Flori, Elisabeth; Fradin, Mélanie; Francannet, Christine; Goldenberg, Alice; Lumbroso, Serge; Mathieu-Dramard, Michèle; Martin-Coignard, Dominique; Lacombe, Didier; Morin, Gilles; Polge, Anne; Sukno, Sylvie; Thauvin-Robinet, Christel; Thevenon, Julien; Doco-Fenzy, Martine; Genevieve, David; Sarda, Pierre; Edery, Patrick; Isidor, Bertrand; Jost, Bernard; Olivier-Faivre, Laurence; Mandel, Jean-Louis; Piton, Amélie

2014-11-01

Intellectual disability (ID) is characterised by an extreme genetic heterogeneity. Several hundred genes have been associated to monogenic forms of ID, considerably complicating molecular diagnostics. Trio-exome sequencing was recently proposed as a diagnostic approach, yet remains costly for a general implementation. We report the alternative strategy of targeted high-throughput sequencing of 217 genes in which mutations had been reported in patients with ID or autism as the major clinical concern. We analysed 106 patients with ID of unknown aetiology following array-CGH analysis and other genetic investigations. Ninety per cent of these patients were males, and 75% sporadic cases. We identified 26 causative mutations: 16 in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA, MECP2, SLC9A6, SLC16A2, PHF8) and 10 de novo in autosomal-dominant genes (DYRK1A, GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We also detected four possibly causative mutations (eg, in NLGN3) requiring further investigations. We present detailed reasoning for assigning causality for each mutation, and associated patients' clinical information. Some genes were hit more than once in our cohort, suggesting they correspond to more frequent ID-associated conditions (KDM5C, MECP2, DYRK1A, TCF4). We highlight some unexpected genotype to phenotype correlations, with causative mutations being identified in genes associated to defined syndromes in patients deviating from the classic phenotype (DMD, TCF4, MECP2). We also bring additional supportive (HCFC1, MED13L) or unsupportive (SHROOM4, SRPX2) evidences for the implication of previous candidate genes or mutations in cognitive disorders. With a diagnostic yield of 25% targeted sequencing appears relevant as a first intention test for the diagnosis of ID, but importantly will also contribute to a better understanding regarding the specific contribution of the many genes implicated in ID and autism. Published by the

Gain-of-function mutations identify amino acids within transmembrane domains of the yeast vacuolar transporter Zrc1 that determine metal specificity

PubMed Central

Lin, Huilan; Burton, Damali; Li, Liangtao; Warner, David E.; Phillips, John D.; Ward, Diane McVEY; Kaplan, Jerry

2015-01-01

Cation diffusion facilitator transporters are found in all three Kingdoms of life and are involved in transporting transition metals out of the cytosol. The metals they transport include Zn2+, Co2+, Fe2+, Cd2+, Ni2+ and Mn2+; however, no single transporter transports all metals. Previously we showed that a single amino acid mutation in the yeast vacuolar zinc transporter Zrc1 changed its substrate specificity from Zn2+ to Fe2+ and Mn2+ [Lin, Kumanovics, Nelson, Warner, Ward and Kaplan (2008) J. Biol. Chem. 283, 33865–33873]. Mutant Zrc1 that gained iron transport activity could protect cells with a deletion in the vacuolar iron transporter (CCC1) from high iron toxicity. Utilizing suppression of high iron toxicity and PCR mutagenesis of ZRC1, we identified other amino acid substitutions within ZRC1 that changed its metal specificity. All Zrc1 mutants that transported Fe2+ could also transport Mn2+. Some Zrc1 mutants lost the ability to transport Zn2+, but others retained the ability to transport Zn2+. All of the amino acid substitutions that resulted in a gain in Fe2+ transport activity were found in transmembrane domains. In addition to alteration of residues adjacent to the putative metal-binding site in two transmembrane domains, alteration of residues distant from the binding site affected substrate specificity. These results suggest that substrate selection involves co-operativity between transmembrane domains. PMID:19538181

Conserved sequences in the current strains of HIV-1 subtype A in Russia are effectively targeted by artificial RNAi in vitro.

PubMed

Tchurikov, Nickolai A; Fedoseeva, Daria M; Gashnikova, Natalya M; Sosin, Dmitri V; Gorbacheva, Maria A; Alembekov, Ildar R; Chechetkin, Vladimir R; Kravatsky, Yuri V; Kretova, Olga V

2016-05-25

Highly active antiretroviral therapy has greatly reduced the morbidity and mortality of AIDS. However, many of the antiretroviral drugs are toxic with long-term use, and all currently used anti-HIV agents generate drug-resistant mutants. Therefore, there is a great need for new approaches to AIDS therapy. RNAi is a powerful means of inhibiting HIV-1 production in human cells. We propose to use RNAi for gene therapy of HIV/AIDS. Previously we identified a number of new biologically active siRNAs targeting several moderately conserved regions in HIV-1 transcripts. Here we analyze the heterogeneity of nucleotide sequences in three RNAi targets in sequences encoding the reverse transcriptase and integrase domains of current isolates of HIV-1 subtype A in Russia. These data were used to generate genetic constructs expressing short hairpin RNAs 28-30-bp in length that could be processed in cells into siRNAs. After transfection of the constructs we observed siRNAs that efficiently attacked the selected targets. We expect that targeting several viral genes important for HIV-1 reproduction will help overcome the problem of viral adaptation and will prevent the appearance of RNAi escape mutants in current virus strains, an important feature of gene therapy of HIV/AIDS. Copyright © 2016 Elsevier B.V. All rights reserved.

Targeted sequencing-based analyses of candidate gene variants in ulcerative colitis-associated colorectal neoplasia.

PubMed

Chakrabarty, Sanjiban; Varghese, Vinay Koshy; Sahu, Pranoy; Jayaram, Pradyumna; Shivakumar, Bhadravathi M; Pai, Cannanore Ganesh; Satyamoorthy, Kapaettu

2017-06-27

Long-standing ulcerative colitis (UC) leading to colorectal cancer (CRC) is one of the most serious and life-threatening consequences acknowledged globally. Ulcerative colitis-associated colorectal carcinogenesis showed distinct molecular alterations when compared with sporadic colorectal carcinoma. Targeted sequencing of 409 genes in tissue samples of 18 long-standing UC subjects at high risk of colorectal carcinoma (UCHR) was performed to identify somatic driver mutations, which may be involved in the molecular changes during the transformation of non-dysplastic mucosa to high-grade dysplasia. Findings from the study are also compared with previously published genome wide and exome sequencing data in inflammatory bowel disease-associated and sporadic colorectal carcinoma. Next-generation sequencing analysis identified 1107 mutations in 275 genes in UCHR subjects. In addition to TP53 (17%) and KRAS (22%) mutations, recurrent mutations in APC (33%), ACVR2A (61%), ARID1A (44%), RAF1 (39%) and MTOR (61%) were observed in UCHR subjects. In addition, APC, FGFR3, FGFR2 and PIK3CA driver mutations were identified in UCHR subjects. Recurrent mutations in ARID1A (44%), SMARCA4 (17%), MLL2 (44%), MLL3 (67%), SETD2 (17%) and TET2 (50%) genes involved in histone modification and chromatin remodelling were identified in UCHR subjects. Our study identifies new oncogenic driver mutations which may be involved in the transition of non-dysplastic cells to dysplastic phenotype in the subjects with long-standing UC with high risk of progression into colorectal neoplasia.

Expression, crystallization and phasing of vacuolar H(+)-ATPase subunit C (Vma5p) of Saccharomyces cerevisiae.

PubMed

Drory, Omri; Mor, Adi; Frolow, Felix; Nelson, Nathan

2004-10-01

The expression, crystallization and phasing of subunit C (Vma5p) of the yeast (Saccharomyces cerevisiae) vacuolar proton-translocating ATPase (V-ATPase) is described. The expressed protein consists of 412 residues: 392 from the reading frame of Vma5p and 20 N-terminal residues originating from the plasmid. Diffraction-quality crystals were obtained using the hanging-drop and sitting-drop vapour-diffusion methods assisted by streak-seeding, with PEG 3350 as precipitant. The crystals formed in hanging drops diffracted to 1.80 A and belong to space group P4(3)2(1)2(1), with unit-cell parameters a = b = 62.54, c = 327.37 A, alpha = beta = gamma = 90 degrees. The structure was solved using SIRAS with a Lu(O2C2H3)2 heavy-atom derivative.

ampliMethProfiler: a pipeline for the analysis of CpG methylation profiles of targeted deep bisulfite sequenced amplicons.

PubMed

Scala, Giovanni; Affinito, Ornella; Palumbo, Domenico; Florio, Ermanno; Monticelli, Antonella; Miele, Gennaro; Chiariotti, Lorenzo; Cocozza, Sergio

2016-11-25

CpG sites in an individual molecule may exist in a binary state (methylated or unmethylated) and each individual DNA molecule, containing a certain number of CpGs, is a combination of these states defining an epihaplotype. Classic quantification based approaches to study DNA methylation are intrinsically unable to fully represent the complexity of the underlying methylation substrate. Epihaplotype based approaches, on the other hand, allow methylation profiles of cell populations to be studied at the single molecule level. For such investigations, next-generation sequencing techniques can be used, both for quantitative and for epihaplotype analysis. Currently available tools for methylation analysis lack output formats that explicitly report CpG methylation profiles at the single molecule level and that have suited statistical tools for their interpretation. Here we present ampliMethProfiler, a python-based pipeline for the extraction and statistical epihaplotype analysis of amplicons from targeted deep bisulfite sequencing of multiple DNA regions. ampliMethProfiler tool provides an easy and user friendly way to extract and analyze the epihaplotype composition of reads from targeted bisulfite sequencing experiments. ampliMethProfiler is written in python language and requires a local installation of BLAST and (optionally) QIIME tools. It can be run on Linux and OS X platforms. The software is open source and freely available at http://amplimethprofiler.sourceforge.net .

Sequence-Specific Targeting of Bacterial Resistance Genes Increases Antibiotic Efficacy

PubMed Central

Wong, Michael; Daly, Seth M.; Greenberg, David E.; Toprak, Erdal

2016-01-01

The lack of effective and well-tolerated therapies against antibiotic-resistant bacteria is a global public health problem leading to prolonged treatment and increased mortality. To improve the efficacy of existing antibiotic compounds, we introduce a new method for strategically inducing antibiotic hypersensitivity in pathogenic bacteria. Following the systematic verification that the AcrAB-TolC efflux system is one of the major determinants of the intrinsic antibiotic resistance levels in Escherichia coli, we have developed a short antisense oligomer designed to inhibit the expression of acrA and increase antibiotic susceptibility in E. coli. By employing this strategy, we can inhibit E. coli growth using 2- to 40-fold lower antibiotic doses, depending on the antibiotic compound utilized. The sensitizing effect of the antisense oligomer is highly specific to the targeted gene’s sequence, which is conserved in several bacterial genera, and the oligomer does not have any detectable toxicity against human cells. Finally, we demonstrate that antisense oligomers improve the efficacy of antibiotic combinations, allowing the combined use of even antagonistic antibiotic pairs that are typically not favored due to their reduced activities. PMID:27631336

A vacuolar H(+)-pyrophosphatase differential activation and energy coupling integrate the responses of weeds and crops to drought stress.

PubMed

Venancio, Josimara Barcelos; Catunda, Michelle Guedes; Ogliari, Juarez; Rima, Janaína Aparecida Hottz; Okorokova-Facanha, Anna Lvovna; Okorokov, Lev Alexandrovitich; Facanha, Arnoldo Rocha

2014-06-01

Cyperus rotundus L. is a C4 weed of large vegetative and reproductive vigor endowed with competitive advantages over most crop species mainly under adverse environmental conditions. Vacuole functions are critical for the mechanisms of drought resistance, and here the modulation of the primary system of vacuolar ion transport is investigated during a transient water stress imposed to this weed and to C4 crop species (Zea mays L.). The vacuolar H(+) pumps, the H(+)-ATPase and H(+)-PPiase, expression, activities and the energy coupling were spectrophotometrically investigated as key elements in the differential drought-resistance mechanisms developed by weeds and crops. In C. rotundus tonoplasts, ATP hydrolysis was more sensitive to drought than its coupled H(+) transport, which was in turn at least 3-folds faster than that mediated by the H(+)-PPiase. Its PPi hydrolysis was only slightly affected by severe water deficit, contrasting with the disruption induced in the PPi-dependent H(+)-gradient. This effect was antagonized by plant rehydration as the H(+)-PPiase activity was highly stimulated, reassuming a coupled PPi-driven H(+) pumping. Maize tonoplasts exhibited 2-4 times lower hydrolytic activities than that of C. rotundus, but were able to overactivate specifically PPi-dependent H(+) pumping in response to stress relief, resulting in an enhanced H(+)-pumps coupling efficiency. These results together with immunoanalysis revealed profiles consistent with pre- and post-translational changes occurring on the tonoplast H(+)-pumps, which differ between weeds and crops upon water deficit. The evidences highlight an unusual modulation of the H(+)-PPiase energy coupling as a key biochemical change related to environmental stresses adaptive capacity of plants. Copyright © 2014 Elsevier B.V. All rights reserved.

The C-terminal propeptide of a plant defensin confers cytoprotective and subcellular targeting functions

PubMed Central

2014-01-01

Background Plant defensins are small (45–54 amino acids), basic, cysteine-rich proteins that have a major role in innate immunity in plants. Many defensins are potent antifungal molecules and are being evaluated for their potential to create crop plants with sustainable disease resistance. Defensins are produced as precursor molecules which are directed into the secretory pathway and are divided into two classes based on the absence (class I) or presence (class II) of an acidic C-terminal propeptide (CTPP) of about 33 amino acids. The function of this CTPP had not been defined. Results By transgenically expressing the class II plant defensin NaD1 with and without its cognate CTPP we have demonstrated that NaD1 is phytotoxic to cotton plants when expressed without its CTPP. Transgenic cotton plants expressing constructs encoding the NaD1 precursor with the CTPP had the same morphology as non-transgenic plants but expression of NaD1 without the CTPP led to plants that were stunted, had crinkled leaves and were less viable. Immunofluorescence microscopy and transient expression of a green fluorescent protein (GFP)-CTPP chimera were used to confirm that the CTPP is sufficient for vacuolar targeting. Finally circular dichroism and NMR spectroscopy were used to show that the CTPP adopts a helical confirmation. Conclusions In this report we have described the role of the CTPP on NaD1, a class II defensin from Nicotiana alata flowers. The CTPP of NaD1 is sufficient for vacuolar targeting and plays an important role in detoxification of the defensin as it moves through the plant secretory pathway. This work may have important implications for the use of defensins for disease protection in transgenic crops. PMID:24495600

Targeted sequencing of clade-specific markers from skin microbiomes for forensic human identification.

PubMed

Schmedes, Sarah E; Woerner, August E; Novroski, Nicole M M; Wendt, Frank R; King, Jonathan L; Stephens, Kathryn M; Budowle, Bruce

2018-01-01

The human skin microbiome is comprised of diverse communities of bacterial, eukaryotic, and viral taxa and contributes millions of additional genes to the repertoire of human genes, affecting human metabolism and immune response. Numerous genetic and environmental factors influence the microbiome composition and as such contribute to individual-specific microbial signatures which may be exploited for forensic applications. Previous studies have demonstrated the potential to associate skin microbial profiles collected from touched items to their individual owner, mainly using unsupervised methods from samples collected over short time intervals. Those studies utilize either targeted 16S rRNA or shotgun metagenomic sequencing to characterize skin microbiomes; however, these approaches have limited species and strain resolution and susceptibility to stochastic effects, respectively. Clade-specific markers from the skin microbiome, using supervised learning, can predict individual identity using skin microbiomes from their respective donors with high accuracy. In this study the hidSkinPlex is presented, a novel targeted sequencing method using skin microbiome markers developed for human identification. The hidSkinPlex (comprised of 286 bacterial (and phage) family-, genus-, species-, and subspecies-level markers), initially was evaluated on three bacterial control samples represented in the panel (i.e., Propionibacterium acnes, Propionibacterium granulosum, and Rothia dentocariosa) to assess the performance of the multiplex. The hidSkinPlex was further evaluated for prediction purposes. The hidSkinPlex markers were used to attribute skin microbiomes collected from eight individuals from three body sites (i.e., foot (Fb), hand (Hp) and manubrium (Mb)) to their host donor. Supervised learning, specifically regularized multinomial logistic regression and 1-nearest-neighbor classification were used to classify skin microbiomes to their hosts with up to 92% (Fb), 96% (Mb

[Using exon combined target region capture sequencing chip to detect the disease-causing genes of retinitis pigmentosa].

PubMed

Rong, Weining; Chen, Xuejuan; Li, Huiping; Liu, Yani; Sheng, Xunlun

2014-06-01

To detect the disease-causing genes of 10 retinitis pigmentosa pedigrees by using exon combined target region capture sequencing chip. Pedigree investigation study. From October 2010 to December 2013, 10 RP pedigrees were recruited for this study in Ningxia Eye Hospital. All the patients and family members received complete ophthalmic examinations. DNA was abstracted from patients, family members and controls. Using exon combined target region capture sequencing chip to screen the candidate disease-causing mutations. Polymerase chain reaction (PCR) and direct sequencing were used to confirm the disease-causing mutations. Seventy patients and 23 normal family members were recruited from 10 pedigrees. Among 10 RP pedigrees, 1 was autosomal dominant pedigrees and 9 were autosomal recessive pedigrees. 7 mutations related to 5 genes of 5 pedigrees were detected. A frameshift mutation on BBS7 gene was detected in No.2 pedigree, the patients of this pedigree combined with central obesity, polydactyly and mental handicap. No.2 pedigree was diagnosed as Bardet-Biedl syndrome finally. A missense mutation was detected in No.7 and No.10 pedigrees respectively. Because the patients suffered deafness meanwhile, the final diagnosis was Usher syndrome. A missense mutation on C3 gene related to age-related macular degeneration was also detected in No. 7 pedigrees. A nonsense mutation and a missense mutation on CRB1 gene were detected in No. 1 pedigree and a splicesite mutation on PROM1 gene was detected in No. 5 pedigree. Retinitis pigmentosa is a kind of genetic eye disease with diversity clinical phenotypes. Rapid and effective genetic diagnosis technology combined with clinical characteristics analysis is helpful to improve the level of clinical diagnosis of RP.

Sequence-defined cMET/HGFR-targeted Polymers as Gene Delivery Vehicles for the Theranostic Sodium Iodide Symporter (NIS) Gene

PubMed Central

Urnauer, Sarah; Morys, Stephan; Krhac Levacic, Ana; Müller, Andrea M; Schug, Christina; Schmohl, Kathrin A; Schwenk, Nathalie; Zach, Christian; Carlsen, Janette; Bartenstein, Peter; Wagner, Ernst; Spitzweg, Christine

2016-01-01

The sodium iodide symporter (NIS) as well-characterized theranostic gene represents an outstanding tool to target different cancer types allowing noninvasive imaging of functional NIS expression and therapeutic radioiodide application. Based on its overexpression on the surface of most cancer types, the cMET/hepatocyte growth factor receptor serves as ideal target for tumor-selective gene delivery. Sequence-defined polymers as nonviral gene delivery vehicles comprising polyethylene glycol (PEG) and cationic (oligoethanoamino) amide cores coupled with a cMET-binding peptide (cMBP2) were complexed with NIS-DNA and tested for receptor-specificity, transduction efficiency, and therapeutic efficacy in hepatocellular cancer cells HuH7. In vitro iodide uptake studies demonstrated high transduction efficiency and cMET-specificity of NIS-encoding polyplexes (cMBP2-PEG-Stp/NIS) compared to polyplexes without targeting ligand (Ala-PEG-Stp/NIS) and without coding DNA (cMBP2-PEG-Stp/Antisense-NIS). Tumor recruitment and vector biodistribution were investigated in vivo in a subcutaneous xenograft mouse model showing high tumor-selective iodide accumulation in cMBP2-PEG-Stp/NIS-treated mice (6.6 ± 1.6% ID/g 123I, biological half-life 3 hours) by 123I-scintigraphy. Therapy studies with three cycles of polyplexes and 131I application resulted in significant delay in tumor growth and prolonged survival. These data demonstrate the enormous potential of cMET-targeted sequence-defined polymers combined with the unique theranostic function of NIS allowing for optimized transfection efficiency while eliminating toxicity. PMID:27157666

Diversity, expression and mRNA targeting abilities of Argonaute-targeting miRNAs among selected vascular plants.

PubMed

Jagtap, Soham; Shivaprasad, Padubidri V

2014-12-02

Micro (mi)RNAs are important regulators of plant development. Across plant lineages, Dicer-like 1 (DCL1) proteins process long ds-like structures to produce micro (mi) RNA duplexes in a stepwise manner. These miRNAs are incorporated into Argonaute (AGO) proteins and influence expression of RNAs that have sequence complementarity with miRNAs. Expression levels of AGOs are greatly regulated by plants in order to minimize unwarranted perturbations using miRNAs to target mRNAs coding for AGOs. AGOs may also have high promoter specificity-sometimes expression of AGO can be limited to just a few cells in a plant. Viral pathogens utilize various means to counter antiviral roles of AGOs including hijacking the host encoded miRNAs to target AGOs. Two host encoded miRNAs namely miR168 and miR403 that target AGOs have been described in the model plant Arabidopsis and such a mechanism is thought to be well conserved across plants because AGO sequences are well conserved. We show that the interaction between AGO mRNAs and miRNAs is species-specific due to the diversity in sequences of two miRNAs that target AGOs, sequence diversity among corresponding target regions in AGO mRNAs and variable expression levels of these miRNAs among vascular plants. We used miRNA sequences from 68 plant species representing 31 plant families for this analysis. Sequences of miR168 and miR403 are not conserved among plant lineages, but surprisingly they differ drastically in their sequence diversity and expression levels even among closely related plants. Variation in miR168 expression among plants correlates well with secondary structures/length of loop sequences of their precursors. Our data indicates a complex AGO targeting interaction among plant lineages due to miRNA sequence diversity and sequences of miRNA targeting regions among AGO mRNAs, thus leading to the assumption that the perturbations by viruses that use host miRNAs to target antiviral AGOs can only be species-specific. We also show

A Phylogenomic Perspective on the Radiation of Ray-Finned Fishes Based upon Targeted Sequencing of Ultraconserved Elements (UCEs)

PubMed Central

Sorenson, Laurie; Santini, Francesco

2013-01-01

Ray-finned fishes constitute the dominant radiation of vertebrates with over 32,000 species. Although molecular phylogenetics has begun to disentangle major evolutionary relationships within this vast section of the Tree of Life, there is no widely available approach for efficiently collecting phylogenomic data within fishes, leaving much of the enormous potential of massively parallel sequencing technologies for resolving major radiations in ray-finned fishes unrealized. Here, we provide a genomic perspective on longstanding questions regarding the diversification of major groups of ray-finned fishes through targeted enrichment of ultraconserved nuclear DNA elements (UCEs) and their flanking sequence. Our workflow efficiently and economically generates data sets that are orders of magnitude larger than those produced by traditional approaches and is well-suited to working with museum specimens. Analysis of the UCE data set recovers a well-supported phylogeny at both shallow and deep time-scales that supports a monophyletic relationship between Amia and Lepisosteus (Holostei) and reveals elopomorphs and then osteoglossomorphs to be the earliest diverging teleost lineages. Our approach additionally reveals that sequence capture of UCE regions and their flanking sequence offers enormous potential for resolving phylogenetic relationships within ray-finned fishes. PMID:23824177

TargetM6A: Identifying N6-Methyladenosine Sites From RNA Sequences via Position-Specific Nucleotide Propensities and a Support Vector Machine.

PubMed

Li, Guang-Qing; Liu, Zi; Shen, Hong-Bin; Yu, Dong-Jun

2016-10-01

As one of the most ubiquitous post-transcriptional modifications of RNA, N 6 -methyladenosine ( [Formula: see text]) plays an essential role in many vital biological processes. The identification of [Formula: see text] sites in RNAs is significantly important for both basic biomedical research and practical drug development. In this study, we designed a computational-based method, called TargetM6A, to rapidly and accurately target [Formula: see text] sites solely from the primary RNA sequences. Two new features, i.e., position-specific nucleotide/dinucleotide propensities (PSNP/PSDP), are introduced and combined with the traditional nucleotide composition (NC) feature to formulate RNA sequences. The extracted features are further optimized to obtain a much more compact and discriminative feature subset by applying an incremental feature selection (IFS) procedure. Based on the optimized feature subset, we trained TargetM6A on the training dataset with a support vector machine (SVM) as the prediction engine. We compared the proposed TargetM6A method with existing methods for predicting [Formula: see text] sites by performing stringent jackknife tests and independent validation tests on benchmark datasets. The experimental results show that the proposed TargetM6A method outperformed the existing methods for predicting [Formula: see text] sites and remarkably improved the prediction performances, with MCC = 0.526 and AUC = 0.818. We also provided a user-friendly web server for TargetM6A, which is publicly accessible for academic use at http://csbio.njust.edu.cn/bioinf/TargetM6A.

Silencing of vacuolar invertase and asparagine synthetase genes and its impact on acrylamide formation of fried potato products.

PubMed

Zhu, Xiaobiao; Gong, Huiling; He, Qunyan; Zeng, Zixian; Busse, James S; Jin, Weiwei; Bethke, Paul C; Jiang, Jiming

2016-02-01

Acrylamide is produced in a wide variety of carbohydrate-rich foods during high-temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to the average daily intake of acrylamide, especially in developed countries. One way to mitigate health concerns related to acrylamide is to develop potato cultivars that have reduced contents of the acrylamide precursors asparagine, glucose and fructose in tubers. We generated a large number of silencing lines of potato cultivar Russet Burbank by targeting the vacuolar invertase gene VInv and the asparagine synthetase genes StAS1 and StAS2 with a single RNA interference construct. The transcription levels of these three genes were correlated with reducing sugar (glucose and fructose) and asparagine content in tubers. Fried potato products from the best VInv/StAS1/StAS2-triple silencing lines contained only one-fifteenth of the acrylamide content of the controls. Interestingly, the extent of acrylamide reduction of the best triple silencing lines was similar to that of the best VInv-single silencing lines developed previously from the same potato cultivar Russet Burbank. These results show that an acrylamide mitigation strategy focused on developing potato cultivars with low reducing sugars is likely to be an effective and sufficient approach for minimizing the acrylamide-forming potential of French fry processing potatoes. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.