Discovery-2: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria

PubMed Central

2013-01-01

Background Drug resistance to anti-malarial compounds remains a serious problem, with resistance to newer pharmaceuticals developing at an alarming rate. The development of new anti-malarials remains a priority, and the rational selection of putative targets is a key element of this process. Discovery-2 is an update of the original Discovery in silico resource for the rational selection of putative drug target proteins, enabling researchers to obtain information for a protein which may be useful for the selection of putative drug targets, and to perform advanced filtering of proteins encoded by the malaria genome based on a series of molecular properties. Methods An updated in silico resource has been developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein properties used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions. Newly added features include drugability measures from ChEMBL, automated literature relations and links to clinical trial information. Searching by chemical structure is also available. Results The updated functionality of the Discovery-2 resource is presented, together with a detailed case study of the Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase (PfSAHH) protein. A short example of a chemical search with pyrimethamine is also illustrated. Conclusion The updated Discovery-2 resource allows researchers to obtain detailed properties of proteins from the malaria genome, which may be of interest in the target selection process, and to perform advanced filtering and selection of proteins based on a relevant range of molecular characteristics. PMID:23537208

A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18*

PubMed Central

Yang, Zhaoshou; Hou, Yongheng; Hao, Taofang; Rho, Hee-Sool; Wan, Jun; Luan, Yizhao; Gao, Xin; Yao, Jianping; Pan, Aihua; Xie, Zhi; Qian, Jiang; Liao, Wanqin; Zhu, Heng; Zhou, Xingwang

2017-01-01

Toxoplasma kinase ROP18 is a key molecule responsible for the virulence of Toxoplasma gondii; however, the mechanisms by which ROP18 exerts parasite virulence via interaction with host proteins remain limited to a small number of identified substrates. To identify a broader array of ROP18 substrates, we successfully purified bioactive mature ROP18 and used it to probe a human proteome array. Sixty eight new putative host targets were identified. Functional annotation analysis suggested that these proteins have a variety of functions, including metabolic process, kinase activity and phosphorylation, cell growth, apoptosis and cell death, and immunity, indicating a pleiotropic role of ROP18 kinase. Among these proteins, four candidates, p53, p38, UBE2N, and Smad1, were further validated. We demonstrated that ROP18 targets p53, p38, UBE2N, and Smad1 for degradation. Importantly, we demonstrated that ROP18 phosphorylates Smad1 Ser-187 to trigger its proteasome-dependent degradation. Further functional characterization of the substrates of ROP18 may enhance understanding of the pathogenesis of Toxoplasma infection and provide new therapeutic targets. Similar strategies could be used to identify novel host targets for other microbial kinases functioning at the pathogen-host interface. PMID:28087594

Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements

PubMed Central

Mumbach, Maxwell R; Satpathy, Ansuman T; Boyle, Evan A; Dai, Chao; Gowen, Benjamin G; Cho, Seung Woo; Nguyen, Michelle L; Rubin, Adam J; Granja, Jeffrey M; Kazane, Katelynn R; Wei, Yuning; Nguyen, Trieu; Greenside, Peyton G; Corces, M Ryan; Tycko, Josh; Simeonov, Dimitre R; Suliman, Nabeela; Li, Rui; Xu, Jin; Flynn, Ryan A; Kundaje, Anshul; Khavari, Paul A; Marson, Alexander; Corn, Jacob E; Quertermous, Thomas; Greenleaf, William J; Chang, Howard Y

2018-01-01

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer–promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases. PMID:28945252

TLR4, NOD1 and NOD2 mediate immune recognition of putative newly identified periodontal pathogens.

PubMed

Marchesan, Julie; Jiao, Yizu; Schaff, Riley A; Hao, Jie; Morelli, Thiago; Kinney, Janet S; Gerow, Elizabeth; Sheridan, Rachel; Rodrigues, Vinicius; Paster, Bruce J; Inohara, Naohiro; Giannobile, William V

2016-06-01

Periodontitis is a polymicrobial inflammatory disease that results from the interaction between the oral microbiota and the host immunity. Although the innate immune response is important for disease initiation and progression, the innate immune receptors that recognize both classical and putative periodontal pathogens that elicit an immune response have not been elucidated. By using the Human Oral Microbe Identification Microarray (HOMIM), we identified multiple predominant oral bacterial species in human plaque biofilm that strongly associate with severe periodontitis. Ten of the identified species were evaluated in greater depth, six being classical pathogens and four putative novel pathogens. Using human peripheral blood monocytes (HPBM) and murine bone-marrow-derived macrophages (BMDM) from wild-type (WT) and Toll-like receptor (TLR)-specific and MyD88 knockouts (KOs), we demonstrated that heat-killed Campylobacter concisus, Campylobacter rectus, Selenomonas infelix, Porphyromonas endodontalis, Porphyromonas gingivalis, and Tannerella forsythia mediate high immunostimulatory activity. Campylobacter concisus, C. rectus, and S. infelix exhibited robust TLR4 stimulatory activity. Studies using mesothelial cells from WT and NOD1-specific KOs and NOD2-expressing human embryonic kidney cells demonstrated that Eubacterium saphenum, Eubacterium nodatum and Filifactor alocis exhibit robust NOD1 stimulatory activity, and that Porphyromonas endodontalis and Parvimonas micra have the highest NOD2 stimulatory activity. These studies allowed us to provide important evidence on newly identified putative pathogens in periodontal disease pathogenesis showing that these bacteria exhibit different immunostimulatory activity via TLR4, NOD1, and NOD2 (Clinicaltrials.gov NCT01154855). © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

TLR4, NOD1 and NOD2 Mediate Immune Recognition of Putative Newly-Identified Periodontal Pathogens

PubMed Central

Schaff, Riley A.; Hao, Jie; Morelli, Thiago; Kinney, Janet S.; Gerow, Elizabeth; Sheridan, Rachel; Rodrigues, Vinicius; Paster, Bruce J.; Inohara, Naohiro; Giannobile, William V.

2015-01-01

SUMMARY Periodontitis is a polymicrobial inflammatory disease that results from the interaction between the oral microbiota and the host immunity. While the innate immune response is important for disease initiation and progression, the innate immune receptors that recognize both classical and putative periodontal pathogens that elicit an immune response have not been elucidated. By using the Human Oral Microbe Identification Microarray (HOMIM), we identified multiple predominant oral bacterial species in human plaque biofilm that strongly associate with severe periodontitis. Ten of the identified species were evaluated in greater depth, 6 being classical pathogens and 4 putative novel pathogens. Using human peripheral blood monocytes (HPBM) and murine bone marrow–derived macrophages (BMDM) from wild-type (WT) and toll-like receptor (TLR)-specific and MyD88 knockouts (KOs), we demonstrated that heat-killed Campylobacter concisus, Campylobacter rectus, Selenomonas infelix, Porphyromonas endodontalis, Porphyromonas gingivalis, and Tannerella forsythia mediate high immunostimulatory activity. C. concisus, C. rectus, and S. infelix exhibited robust TLR4 stimulatory activity. Studies using mesothelial cells from WT and NOD1-specific KOs and NOD2-expressing human embryonic kidney (HEK) cells demonstrated that Eubacterium saphenum, Eubacterium nodatum and Filifactor alocis exhibit robust NOD1 stimulatory activity, and that Porphyromonas endodontalis and Parvimonas micra have the highest NOD2-stimulatory activity. These studies allowed us to provide important evidence on newly-identified putative pathogens in periodontal disease pathogenesis showing that these bacteria exhibit different immunostimulatory activity via TLR4, NOD1, and NOD2 (Clinicaltrials.gov NCT01154855). PMID:26177212

Target-similarity search using Plasmodium falciparum proteome identifies approved drugs with anti-malarial activity and their possible targets

PubMed Central

Akala, Hoseah M.; Macharia, Rosaline W.; Juma, Dennis W.; Cheruiyot, Agnes C.; Andagalu, Ben; Brown, Mathew L.; El-Shemy, Hany A.; Nyanjom, Steven G.

2017-01-01

Malaria causes about half a million deaths annually, with Plasmodium falciparum being responsible for 90% of all the cases. Recent reports on artemisinin resistance in Southeast Asia warrant urgent discovery of novel drugs for the treatment of malaria. However, most bioactive compounds fail to progress to treatments due to safety concerns. Drug repositioning offers an alternative strategy where drugs that have already been approved as safe for other diseases could be used to treat malaria. This study screened approved drugs for antimalarial activity using an in silico chemogenomics approach prior to in vitro verification. All the P. falciparum proteins sequences available in NCBI RefSeq were mined and used to perform a similarity search against DrugBank, TTD and STITCH databases to identify similar putative drug targets. Druggability indices of the potential P. falciparum drug targets were obtained from TDR targets database. Functional amino acid residues of the drug targets were determined using ConSurf server which was used to fine tune the similarity search. This study predicted 133 approved drugs that could target 34 P. falciparum proteins. A literature search done at PubMed and Google Scholar showed 105 out of the 133 drugs to have been previously tested against malaria, with most showing activity. For further validation, drug susceptibility assays using SYBR Green I method were done on a representative group of 10 predicted drugs, eight of which did show activity against P. falciparum 3D7 clone. Seven had IC50 values ranging from 1 μM to 50 μM. This study also suggests drug-target association and hence possible mechanisms of action of drugs that did show antiplasmodial activity. The study results validate the use of proteome-wide target similarity approach in identifying approved drugs with activity against P. falciparum and could be adapted for other pathogens. PMID:29088219

Discovery: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria

PubMed Central

Joubert, Fourie; Harrison, Claudia M; Koegelenberg, Riaan J; Odendaal, Christiaan J; de Beer, Tjaart AP

2009-01-01

Background Up to half a billion human clinical cases of malaria are reported each year, resulting in about 2.7 million deaths, most of which occur in sub-Saharan Africa. Due to the over-and misuse of anti-malarials, widespread resistance to all the known drugs is increasing at an alarming rate. Rational methods to select new drug target proteins and lead compounds are urgently needed. The Discovery system provides data mining functionality on extensive annotations of five malaria species together with the human and mosquito hosts, enabling the selection of new targets based on multiple protein and ligand properties. Methods A web-based system was developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein features used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions and host-pathogen interactions among others. Searching by chemical structure is also available. Results An in silico system for the selection of putative drug targets and lead compounds is presented, together with an example study on the bifunctional DHFR-TS from Plasmodium falciparum. Conclusion The Discovery system allows for the identification of putative drug targets and lead compounds in Plasmodium species based on the filtering of protein and chemical properties. PMID:19642978

Whole exome sequencing for familial bicuspid aortic valve identifies putative variants.

PubMed

Martin, Lisa J; Pilipenko, Valentina; Kaufman, Kenneth M; Cripe, Linda; Kottyan, Leah C; Keddache, Mehdi; Dexheimer, Phillip; Weirauch, Matthew T; Benson, D Woodrow

2014-10-01

Bicuspid aortic valve (BAV) is the most common congenital cardiovascular malformation. Although highly heritable, few causal variants have been identified. The purpose of this study was to identify genetic variants underlying BAV by whole exome sequencing a multiplex BAV kindred. Whole exome sequencing was performed on 17 individuals from a single family (BAV=3; other cardiovascular malformation, 3). Postvariant calling error control metrics were established after examining the relationship between Mendelian inheritance error rate and coverage, quality score, and call rate. To determine the most effective approach to identifying susceptibility variants from among 54 674 variants passing error control metrics, we evaluated 3 variant selection strategies frequently used in whole exome sequencing studies plus extended family linkage. No putative rare, high-effect variants were identified in all affected but no unaffected individuals. Eight high-effect variants were identified by ≥2 of the commonly used selection strategies; however, these were either common in the general population (>10%) or present in the majority of the unaffected family members. However, using extended family linkage, 3 synonymous variants were identified; all 3 variants were identified by at least one other strategy. These results suggest that traditional whole exome sequencing approaches, which assume causal variants alter coding sense, may be insufficient for BAV and other complex traits. Identification of disease-associated variants is facilitated by the use of segregation within families. © 2014 American Heart Association, Inc.

Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2-Monoacylglycerols and N-acyl Amides

PubMed Central

Takacs, Sara M.; Stuart, Jordyn M.; Basnet, Arjun; Raboune, Siham; Widlanski, Theodore S.; Doherty, Patrick; Bradshaw, Heather B.

2013-01-01

Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling. PMID:23874457

Semi-automated literature mining to identify putative biomarkers of disease from multiple biofluids

PubMed Central

2014-01-01

Background Computational methods for mining of biomedical literature can be useful in augmenting manual searches of the literature using keywords for disease-specific biomarker discovery from biofluids. In this work, we develop and apply a semi-automated literature mining method to mine abstracts obtained from PubMed to discover putative biomarkers of breast and lung cancers in specific biofluids. Methodology A positive set of abstracts was defined by the terms ‘breast cancer’ and ‘lung cancer’ in conjunction with 14 separate ‘biofluids’ (bile, blood, breastmilk, cerebrospinal fluid, mucus, plasma, saliva, semen, serum, synovial fluid, stool, sweat, tears, and urine), while a negative set of abstracts was defined by the terms ‘(biofluid) NOT breast cancer’ or ‘(biofluid) NOT lung cancer.’ More than 5.3 million total abstracts were obtained from PubMed and examined for biomarker-disease-biofluid associations (34,296 positive and 2,653,396 negative for breast cancer; 28,355 positive and 2,595,034 negative for lung cancer). Biological entities such as genes and proteins were tagged using ABNER, and processed using Python scripts to produce a list of putative biomarkers. Z-scores were calculated, ranked, and used to determine significance of putative biomarkers found. Manual verification of relevant abstracts was performed to assess our method’s performance. Results Biofluid-specific markers were identified from the literature, assigned relevance scores based on frequency of occurrence, and validated using known biomarker lists and/or databases for lung and breast cancer [NCBI’s On-line Mendelian Inheritance in Man (OMIM), Cancer Gene annotation server for cancer genomics (CAGE), NCBI’s Genes & Disease, NCI’s Early Detection Research Network (EDRN), and others]. The specificity of each marker for a given biofluid was calculated, and the performance of our semi-automated literature mining method assessed for breast and lung cancer

Prioritizing and modelling of putative drug target proteins of Candida albicans by systems biology approach.

PubMed

Ismail, Tariq; Fatima, Nighat; Muhammad, Syed Aun; Zaidi, Syed Saoud; Rehman, Nisar; Hussain, Izhar; Tariq, Najam Us Sahr; Amirzada, Imran; Mannan, Abdul

2018-01-01

Candida albicans (Candida albicans) is one of the major sources of nosocomial infections in humans which may prove fatal in 30% of cases. The hospital acquired infection is very difficult to treat affectively due to the presence of drug resistant pathogenic strains, therefore there is a need to find alternative drug targets to cure this infection. In silico and computational level frame work was used to prioritize and establish antifungal drug targets of Candida albicans. The identification of putative drug targets was based on acquiring 5090 completely annotated genes of Candida albicans from available databases which were categorized into essential and non-essential genes. The result indicated that 9% of proteins were essential and could become potential candidates for intervention which might result in pathogen eradication. We studied cluster of orthologs and the subtractive genomic analysis of these essential proteins against human genome was made as a reference to minimize the side effects. It was seen that 14% of Candida albicans proteins were evolutionary related to the human proteins while 86% are non-human homologs. In the next step of compatible drug target selections, the non-human homologs were sequentially compared to the human microbiome data to minimize the potential effects against gut flora which accumulated to 38% of the essential genome. The sub-cellular localization of these candidate proteins in fungal cellular systems indicated that 80% of them are cytoplasmic, 10% are mitochondrial and the remaining 10% are associated with the cell wall. The role of these non-human and non-gut flora putative target proteins in Candida albicans biological pathways was studied. Due to their integrated and critical role in Candida albicans replication cycle, four proteins were selected for molecular modeling. For drug designing and development, four high quality and reliable protein models with more than 70% sequence identity were constructed. These proteins are

Cadherin-6 is a putative tumor suppressor and target of epigenetically dysregulated miR-429 in cholangiocarcinoma

PubMed Central

Goeppert, Benjamin; Ernst, Christina; Baer, Constance; Roessler, Stephanie; Renner, Marcus; Mehrabi, Arianeb; Hafezi, Mohammadreza; Pathil, Anita; Warth, Arne; Stenzinger, Albrecht; Weichert, Wilko; Bähr, Marion; Will, Rainer; Schirmacher, Peter; Plass, Christoph; Weichenhan, Dieter

2016-01-01

ABSTRACT Cholangiocarcinoma (CC) is a rare malignancy of the extrahepatic or intrahepatic biliary tract with an outstanding poor prognosis. Non-surgical therapeutic regimens result in minimally improved survival of CC patients. Global genomic analyses identified a few recurrently mutated genes, some of them in genes involved in epigenetic patterning. In a previous study, we demonstrated global DNA methylation changes in CC, indicating major contribution of epigenetic alterations to cholangiocarcinogenesis. Here, we aimed at the identification and characterization of CC-related, differentially methylated regions (DMRs) in potential microRNA promoters and of genes targeted by identified microRNAs. Twenty-seven hypermethylated and 13 hypomethylated potential promoter regions of microRNAs, known to be associated with cancer-related pathways like Wnt, ErbB, and PI3K-Akt signaling, were identified. Selected DMRs were confirmed in 2 independent patient cohorts. Inverse correlation between promoter methylation and expression suggested miR-129-2 and members of the miR-200 family (miR-200a, miR-200b, and miR-429) as novel tumor suppressors and oncomiRs, respectively, in CC. Tumor suppressor genes deleted in liver cancer 1 (DLC1), F-box/WD-repeat-containing protein 7 (FBXW7), and cadherin-6 (CDH6) were identified as presumed targets in CC. Tissue microarrays of a representative and well-characterized cohort of biliary tract cancers (n=212) displayed stepwise downregulation of CDH6 and association with poor patient outcome. Ectopic expression of CDH6 on the other hand, delayed growth in the CC cell lines EGI-1 and TFK-1, together suggesting a tumor suppressive function of CDH6. Our work represents a valuable repository for the study of epigenetically altered miRNAs in cholangiocarcinogenesis and novel putative, CC-related tumor suppressive miRNAs and oncomiRs. PMID:27593557

Assessment of deoxyhypusine hydroxylase as a putative, novel drug target.

PubMed

Kerscher, B; Nzukou, E; Kaiser, A

2010-02-01

Antimalarial drug resistance has nowadays reached each drug class on the market for longer than 10 years. The focus on validated, classical targets has severe drawbacks. If resistance is arising or already present in the field, a target-based High-Throughput-Screening (HTS) with the respective target involves the risk of identifying compounds to which field populations are also resistant. Thus, it appears that a rewarding albeit demanding challenge for target-based drug discovery is to identify novel drug targets. In the search for new targets for antimalarials, we have investigated the biosynthesis of hypusine, present in eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine hydroxylase (DOHH), which has recently been cloned and expressed from P. falciparum, completes the modification of eIF5A through hydroxylation. Here, we assess the present druggable data on Plasmodium DOHH and its human counterpart. Plasmodium DOHH arose from a cyanobacterial phycobilin lyase by loss of function. It has a low FASTA score of 27 to its human counterpart. The HEAT-like repeats present in the parasite DOHH differ in number and amino acid identity from its human ortholog and might be of considerable interest for inhibitor design.

Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer

PubMed Central

Azorsa, David O; Gonzales, Irma M; Basu, Gargi D; Choudhary, Ashish; Arora, Shilpi; Bisanz, Kristen M; Kiefer, Jeffrey A; Henderson, Meredith C; Trent, Jeffrey M; Von Hoff, Daniel D; Mousses, Spyro

2009-01-01

Background Pancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement. Methods In order to improve gemcitabine response in pancreatic cancer cells, we utilized a synthetic lethal RNAi screen targeting 572 known kinases to identify genes that when silenced would sensitize pancreatic cancer cells to gemcitabine. Results Results from the RNAi screens identified several genes that, when silenced, potentiated the growth inhibitory effects of gemcitabine in pancreatic cancer cells. The greatest potentiation was shown by siRNA targeting checkpoint kinase 1 (CHK1). Validation of the screening results was performed in MIA PaCa-2 and BxPC3 pancreatic cancer cells by examining the dose response of gemcitabine treatment in the presence of either CHK1 or CHK2 siRNA. These results showed a three to ten-fold decrease in the EC50 for CHK1 siRNA-treated cells versus control siRNA-treated cells while treatment with CHK2 siRNA resulted in no change compared to controls. CHK1 was further targeted with specific small molecule inhibitors SB 218078 and PD 407824 in combination with gemcitabine. Results showed that treatment of MIA PaCa-2 cells with either of the CHK1 inhibitors SB 218078 or PD 407824 led to sensitization of the pancreatic cancer cells to gemcitabine. Conclusion These findings demonstrate the effectiveness of synthetic lethal RNAi screening as a tool for identifying sensitizing targets to chemotherapeutic agents. These results also indicate that CHK1 could serve as a putative therapeutic target for sensitizing pancreatic cancer cells to gemcitabine. PMID

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.

Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

PubMed

Li, Yuanjun; Gou, Junbo; Chen, Fangfang; Li, Changfu; Zhang, Yansheng

2016-01-01

Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones (STLs), which include the xanthanolides. To date, the biogenesis of xanthanolides, especially their downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that are highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of STLs are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides.

Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis.

PubMed

Cheng, Feixiong; Murray, James L; Zhao, Junfei; Sheng, Jinsong; Zhao, Zhongming; Rubin, Donald H

2016-09-01

Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap) host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase). Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B) identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline) that may be potential for antiviral indication (e.g. anti-Ebola). In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.

Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

PubMed Central

Li, Yuanjun; Gou, Junbo; Chen, Fangfang; Li, Changfu; Zhang, Yansheng

2016-01-01

Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones (STLs), which include the xanthanolides. To date, the biogenesis of xanthanolides, especially their downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that are highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of STLs are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides. PMID:27625674

Study Identifies New Lymphoma Treatment Target

Cancer.gov

NCI researchers have identified new therapeutic targets for diffuse large B-cell lymphoma. Drugs that hit these targets are under clinical development and the researchers hope to begin testing them in clinical trials of patients with DLBCL.

PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

EPA Science Inventory

THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM
IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2

* Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue1
1The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

Systems analysis identifies miR-29b regulation of invasiveness in melanoma.

PubMed

Andrews, Miles C; Cursons, Joseph; Hurley, Daniel G; Anaka, Matthew; Cebon, Jonathan S; Behren, Andreas; Crampin, Edmund J

2016-11-16

In many cancers, microRNAs (miRs) contribute to metastatic progression by modulating phenotypic reprogramming processes such as epithelial-mesenchymal plasticity. This can be driven by miRs targeting multiple mRNA transcripts, inducing regulated changes across large sets of genes. The miR-target databases TargetScan and DIANA-microT predict putative relationships by examining sequence complementarity between miRs and mRNAs. However, it remains a challenge to identify which miR-mRNA interactions are active at endogenous expression levels, and of biological consequence. We developed a workflow to integrate TargetScan and DIANA-microT predictions into the analysis of data-driven associations calculated from transcript abundance (RNASeq) data, specifically the mutual information and Pearson's correlation metrics. We use this workflow to identify putative relationships of miR-mediated mRNA repression with strong support from both lines of evidence. Applying this approach systematically to a large, published collection of unique melanoma cell lines - the Ludwig Melbourne melanoma (LM-MEL) cell line panel - we identified putative miR-mRNA interactions that may contribute to invasiveness. This guided the selection of interactions of interest for further in vitro validation studies. Several miR-mRNA regulatory relationships supported by TargetScan and DIANA-microT demonstrated differential activity across cell lines of varying matrigel invasiveness. Strong negative statistical associations for these putative regulatory relationships were consistent with target mRNA inhibition by the miR, and suggest that differential activity of such miR-mRNA relationships contribute to differences in melanoma invasiveness. Many of these relationships were reflected across the skin cutaneous melanoma TCGA dataset, indicating that these observations also show graded activity across clinical samples. Several of these miRs are implicated in cancer progression (miR-211, -340, -125b, -221, and

Proteomic Identification of Putative MicroRNA394 Target Genes in Arabidopsis thaliana Identifies Major Latex Protein Family Members Critical for Normal Development.

PubMed

Litholdo, Celso G; Parker, Benjamin L; Eamens, Andrew L; Larsen, Martin R; Cordwell, Stuart J; Waterhouse, Peter M

2016-06-01

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomic Identification of Putative MicroRNA394 Target Genes in Arabidopsis thaliana Identifies Major Latex Protein Family Members Critical for Normal Development*

PubMed Central

Litholdo, Celso G.; Parker, Benjamin L.; Eamens, Andrew L.; Larsen, Martin R.; Cordwell, Stuart J.; Waterhouse, Peter M.

2016-01-01

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR. Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development. PMID:27067051

Integrated microarray and ChIP analysis identifies multiple Foxa2 dependent target genes in the notochord.

PubMed

Tamplin, Owen J; Cox, Brian J; Rossant, Janet

2011-12-15

The node and notochord are key tissues required for patterning of the vertebrate body plan. Understanding the gene regulatory network that drives their formation and function is therefore important. Foxa2 is a key transcription factor at the top of this genetic hierarchy and finding its targets will help us to better understand node and notochord development. We performed an extensive microarray-based gene expression screen using sorted embryonic notochord cells to identify early notochord-enriched genes. We validated their specificity to the node and notochord by whole mount in situ hybridization. This provides the largest available resource of notochord-expressed genes, and therefore candidate Foxa2 target genes in the notochord. Using existing Foxa2 ChIP-seq data from adult liver, we were able to identify a set of genes expressed in the notochord that had associated regions of Foxa2-bound chromatin. Given that Foxa2 is a pioneer transcription factor, we reasoned that these sites might represent notochord-specific enhancers. Candidate Foxa2-bound regions were tested for notochord specific enhancer function in a zebrafish reporter assay and 7 novel notochord enhancers were identified. Importantly, sequence conservation or predictive models could not have readily identified these regions. Mutation of putative Foxa2 binding elements in two of these novel enhancers abrogated reporter expression and confirmed their Foxa2 dependence. The combination of highly specific gene expression profiling and genome-wide ChIP analysis is a powerful means of understanding developmental pathways, even for small cell populations such as the notochord. Copyright © 2011 Elsevier Inc. All rights reserved.

Characterization of putative class II bacteriocins identified from a non-bacteriocin-producing strain Lactobacillus casei ATCC 334.

PubMed

Kuo, Yang-Cheng; Liu, Cheng-Feng; Lin, Jhao-Fen; Li, An-Chieh; Lo, Ta-Chun; Lin, Thy-Hou

2013-01-01

Several putative class II bacteriocin-like genes were identified in Lactobacillus casei ATCC 334, all of which might encode peptides with a double-glycine leader. Six peptides encoded by these genes were heterologously expressed in Escherichia coli and then partially purified in order to test their bacteriocin activity. The results revealed that the mature LSEI_2163 peptide was a class IId bacteriocin that exhibited antimicrobial activity against some lactobacilli and several Listeria species. Similarly, mature LSEI_2386 was a putative pheromone peptide that also had significant bacteriocin activity against several Listeria species. The activities of both peptides tolerated 121°C for 30 min but not treatment with proteinase K or trypsin. The two Cys residues located at positions 4 and 24 in the mature LSEI_2163 peptide were shown by mass spectrometry to form a disulfide bridge, which was required for optimal antibacterial activity. However, replacement of one or both Cys with Ser would cause significant reduction of the antibacterial activity, the reduction being greater when only one of the Cys residues (C4S) was replaced than when both (C4S/C24S) were replaced.

Identification of inhibitors for putative malaria drug targets amongst novel antimalarial compounds

PubMed Central

Crowther, Gregory J.; Napuli, Alberto J.; Gilligan, James H.; Gagaring, Kerstin; Borboa, Rachel; Francek, Carolyn; Chen, Zhong; Dagostino, Eleanor F.; Stockmyer, Justin B.; Wang, Yu; Rodenbough, Philip P.; Castaneda, Lisa J.; Leibly, David J.; Bhandari, Janhavi; Gelb, Michael H.; Brinker, Achim; Engels, Ingo; Taylor, Jennifer; Chatterjee, Arnab K.; Fantauzzi, Pascal; Glynne, Richard J.; Van Voorhis, Wesley C.; Kuhen, Kelli L.

2011-01-01

The efficacy of most marketed antimalarial drugs has been compromised by evolution of parasite resistance, underscoring an urgent need to find new drugs with new mechanisms of action. We have taken a high-throughput approach toward identifying novel antimalarial chemical inhibitors of prioritized drug targets for P. falciparum, excluding targets which are inhibited by currently used drugs. A screen of commercially available libraries identified 5,655 low molecular weight compounds that inhibit growth of P. falciparum cultures with EC50 values below 1.25 μM. These compounds were then tested in 384- or 1536-well biochemical assays for activity against nine Plasmodium enzymes: adenylosuccinate synthetase (AdSS), choline kinase (CK), deoxyuridine triphosphate nucleotidohydrolase (dUTPase), glutamate dehydrogenase (GDH), guanylate kinase (GK), N-myristoyltransferase (NMT), orotidine 5′-monophosphate decarboxylase (OMPDC), farnesyl pyrophosphate synthase (FPPS) and S-adenosylhomocysteine hydrolase (SAHH). These enzymes were selected using TDRtargets.org, and are believed to have excellent potential as drug targets based on criteria such as their likely essentiality, druggability, and amenability to high-throughput biochemical screening. Six of these targets were inhibited by one or more of the antimalarial scaffolds and may have potential use in drug development, further target validation studies and exploration of P. falciparum biochemistry and biology. PMID:20813141

Identification of inhibitors for putative malaria drug targets among novel antimalarial compounds.

PubMed

Crowther, Gregory J; Napuli, Alberto J; Gilligan, James H; Gagaring, Kerstin; Borboa, Rachel; Francek, Carolyn; Chen, Zhong; Dagostino, Eleanor F; Stockmyer, Justin B; Wang, Yu; Rodenbough, Philip P; Castaneda, Lisa J; Leibly, David J; Bhandari, Janhavi; Gelb, Michael H; Brinker, Achim; Engels, Ingo H; Taylor, Jennifer; Chatterjee, Arnab K; Fantauzzi, Pascal; Glynne, Richard J; Van Voorhis, Wesley C; Kuhen, Kelli L

2011-01-01

The efficacy of most marketed antimalarial drugs has been compromised by evolution of parasite resistance, underscoring an urgent need to find new drugs with new mechanisms of action. We have taken a high-throughput approach toward identifying novel antimalarial chemical inhibitors of prioritized drug targets for Plasmodium falciparum, excluding targets which are inhibited by currently used drugs. A screen of commercially available libraries identified 5655 low molecular weight compounds that inhibit growth of P. falciparum cultures with EC(50) values below 1.25μM. These compounds were then tested in 384- or 1536-well biochemical assays for activity against nine Plasmodium enzymes: adenylosuccinate synthetase (AdSS), choline kinase (CK), deoxyuridine triphosphate nucleotidohydrolase (dUTPase), glutamate dehydrogenase (GDH), guanylate kinase (GK), N-myristoyltransferase (NMT), orotidine 5'-monophosphate decarboxylase (OMPDC), farnesyl pyrophosphate synthase (FPPS) and S-adenosylhomocysteine hydrolase (SAHH). These enzymes were selected using TDRtargets.org, and are believed to have excellent potential as drug targets based on criteria such as their likely essentiality, druggability, and amenability to high-throughput biochemical screening. Six of these targets were inhibited by one or more of the antimalarial scaffolds and may have potential use in drug development, further target validation studies and exploration of P. falciparum biochemistry and biology. Copyright © 2010 Elsevier B.V. All rights reserved.

A systems biology approach identified different regulatory networks targeted by KSHV miR-K12-11 in B cells and endothelial cells.

PubMed

Yang, Yajie; Boss, Isaac W; McIntyre, Lauren M; Renne, Rolf

2014-08-08

Kaposi's sarcoma associated herpes virus (KSHV) is associated with tumors of endothelial and lymphoid origin. During latent infection, KSHV expresses miR-K12-11, an ortholog of the human tumor gene hsa-miR-155. Both gene products are microRNAs (miRNAs), which are important post-transcriptional regulators that contribute to tissue specific gene expression. Advances in target identification technologies and molecular interaction databases have allowed a systems biology approach to unravel the gene regulatory networks (GRNs) triggered by miR-K12-11 in endothelial and lymphoid cells. Understanding the tissue specific function of miR-K12-11 will help to elucidate underlying mechanisms of KSHV pathogenesis. Ectopic expression of miR-K12-11 differentially affected gene expression in BJAB cells of lymphoid origin and TIVE cells of endothelial origin. Direct miRNA targeting accounted for a small fraction of the observed transcriptome changes: only 29 genes were identified as putative direct targets of miR-K12-11 in both cell types. However, a number of commonly affected biological pathways, such as carbohydrate metabolism and interferon response related signaling, were revealed by gene ontology analysis. Integration of transcriptome profiling, bioinformatic algorithms, and databases of protein-protein interactome from the ENCODE project identified different nodes of GRNs utilized by miR-K12-11 in a tissue-specific fashion. These effector genes, including cancer associated transcription factors and signaling proteins, amplified the regulatory potential of a single miRNA, from a small set of putative direct targets to a larger set of genes. This is the first comparative analysis of miRNA-K12-11's effects in endothelial and B cells, from tissues infected with KSHV in vivo. MiR-K12-11 was able to broadly modulate gene expression in both cell types. Using a systems biology approach, we inferred that miR-K12-11 establishes its GRN by both repressing master TFs and influencing

Transcriptome Analysis of Mango (Mangifera indica L.) Fruit Epidermal Peel to Identify Putative Cuticle-Associated Genes

PubMed Central

Tafolla-Arellano, Julio C.; Zheng, Yi; Sun, Honghe; Jiao, Chen; Ruiz-May, Eliel; Hernández-Oñate, Miguel A.; González-León, Alberto; Báez-Sañudo, Reginaldo; Fei, Zhangjun; Domozych, David; Rose, Jocelyn K. C.; Tiznado-Hernández, Martín E.

2017-01-01

Mango fruit (Mangifera indica L.) are highly perishable and have a limited shelf life, due to postharvest desiccation and senescence, which limits their global distribution. Recent studies of tomato fruit suggest that these traits are influenced by the expression of genes that are associated with cuticle metabolism. However, studies of these phenomena in mango fruit are limited by the lack of genome-scale data. In order to gain insight into the mango cuticle biogenesis and identify putative cuticle-associated genes, we analyzed the transcriptomes of peels from ripe and overripe mango fruit using RNA-Seq. Approximately 400 million reads were generated and de novo assembled into 107,744 unigenes, with a mean length of 1,717 bp and with this information an online Mango RNA-Seq Database (http://bioinfo.bti.cornell.edu/cgi-bin/mango/index.cgi) which is a valuable genomic resource for molecular research into the biology of mango fruit was created. RNA-Seq analysis suggested that the pathway leading to biosynthesis of the cuticle component, cutin, is up-regulated during overripening. This data was supported by analysis of the expression of several putative cuticle-associated genes and by gravimetric and microscopic studies of cuticle deposition, revealing a complex continuous pattern of cuticle deposition during fruit development and involving substantial accumulation during ripening/overripening. PMID:28425468

Transcriptome Analysis of Mango (Mangifera indica L.) Fruit Epidermal Peel to Identify Putative Cuticle-Associated Genes.

PubMed

Tafolla-Arellano, Julio C; Zheng, Yi; Sun, Honghe; Jiao, Chen; Ruiz-May, Eliel; Hernández-Oñate, Miguel A; González-León, Alberto; Báez-Sañudo, Reginaldo; Fei, Zhangjun; Domozych, David; Rose, Jocelyn K C; Tiznado-Hernández, Martín E

2017-04-20

Mango fruit (Mangifera indica L.) are highly perishable and have a limited shelf life, due to postharvest desiccation and senescence, which limits their global distribution. Recent studies of tomato fruit suggest that these traits are influenced by the expression of genes that are associated with cuticle metabolism. However, studies of these phenomena in mango fruit are limited by the lack of genome-scale data. In order to gain insight into the mango cuticle biogenesis and identify putative cuticle-associated genes, we analyzed the transcriptomes of peels from ripe and overripe mango fruit using RNA-Seq. Approximately 400 million reads were generated and de novo assembled into 107,744 unigenes, with a mean length of 1,717 bp and with this information an online Mango RNA-Seq Database (http://bioinfo.bti.cornell.edu/cgi-bin/mango/index.cgi) which is a valuable genomic resource for molecular research into the biology of mango fruit was created. RNA-Seq analysis suggested that the pathway leading to biosynthesis of the cuticle component, cutin, is up-regulated during overripening. This data was supported by analysis of the expression of several putative cuticle-associated genes and by gravimetric and microscopic studies of cuticle deposition, revealing a complex continuous pattern of cuticle deposition during fruit development and involving substantial accumulation during ripening/overripening.

Transcriptome Analysis of Mango (Mangifera indica L.) Fruit Epidermal Peel to Identify Putative Cuticle-Associated Genes

NASA Astrophysics Data System (ADS)

Tafolla-Arellano, Julio C.; Zheng, Yi; Sun, Honghe; Jiao, Chen; Ruiz-May, Eliel; Hernández-Oñate, Miguel A.; González-León, Alberto; Báez-Sañudo, Reginaldo; Fei, Zhangjun; Domozych, David; Rose, Jocelyn K. C.; Tiznado-Hernández, Martín E.

2017-04-01

Mango fruit (Mangifera indica L.) are highly perishable and have a limited shelf life, due to postharvest desiccation and senescence, which limits their global distribution. Recent studies of tomato fruit suggest that these traits are influenced by the expression of genes that are associated with cuticle metabolism. However, studies of these phenomena in mango fruit are limited by the lack of genome-scale data. In order to gain insight into the mango cuticle biogenesis and identify putative cuticle-associated genes, we analyzed the transcriptomes of peels from ripe and overripe mango fruit using RNA-Seq. Approximately 400 million reads were generated and de novo assembled into 107,744 unigenes, with a mean length of 1,717 bp and with this information an online Mango RNA-Seq Database (http://bioinfo.bti.cornell.edu/cgi-bin/mango/index.cgi) which is a valuable genomic resource for molecular research into the biology of mango fruit was created. RNA-Seq analysis suggested that the pathway leading to biosynthesis of the cuticle component, cutin, is up-regulated during overripening. This data was supported by analysis of the expression of several putative cuticle-associated genes and by gravimetric and microscopic studies of cuticle deposition, revealing a complex continuous pattern of cuticle deposition during fruit development and involving substantial accumulation during ripening/overripening.

Plant-Derived Polyphenols in Human Health: Biological Activity, Metabolites and Putative Molecular Targets.

PubMed

Olivares-Vicente, Marilo; Barrajon-Catalan, Enrique; Herranz-Lopez, Maria; Segura-Carretero, Antonio; Joven, Jorge; Encinar, Jose Antonio; Micol, Vicente

2018-01-01

Hibiscus sabdariffa, Lippia citriodora, Rosmarinus officinalis and Olea europaea, are rich in bioactive compounds that represent most of the phenolic compounds' families and have exhibited potential benefits in human health. These plants have been used in folk medicine for their potential therapeutic properties in human chronic diseases. Recent evidence leads to postulate that polyphenols may account for such effects. Nevertheless, the compounds or metabolites that are responsible for reaching the molecular targets are unknown. data based on studies directly using complex extracts on cellular models, without considering metabolic aspects, have limited applicability. In contrast, studies exploring the absorption process, metabolites in the blood circulation and tissues have become essential to identify the intracellular final effectors that are responsible for extracts bioactivity. Once the cellular metabolites are identified using high-resolution mass spectrometry, docking techniques suppose a unique tool for virtually screening a large number of compounds on selected targets in order to elucidate their potential mechanisms. we provide an updated overview of the in vitro and in vivo studies on the toxicity, absorption, permeability, pharmacokinetics and cellular metabolism of bioactive compounds derived from the abovementioned plants to identify the potential compounds that are responsible for the observed health effects. we propose the use of targeted metabolomics followed by in silico studies to virtually screen identified metabolites on selected protein targets, in combination with the use of the candidate metabolites in cellular models, as the methods of choice for elucidating the molecular mechanisms of these compounds. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

HaploReg v4: systematic mining of putative causal variants, cell types, regulators and target genes for human complex traits and disease.

PubMed

Ward, Lucas D; Kellis, Manolis

2016-01-04

More than 90% of common variants associated with complex traits do not affect proteins directly, but instead the circuits that control gene expression. This has increased the urgency of understanding the regulatory genome as a key component for translating genetic results into mechanistic insights and ultimately therapeutics. To address this challenge, we developed HaploReg (http://compbio.mit.edu/HaploReg) to aid the functional dissection of genome-wide association study (GWAS) results, the prediction of putative causal variants in haplotype blocks, the prediction of likely cell types of action, and the prediction of candidate target genes by systematic mining of comparative, epigenomic and regulatory annotations. Since first launching the website in 2011, we have greatly expanded HaploReg, increasing the number of chromatin state maps to 127 reference epigenomes from ENCODE 2012 and Roadmap Epigenomics, incorporating regulator binding data, expanding regulatory motif disruption annotations, and integrating expression quantitative trait locus (eQTL) variants and their tissue-specific target genes from GTEx, Geuvadis, and other recent studies. We present these updates as HaploReg v4, and illustrate a use case of HaploReg for attention deficit hyperactivity disorder (ADHD)-associated SNPs with putative brain regulatory mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Systems level mapping of metabolic complexity in Mycobacterium tuberculosis to identify high-value drug targets.

PubMed

Vashisht, Rohit; Bhat, Ashwini G; Kushwaha, Shreeram; Bhardwaj, Anshu; Brahmachari, Samir K

2014-10-11

The effectiveness of current therapeutic regimens for Mycobacterium tuberculosis (Mtb) is diminished by the need for prolonged therapy and the rise of drug resistant/tolerant strains. This global health threat, despite decades of basic research and a wealth of legacy knowledge, is due to a lack of systems level understanding that can innovate the process of fast acting and high efficacy drug discovery. The enhanced functional annotations of the Mtb genome, which were previously obtained through a crowd sourcing approach was used to reconstruct the metabolic network of Mtb in a bottom up manner. We represent this information by developing a novel Systems Biology Spindle Map of Metabolism (SBSM) and comprehend its static and dynamic structure using various computational approaches based on simulation and design. The reconstructed metabolism of Mtb encompasses 961 metabolites, involved in 1152 reactions catalyzed by 890 protein coding genes, organized into 50 pathways. By accounting for static and dynamic analysis of SBSM in Mtb we identified various critical proteins required for the growth and survival of bacteria. Further, we assessed the potential of these proteins as putative drug targets that are fast acting and less toxic. Further, we formulate a novel concept of metabolic persister genes (MPGs) and compared our predictions with published in vitro and in vivo experimental evidence. Through such analyses, we report for the first time that de novo biosynthesis of NAD may give rise to bacterial persistence in Mtb under conditions of metabolic stress induced by conventional anti-tuberculosis therapy. We propose such MPG's as potential combination of drug targets for existing antibiotics that can improve their efficacy and efficiency for drug tolerant bacteria. The systems level framework formulated by us to identify potential non-toxic drug targets and strategies to circumvent the issue of bacterial persistence can substantially aid in the process of TB drug

An analysis of possible off target effects following CAS9/CRISPR targeted deletions of neuropeptide gene enhancers from the mouse genome.

PubMed

Hay, Elizabeth Anne; Khalaf, Abdulla Razak; Marini, Pietro; Brown, Andrew; Heath, Karyn; Sheppard, Darrin; MacKenzie, Alasdair

2017-08-01

We have successfully used comparative genomics to identify putative regulatory elements within the human genome that contribute to the tissue specific expression of neuropeptides such as galanin and receptors such as CB1. However, a previous inability to rapidly delete these elements from the mouse genome has prevented optimal assessment of their function in-vivo. This has been solved using CAS9/CRISPR genome editing technology which uses a bacterial endonuclease called CAS9 that, in combination with specifically designed guide RNA (gRNA) molecules, cuts specific regions of the mouse genome. However, reports of "off target" effects, whereby the CAS9 endonuclease is able to cut sites other than those targeted, limits the appeal of this technology. We used cytoplasmic microinjection of gRNA and CAS9 mRNA into 1-cell mouse embryos to rapidly generate enhancer knockout mouse lines. The current study describes our analysis of the genomes of these enhancer knockout lines to detect possible off-target effects. Bioinformatic analysis was used to identify the most likely putative off-target sites and to design PCR primers that would amplify these sequences from genomic DNA of founder enhancer deletion mouse lines. Amplified DNA was then sequenced and blasted against the mouse genome sequence to detect off-target effects. Using this approach we were unable to detect any evidence of off-target effects in the genomes of three founder lines using any of the four gRNAs used in the analysis. This study suggests that the problem of off-target effects in transgenic mice have been exaggerated and that CAS9/CRISPR represents a highly effective and accurate method of deleting putative neuropeptide gene enhancer sequences from the mouse genome. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

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.

Putative archaeal viruses from the mesopelagic ocean.

PubMed

Vik, Dean R; Roux, Simon; Brum, Jennifer R; Bolduc, Ben; Emerson, Joanne B; Padilla, Cory C; Stewart, Frank J; Sullivan, Matthew B

2017-01-01

Oceanic viruses that infect bacteria, or phages, are known to modulate host diversity, metabolisms, and biogeochemical cycling, while the viruses that infect marine Archaea remain understudied despite the critical ecosystem roles played by their hosts. Here we introduce "MArVD", for Metagenomic Archaeal Virus Detector, an annotation tool designed to identify putative archaeal virus contigs in metagenomic datasets. MArVD is made publicly available through the online iVirus analytical platform. Benchmarking analysis of MArVD showed it to be >99% accurate and 100% sensitive in identifying the 127 known archaeal viruses among the 12,499 viruses in the VirSorter curated dataset. Application of MArVD to 10 viral metagenomes from two depth profiles in the Eastern Tropical North Pacific (ETNP) oxygen minimum zone revealed 43 new putative archaeal virus genomes and large genome fragments ranging in size from 10 to 31 kb. Network-based classifications, which were consistent with marker gene phylogenies where available, suggested that these putative archaeal virus contigs represented six novel candidate genera. Ecological analyses, via fragment recruitment and ordination, revealed that the diversity and relative abundances of these putative archaeal viruses were correlated with oxygen concentration and temperature along two OMZ-spanning depth profiles, presumably due to structuring of the host Archaea community. Peak viral diversity and abundances were found in surface waters, where Thermoplasmata 16S rRNA genes are prevalent, suggesting these archaea as hosts in the surface habitats. Together these findings provide a baseline for identifying archaeal viruses in sequence datasets, and an initial picture of the ecology of such viruses in non-extreme environments.

Systematic MicroRNA Analysis Identifies ATP6V0C as an Essential Host Factor for Human Cytomegalovirus Replication

PubMed Central

Pavelin, Jon; Reynolds, Natalie; Chiweshe, Stephen; Wu, Guanming; Tiribassi, Rebecca; Grey, Finn

2013-01-01

Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions. PMID:24385903

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

Conservation of polypyrimidine tract binding proteins and their putative target RNAs in several storage root crops.

PubMed

Kondhare, Kirtikumar R; Kumar, Amit; Hannapel, David J; Banerjee, Anjan K

2018-02-07

Polypyrimidine-tract binding proteins (PTBs) are ubiquitous RNA-binding proteins in plants and animals that play diverse role in RNA metabolic processes. PTB proteins bind to target RNAs through motifs rich in cytosine/uracil residues to fine-tune transcript metabolism. Among tuber and root crops, potato has been widely studied to understand the mobile signals that activate tuber development. Potato PTBs, designated as StPTB1 and StPTB6, function in a long-distance transport system by binding to specific mRNAs (StBEL5 and POTH1) to stabilize them and facilitate their movement from leaf to stolon, the site of tuber induction, where they activate tuber and root growth. Storage tubers and root crops are important sustenance food crops grown throughout the world. Despite the availability of genome sequence for sweet potato, cassava, carrot and sugar beet, the molecular mechanism of root-derived storage organ development remains completely unexplored. Considering the pivotal role of PTBs and their target RNAs in potato storage organ development, we propose that a similar mechanism may be prevalent in storage root crops as well. Through a bioinformatics survey utilizing available genome databases, we identify the orthologues of potato PTB proteins and two phloem-mobile RNAs, StBEL5 and POTH1, in five storage root crops - sweet potato, cassava, carrot, radish and sugar beet. Like potato, PTB1/6 type proteins from these storage root crops contain four conserved RNA Recognition Motifs (characteristic of RNA-binding PTBs) in their protein sequences. Further, 3´ UTR (untranslated region) analysis of BEL5 and POTH1 orthologues revealed the presence of several cytosine/uracil motifs, similar to those present in potato StBEL5 and POTH1 RNAs. Using RT-qPCR assays, we verified the presence of these related transcripts in leaf and root tissues of these five storage root crops. Similar to potato, BEL5-, PTB1/6- and POTH1-like orthologue RNAs from the aforementioned storage root

The Putative Son's Attractiveness Alters the Perceived Attractiveness of the Putative Father.

PubMed

Prokop, Pavol

2015-08-01

A body of literature has investigated female mate choice in the pre-mating context (pre-mating sexual selection). Humans, however, are long-living mammals forming pair-bonds which sequentially produce offspring. Post-mating evaluations of a partner's attractiveness may thus significantly influence the reproductive success of men and women. I tested herein the theory that the attractiveness of putative sons provides extra information about the genetic quality of fathers, thereby influencing fathers' attractiveness across three studies. As predicted, facially attractive boys were more frequently attributed to attractive putative fathers and vice versa (Study 1). Furthermore, priming with an attractive putative son increased the attractiveness of the putative father with the reverse being true for unattractive putative sons. When putative fathers were presented as stepfathers, the effect of the boy's attractiveness on the stepfather's attractiveness was lower and less consistent (Study 2). This suggests that the presence of an attractive boy has the strongest effect on the perceived attractiveness of putative fathers rather than on non-fathers. The generalized effect of priming with beautiful non-human objects also exists, but its effect is much weaker compared with the effects of putative biological sons (Study 3). Overall, this study highlighted the importance of post-mating sexual selection in humans and suggests that the heritable attractive traits of men are also evaluated by females after mating and/or may be used by females in mate poaching.

Tandem application of ligand-based virtual screening and G4-OAS assay to identify novel G-quadruplex-targeting chemotypes.

PubMed

Musumeci, Domenica; Amato, Jussara; Zizza, Pasquale; Platella, Chiara; Cosconati, Sandro; Cingolani, Chiara; Biroccio, Annamaria; Novellino, Ettore; Randazzo, Antonio; Giancola, Concetta; Pagano, Bruno; Montesarchio, Daniela

2017-05-01

G-quadruplex (G4) structures are key elements in the regulation of cancer cell proliferation and their targeting is deemed to be a promising strategy in anticancer therapy. A tandem application of ligand-based virtual screening (VS) calculations together with the experimental G-quadruplex on Oligo Affinity Support (G4-OAS) assay was employed to discover novel G4-targeting compounds. The interaction of the selected compounds with the investigated G4 in solution was analysed through a series of biophysical techniques and their biological activity investigated by immunofluorescence and MTT assays. A focused library of 60 small molecules, designed as putative G4 groove binders, was identified through the VS. The G4-OAS experimental screening led to the selection of 7 ligands effectively interacting with the G4-forming human telomeric DNA. Evaluation of the biological activity of the selected compounds showed that 3 ligands of this sub-library induced a marked telomere-localized DNA damage response in human tumour cells. The combined application of virtual and experimental screening tools proved to be a successful strategy to identify new bioactive chemotypes able to target the telomeric G4 DNA. These compounds may represent useful leads for the development of more potent and selective G4 ligands. Expanding the repertoire of the available G4-targeting chemotypes with improved physico-chemical features, in particular aiming at the discovery of novel, selective G4 telomeric ligands, can help in developing effective anti-cancer drugs with fewer side effects. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2017 Elsevier B.V. All rights reserved.

TargetLink, a new method for identifying the endogenous target set of a specific microRNA in intact living cells.

PubMed

Xu, Yan; Chen, Yan; Li, Daliang; Liu, Qing; Xuan, Zhenyu; Li, Wen-Hong

2017-02-01

MicroRNAs are small non-coding RNAs acting as posttranscriptional repressors of gene expression. Identifying mRNA targets of a given miRNA remains an outstanding challenge in the field. We have developed a new experimental approach, TargetLink, that applied locked nucleic acid (LNA) as the affinity probe to enrich target genes of a specific microRNA in intact cells. TargetLink also consists a rigorous and systematic data analysis pipeline to identify target genes by comparing LNA-enriched sequences between experimental and control samples. Using miR-21 as a test microRNA, we identified 12 target genes of miR-21 in a human colorectal cancer cell by this approach. The majority of the identified targets interacted with miR-21 via imperfect seed pairing. Target validation confirmed that miR-21 repressed the expression of the identified targets. The cellular abundance of the identified miR-21 target transcripts varied over a wide range, with some targets expressed at a rather low level, confirming that both abundant and rare transcripts are susceptible to regulation by microRNAs, and that TargetLink is an efficient approach for identifying the target set of a specific microRNA in intact cells. C20orf111, one of the novel targets identified by TargetLink, was found to reside in the nuclear speckle and to be reliably repressed by miR-21 through the interaction at its coding sequence.

Integration of genetic, genomic and transcriptomic information identifies putative regulators of adventitious root formation in Populus

DOE PAGES

Ribeiro, Cintia L.; Silva, Cynthia M.; Drost, Derek R.; ...

2016-03-16

In this study, adventitious roots (AR) develop from tissues other than the primary root, in a process physiologically regulated by phytohormones. Adventitious roots provide structural support and contribute to water and nutrient absorption, and are critical for commercial vegetative propagation of several crops. Here we quantified the number of AR, root architectural traits and root biomass in cuttings from a pseudo-backcross population of Populus deltoides and Populus trichocarpa. Quantitative trait loci (QTL) mapping and whole-transcriptome analysis of individuals with alternative QTL alleles for AR number were used to identify putative regulators of AR development. As a result, parental individuals andmore » progeny showed extensive segregation for AR developmental traits. Quantitative trait loci for number of AR mapped consistently in the same interval of linkage group (LG) II and LG XIV, explaining 7–10 % of the phenotypic variation. A time series transcriptome analysis identified 26,121 genes differentially expressed during AR development, particularly during the first 24 h after cuttings were harvested. Of those, 1929 genes were differentially regulated between individuals carrying alternative alleles for the two QTL for number of AR, in one or more time point. Eighty-one of these genes were physically located within the QTL intervals for number of AR, including putative homologs of the Arabidopsis genes SUPERROOT2 (SUR2) and TRYPTOPHAN SYNTHASE ALPHA CHAIN (TSA1), both of which are involved in the auxin indole-3-acetic acid (IAA) biosynthesis pathway. In conclusion, this study suggests the involvement of two genes of the tryptophan-dependent auxin biosynthesis pathway, SUR2 and TSA1, in the regulation of a critical trait for the clonal propagation of woody species. A possible model for this regulation is that poplar individuals that have poor AR formation synthesize auxin indole-3-acetic acid (IAA) primarily through the tryptophan (Trp) pathway. Much of

Putative pyramidal neurons and interneurons in the monkey parietal cortex make different contributions to the performance of a visual grouping task.

PubMed

Yokoi, Isao; Komatsu, Hidehiko

2010-09-01

Visual grouping of discrete elements is an important function for object recognition. We recently conducted an experiment to study neural correlates of visual grouping. We recorded neuronal activities while monkeys performed a grouping detection task in which they discriminated visual patterns composed of discrete dots arranged in a cross and detected targets in which dots with the same contrast were aligned horizontally or vertically. We found that some neurons in the lateral bank of the intraparietal sulcus exhibit activity related to visual grouping. In the present study, we analyzed how different types of neurons contribute to visual grouping. We classified the recorded neurons as putative pyramidal neurons or putative interneurons, depending on the duration of their action potentials. We found that putative pyramidal neurons exhibited selectivity for the orientation of the target, and this selectivity was enhanced by attention to a particular target orientation. By contrast, putative interneurons responded more strongly to the target stimuli than to the nontargets, regardless of the orientation of the target. These results suggest that different classes of parietal neurons contribute differently to the grouping of discrete elements.

Identifying transcription factor functions and targets by phenotypic activation

PubMed Central

Chua, Gordon; Morris, Quaid D.; Sopko, Richelle; Robinson, Mark D.; Ryan, Owen; Chan, Esther T.; Frey, Brendan J.; Andrews, Brenda J.; Boone, Charles; Hughes, Timothy R.

2006-01-01

Mapping transcriptional regulatory networks is difficult because many transcription factors (TFs) are activated only under specific conditions. We describe a generic strategy for identifying genes and pathways induced by individual TFs that does not require knowledge of their normal activation cues. Microarray analysis of 55 yeast TFs that caused a growth phenotype when overexpressed showed that the majority caused increased transcript levels of genes in specific physiological categories, suggesting a mechanism for growth inhibition. Induced genes typically included established targets and genes with consensus promoter motifs, if known, indicating that these data are useful for identifying potential new target genes and binding sites. We identified the sequence 5′-TCACGCAA as a binding sequence for Hms1p, a TF that positively regulates pseudohyphal growth and previously had no known motif. The general strategy outlined here presents a straightforward approach to discovery of TF activities and mapping targets that could be adapted to any organism with transgenic technology. PMID:16880382

Fine Time Course Expression Analysis Identifies Cascades of Activation and Repression and Maps a Putative Regulator of Mammalian Sex Determination

PubMed Central

Looger, Loren L.; Ohler, Uwe; Capel, Blanche

2013-01-01

In vertebrates, primary sex determination refers to the decision within a bipotential organ precursor to differentiate as a testis or ovary. Bifurcation of organ fate begins between embryonic day (E) 11.0–E12.0 in mice and likely involves a dynamic transcription network that is poorly understood. To elucidate the first steps of sexual fate specification, we profiled the XX and XY gonad transcriptomes at fine granularity during this period and resolved cascades of gene activation and repression. C57BL/6J (B6) XY gonads showed a consistent ∼5-hour delay in the activation of most male pathway genes and repression of female pathway genes relative to 129S1/SvImJ, which likely explains the sensitivity of the B6 strain to male-to-female sex reversal. Using this fine time course data, we predicted novel regulatory genes underlying expression QTLs (eQTLs) mapped in a previous study. To test predictions, we developed an in vitro gonad primary cell assay and optimized a lentivirus-based shRNA delivery method to silence candidate genes and quantify effects on putative targets. We provide strong evidence that Lmo4 (Lim-domain only 4) is a novel regulator of sex determination upstream of SF1 (Nr5a1), Sox9, Fgf9, and Col9a3. This approach can be readily applied to identify regulatory interactions in other systems. PMID:23874228

De novo sequencing and analysis of the cranberry fruit transcriptome to identify putative genes involved in flavonoid biosynthesis, transport and regulation.

PubMed

Sun, Haiyue; Liu, Yushan; Gai, Yuzhuo; Geng, Jinman; Chen, Li; Liu, Hongdi; Kang, Limin; Tian, Youwen; Li, Yadong

2015-09-02

Cranberries (Vaccinium macrocarpon Ait.), renowned for their excellent health benefits, are an important berry crop. Here, we performed transcriptome sequencing of one cranberry cultivar, from fruits at two different developmental stages, on the Illumina HiSeq 2000 platform. Our main goals were to identify putative genes for major metabolic pathways of bioactive compounds and compare the expression patterns between white fruit (W) and red fruit (R) in cranberry. In this study, two cDNA libraries of W and R were constructed. Approximately 119 million raw sequencing reads were generated and assembled de novo, yielding 57,331 high quality unigenes with an average length of 739 bp. Using BLASTx, 38,460 unigenes were identified as putative homologs of annotated sequences in public protein databases, including NCBI NR, NT, Swiss-Prot, KEGG, COG and GO. Of these, 21,898 unigenes mapped to 128 KEGG pathways, with the metabolic pathways, secondary metabolites, glycerophospholipid metabolism, ether lipid metabolism, starch and sucrose metabolism, purine metabolism, and pyrimidine metabolism being well represented. Among them, many candidate genes were involved in flavonoid biosynthesis, transport and regulation. Furthermore, digital gene expression (DEG) analysis identified 3,257 unigenes that were differentially expressed between the two fruit developmental stages. In addition, 14,473 simple sequence repeats (SSRs) were detected. Our results present comprehensive gene expression information about the cranberry fruit transcriptome that could facilitate our understanding of the molecular mechanisms of fruit development in cranberries. Although it will be necessary to validate the functions carried out by these genes, these results could be used to improve the quality of breeding programs for the cranberry and related species.

Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors.

PubMed

Heisig, Julia; Weber, David; Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

2012-01-01

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.

Target Gene Analysis by Microarrays and Chromatin Immunoprecipitation Identifies HEY Proteins as Highly Redundant bHLH Repressors

PubMed Central

Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

2012-01-01

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression. PMID:22615585

A biochemical approach to identifying microRNA targets

PubMed Central

Karginov, Fedor V.; Conaco, Cecilia; Xuan, Zhenyu; Schmidt, Bryan H.; Parker, Joel S.; Mandel, Gail; Hannon, Gregory J.

2007-01-01

Identifying the downstream targets of microRNAs (miRNAs) is essential to understanding cellular regulatory networks. We devised a direct biochemical method for miRNA target discovery that combined RNA-induced silencing complex (RISC) purification with microarray analysis of bound mRNAs. Because targets of miR-124a have been analyzed, we chose it as our model. We honed our approach both by examining the determinants of stable binding between RISC and synthetic target RNAs in vitro and by determining the dependency of both repression and RISC coimmunoprecipitation on miR-124a seed sites in two of its well characterized targets in vivo. Examining the complete spectrum of miR-124 targets in 293 cells yielded both a set that were down-regulated at the mRNA level, as previously observed, and a set whose mRNA levels were unaffected by miR-124a. Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway. PMID:18042700

In silico Analysis of Toxins of Staphylococcus aureus for Validating Putative Drug Targets.

PubMed

Mohana, Ramadevi; Venugopal, Subhashree

2017-01-01

Toxins are one among the numerous virulence factors produced by the bacteria. These are powerful poisonous substances enabling the bacteria to encounter the defense mechanism of human body. The pathogenic system of Staphylococcus aureus is evolved with various exotoxins that cause detrimental effects on human immune system. Four toxins namely enterotoxin A, exfoliative toxin A, TSST-1 and γ-hemolysin were downloaded from Uniprot database and were analyzed to understand the nature of the toxins and for drug target validation. The results inferred that the toxins were found to interact with many protein partners and no homologous sequences for human proteome were found, and based on similarity search in Drugbank, the targets were identified as novel drug targets. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Use of an activated beta-catenin to identify Wnt pathway target genes in caenorhabditis elegans, including a subset of collagen genes expressed in late larval development.

PubMed

Jackson, Belinda M; Abete-Luzi, Patricia; Krause, Michael W; Eisenmann, David M

2014-04-16

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

Selecting a Targeting Method to Identify BPL Households in India

ERIC Educational Resources Information Center

Alkire, Sabina; Seth, Suman

2013-01-01

This paper proposes how to select a methodology to target multidimensionally poor households, and how to update that targeting exercise periodically. We present this methodology in the context of discussions regarding the selection of a targeting methodology in India. In 1992, 1997, and 2002 the Indian government identified households that are…

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

Identifying Molecular Targets for PTSD Treatment Using Single Prolonged Stress

DTIC Science & Technology

2015-10-01

1 AWARD NUMBER: W81XWH-13-1-0377 TITLE: Identifying Molecular Targets For PTSD Treatment Using Single Prolonged Stress PRINCIPAL...TITLE AND SUBTITLE 5a. CONTRACT NUMBER W81XWH-13-1-0377 Identifying Molecular Targets For PTSD Treatment Using Single Prolonged Stress 5b. GRANT...brain GR and β-AR expression alters glutamatergic and GABAergic function in neural circuits that mediate SPS-induced deficits in extinction retention

Genome-Wide Association Study of d-Amphetamine Response in Healthy Volunteers Identifies Putative Associations, Including Cadherin 13 (CDH13)

PubMed Central

Wardle, Margaret C.; Sokoloff, Greta; Stephens, Matthew; de Wit, Harriet; Palmer, Abraham A.

2012-01-01

Both the subjective response to d-amphetamine and the risk for amphetamine addiction are known to be heritable traits. Because subjective responses to drugs may predict drug addiction, identifying alleles that influence acute response may also provide insight into the genetic risk factors for drug abuse. We performed a Genome Wide Association Study (GWAS) for the subjective responses to amphetamine in 381 non-drug abusing healthy volunteers. Responses to amphetamine were measured using a double-blind, placebo-controlled, within-subjects design. We used sparse factor analysis to reduce the dimensionality of the data to ten factors. We identified several putative associations; the strongest was between a positive subjective drug-response factor and a SNP (rs3784943) in the 8th intron of cadherin 13 (CDH13; P = 4.58×10−8), a gene previously associated with a number of psychiatric traits including methamphetamine dependence. Additionally, we observed a putative association between a factor representing the degree of positive affect at baseline and a SNP (rs472402) in the 1st intron of steroid-5-alpha-reductase-α-polypeptide-1 (SRD5A1; P = 2.53×10−7), a gene whose protein product catalyzes the rate-limiting step in synthesis of the neurosteroid allopregnanolone. This SNP belongs to an LD-block that has been previously associated with the expression of SRD5A1 and differences in SRD5A1 enzymatic activity. The purpose of this study was to begin to explore the genetic basis of subjective responses to stimulant drugs using a GWAS approach in a modestly sized sample. Our approach provides a case study for analysis of high-dimensional intermediate pharmacogenomic phenotypes, which may be more tractable than clinical diagnoses. PMID:22952603

Genome-wide association study of d-amphetamine response in healthy volunteers identifies putative associations, including cadherin 13 (CDH13).

PubMed

Hart, Amy B; Engelhardt, Barbara E; Wardle, Margaret C; Sokoloff, Greta; Stephens, Matthew; de Wit, Harriet; Palmer, Abraham A

2012-01-01

Both the subjective response to d-amphetamine and the risk for amphetamine addiction are known to be heritable traits. Because subjective responses to drugs may predict drug addiction, identifying alleles that influence acute response may also provide insight into the genetic risk factors for drug abuse. We performed a Genome Wide Association Study (GWAS) for the subjective responses to amphetamine in 381 non-drug abusing healthy volunteers. Responses to amphetamine were measured using a double-blind, placebo-controlled, within-subjects design. We used sparse factor analysis to reduce the dimensionality of the data to ten factors. We identified several putative associations; the strongest was between a positive subjective drug-response factor and a SNP (rs3784943) in the 8(th) intron of cadherin 13 (CDH13; P = 4.58×10(-8)), a gene previously associated with a number of psychiatric traits including methamphetamine dependence. Additionally, we observed a putative association between a factor representing the degree of positive affect at baseline and a SNP (rs472402) in the 1(st) intron of steroid-5-alpha-reductase-α-polypeptide-1 (SRD5A1; P = 2.53×10(-7)), a gene whose protein product catalyzes the rate-limiting step in synthesis of the neurosteroid allopregnanolone. This SNP belongs to an LD-block that has been previously associated with the expression of SRD5A1 and differences in SRD5A1 enzymatic activity. The purpose of this study was to begin to explore the genetic basis of subjective responses to stimulant drugs using a GWAS approach in a modestly sized sample. Our approach provides a case study for analysis of high-dimensional intermediate pharmacogenomic phenotypes, which may be more tractable than clinical diagnoses.

Discovery of precursor and mature microRNAs and their putative gene targets using high-throughput sequencing in pineapple (Ananas comosus var. comosus).

PubMed

Yusuf, Noor Hydayaty Md; Ong, Wen Dee; Redwan, Raimi Mohamed; Latip, Mariam Abd; Kumar, S Vijay

2015-10-15

MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that negatively regulate gene expression, resulting in the silencing of target mRNA transcripts through mRNA cleavage or translational inhibition. MiRNAs play significant roles in various biological and physiological processes in plants. However, the miRNA-mediated gene regulatory network in pineapple, the model tropical non-climacteric fruit, remains largely unexplored. Here, we report a complete list of pineapple mature miRNAs obtained from high-throughput small RNA sequencing and precursor miRNAs (pre-miRNAs) obtained from ESTs. Two small RNA libraries were constructed from pineapple fruits and leaves, respectively, using Illumina's Solexa technology. Sequence similarity analysis using miRBase revealed 579,179 reads homologous to 153 miRNAs from 41 miRNA families. In addition, a pineapple fruit transcriptome library consisting of approximately 30,000 EST contigs constructed using Solexa sequencing was used for the discovery of pre-miRNAs. In all, four pre-miRNAs were identified (MIR156, MIR399, MIR444 and MIR2673). Furthermore, the same pineapple transcriptome was used to dissect the function of the miRNAs in pineapple by predicting their putative targets in conjunction with their regulatory networks. In total, 23 metabolic pathways were found to be regulated by miRNAs in pineapple. The use of high-throughput sequencing in pineapples to unveil the presence of miRNAs and their regulatory pathways provides insight into the repertoire of miRNA regulation used exclusively in this non-climacteric model plant. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of Beet necrotic yellow vein virus P25 pathogenicity factor-interacting sugar beet proteins that represent putative virus targets or components of plant resistance.

PubMed

Thiel, Heike; Varrelmann, Mark

2009-08-01

Beet necrotic yellow vein virus (BNYVV) induces the most important disease threatening sugar beet. The growth of partially resistant hybrids carrying monogenic dominant resistance genes stabilize yield but are unable to entirely prevent virus infection and replication. P25 is responsible for symptom development and previous studies have shown that recently occurring resistance-breaking isolates possess increased P25 variability. To better understand the viral pathogenicity factor's interplay with plant proteins and to possibly unravel the molecular basis of sugar beet antivirus resistance, P25 was applied in a yeast two-hybrid screen of a resistant sugar beet cDNA library. This screen identified candidate proteins recognized as orthologues from other plant species which are known to be expressed following pathogen infection and involved in plant defense response. Most of the candidates potentially related to host-pathogen interactions were involved in the ubiquitylation process and plants response to stress, and were part of cell and metabolism components. The interaction of several candidate genes with P25 was confirmed in Nicotiana benthamiana leaf cells by transient agrobacterium-mediated expression applying bimolecular fluorescence complementation assay. The putative functions of several of the candidates identified support previous findings and present first targets for understanding the BNYVV pathogenicity and antivirus resistance mechanism.

A strategy to discover new organizers identifies a putative heart organizer

PubMed Central

Anderson, Claire; Khan, Mohsin A. F.; Wong, Frances; Solovieva, Tatiana; Oliveira, Nidia M. M.; Baldock, Richard A.; Tickle, Cheryll; Burt, Dave W.; Stern, Claudio D.

2016-01-01

Organizers are regions of the embryo that can both induce new fates and impart pattern on other regions. So far, surprisingly few organizers have been discovered, considering the number of patterned tissue types generated during development. This may be because their discovery has relied on transplantation and ablation experiments. Here we describe a new approach, using chick embryos, to discover organizers based on a common gene expression signature, and use it to uncover the anterior intestinal portal (AIP) endoderm as a putative heart organizer. We show that the AIP can induce cardiac identity from non-cardiac mesoderm and that it can pattern this by specifying ventricular and suppressing atrial regional identity. We also uncover some of the signals responsible. The method holds promise as a tool to discover other novel organizers acting during development. PMID:27557800

Indel-seq: a fast-forward genetics approach for identification of trait-associated putative candidate genomic regions and its application in pigeonpea (Cajanus cajan).

PubMed

Singh, Vikas K; Khan, Aamir W; Saxena, Rachit K; Sinha, Pallavi; Kale, Sandip M; Parupalli, Swathi; Kumar, Vinay; Chitikineni, Annapurna; Vechalapu, Suryanarayana; Sameer Kumar, Chanda Venkata; Sharma, Mamta; Ghanta, Anuradha; Yamini, Kalinati Narasimhan; Muniswamy, Sonnappa; Varshney, Rajeev K

2017-07-01

Identification of candidate genomic regions associated with target traits using conventional mapping methods is challenging and time-consuming. In recent years, a number of single nucleotide polymorphism (SNP)-based mapping approaches have been developed and used for identification of candidate/putative genomic regions. However, in the majority of these studies, insertion-deletion (Indel) were largely ignored. For efficient use of Indels in mapping target traits, we propose Indel-seq approach, which is a combination of whole-genome resequencing (WGRS) and bulked segregant analysis (BSA) and relies on the Indel frequencies in extreme bulks. Deployment of Indel-seq approach for identification of candidate genomic regions associated with fusarium wilt (FW) and sterility mosaic disease (SMD) resistance in pigeonpea has identified 16 Indels affecting 26 putative candidate genes. Of these 26 affected putative candidate genes, 24 genes showed effect in the upstream/downstream of the genic region and two genes showed effect in the genes. Validation of these 16 candidate Indels in other FW- and SMD-resistant and FW- and SMD-susceptible genotypes revealed a significant association of five Indels (three for FW and two for SMD resistance). Comparative analysis of Indel-seq with other genetic mapping approaches highlighted the importance of the approach in identification of significant genomic regions associated with target traits. Therefore, the Indel-seq approach can be used for quick and precise identification of candidate genomic regions for any target traits in any crop species. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Wide screening of phage-displayed libraries identifies immune targets in planta.

PubMed

Rioja, Cristina; Van Wees, Saskia C; Charlton, Keith A; Pieterse, Corné M J; Lorenzo, Oscar; García-Sánchez, Susana

2013-01-01

Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 2 × 10(7) different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes, including well

Putative and unique gene sequence utilization for the design of species specific probes as modeled by Lactobacillus plantarum

USDA-ARS?s Scientific Manuscript database

The concept of utilizing putative and unique gene sequences for the design of species specific probes was tested. The abundance profile of assigned functions within the Lactobacillus plantarum genome was used for the identification of the putative and unique gene sequence, csh. The targeted gene (cs...

Complete genome-wide screening and subtractive genomic approach revealed new virulence factors, potential drug targets against bio-war pathogen Brucella melitensis 16M

PubMed Central

Pradeepkiran, Jangampalli Adi; Sainath, Sri Bhashyam; Kumar, Konidala Kranthi; Bhaskar, Matcha

2015-01-01

Brucella melitensis 16M is a Gram-negative coccobacillus that infects both animals and humans. It causes a disease known as brucellosis, which is characterized by acute febrile illness in humans and causes abortions in livestock. To prevent and control brucellosis, identification of putative drug targets is crucial. The present study aimed to identify drug targets in B. melitensis 16M by using a subtractive genomic approach. We used available database repositories (Database of Essential Genes, Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server, and Kyoto Encyclopedia of Genes and Genomes) to identify putative genes that are nonhomologous to humans and essential for pathogen B. melitensis 16M. The results revealed that among 3 Mb genome size of pathogen, 53 putative characterized and 13 uncharacterized hypothetical genes were identified; further, from Basic Local Alignment Search Tool protein analysis, one hypothetical protein showed a close resemblance (50%) to Silicibacter pomeroyi DUF1285 family protein (2RE3). A further homology model of the target was constructed using MODELLER 9.12 and optimized through variable target function method by molecular dynamics optimization with simulating annealing. The stereochemical quality of the restrained model was evaluated by PROCHECK, VERIFY-3D, ERRAT, and WHATIF servers. Furthermore, structure-based virtual screening was carried out against the predicted active site of the respective protein using the glycerol structural analogs from the PubChem database. We identified five best inhibitors with strong affinities, stable interactions, and also with reliable drug-like properties. Hence, these leads might be used as the most effective inhibitors of modeled protein. The outcome of the present work of virtual screening of putative gene targets might facilitate design of potential drugs for better treatment against brucellosis. PMID:25834405

A side-effect free method for identifying cancer drug targets.

PubMed

Ashraf, Md Izhar; Ong, Seng-Kai; Mujawar, Shama; Pawar, Shrikant; More, Pallavi; Paul, Somnath; Lahiri, Chandrajit

2018-04-27

Identifying effective drug targets, with little or no side effects, remains an ever challenging task. A potential pitfall of failing to uncover the correct drug targets, due to side effect of pleiotropic genes, might lead the potential drugs to be illicit and withdrawn. Simplifying disease complexity, for the investigation of the mechanistic aspects and identification of effective drug targets, have been done through several approaches of protein interactome analysis. Of these, centrality measures have always gained importance in identifying candidate drug targets. Here, we put forward an integrated method of analysing a complex network of cancer and depict the importance of k-core, functional connectivity and centrality (KFC) for identifying effective drug targets. Essentially, we have extracted the proteins involved in the pathways leading to cancer from the pathway databases which enlist real experimental datasets. The interactions between these proteins were mapped to build an interactome. Integrative analyses of the interactome enabled us to unearth plausible reasons for drugs being rendered withdrawn, thereby giving future scope to pharmaceutical industries to potentially avoid them (e.g. ESR1, HDAC2, F2, PLG, PPARA, RXRA, etc). Based upon our KFC criteria, we have shortlisted ten proteins (GRB2, FYN, PIK3R1, CBL, JAK2, LCK, LYN, SYK, JAK1 and SOCS3) as effective candidates for drug development.

Chemogenomics profiling of drug targets of peptidoglycan biosynthesis pathway in Leptospira interrogans by virtual screening approaches.

PubMed

Bhattacharjee, Biplab; Simon, Rose Mary; Gangadharaiah, Chaithra; Karunakar, Prashantha

2013-06-28

Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. The availability of ligand libraries has facilitated the search for novel drug targets using chemogenomics approaches, compared with the traditional method of drug discovery, which is time consuming and yields few leads with little intracellular information for guiding target selection. Recent subtractive genomics studies have revealed the putative drug targets in peptidoglycan biosynthesis pathways in Leptospira interrogans. Aligand library for the murD ligase enzyme in the peptidoglycan pathway has also been identified. Our approach in this research involves screening of the pre-existing ligand library of murD with related protein family members in the putative drug target assembly in the peptidoglycan biosynthesis pathway. A chemogenomics approach has been implemented here, which involves screening of known ligands of a protein family having analogous domain architecture for identification of leads for existing druggable protein family members. By means of this approach, one murC and one murF inhibitor were identified, providing a platform for developing an antileptospirosis drug targeting the peptidoglycan biosynthesis pathway. Given that the peptidoglycan biosynthesis pathway is exclusive to bacteria, the in silico identified mur ligase inhibitors are expected to be broad-spectrum Gram-negative inhibitors if synthesized and tested in in vitro and in vivo assays.

Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.

PubMed

Robin, Guillaume P; Kleemann, Jochen; Neumann, Ulla; Cabre, Lisa; Dallery, Jean-Félix; Lapalu, Nicolas; O'Connell, Richard J

2018-01-01

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum , encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium -mediated transformation to transiently express them as N -terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana , and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C -terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.

Molecular Diagnosis of Putative Stargardt Disease by Capture Next Generation Sequencing

PubMed Central

Shi, Wei; Huang, Ping; Min, Qingjie; Li, Minghan; Yu, Xinping; Wu, Yaming; Zhao, Guangyu; Tong, Yi; Jin, Zi-Bing; Qu, Jia; Gu, Feng

2014-01-01

Stargardt Disease (STGD) is the commonest genetic form of juvenile or early adult onset macular degeneration, which is a genetically heterogeneous disease. Molecular diagnosis of STGD remains a challenge in a significant proportion of cases. To address this, seven patients from five putative STGD families were recruited. We performed capture next generation sequencing (CNGS) of the probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Seven disease-causing mutations in ABCA4 and two in PROM1 were identified by CNGS, which provides a confident genetic diagnosis in these five families. We also provided a genetic basis to explain the differences among putative STGD due to various mutations in different genes. Meanwhile, we show for the first time that compound heterozygous mutations in PROM1 gene could cause cone-rod dystrophy. Our findings support the enormous potential of CNGS in putative STGD molecular diagnosis. PMID:24763286

Putative Risk Factors in Developmental Dyslexia: A Case-Control Study of Italian Children

ERIC Educational Resources Information Center

Mascheretti, Sara; Marino, Cecilia; Simone, Daniela; Quadrelli, Ermanno; Riva, Valentina; Cellino, Maria Rosaria; Maziade, Michel; Brombin, Chiara; Battaglia, Marco

2015-01-01

Although dyslexia runs in families, several putative risk factors that cannot be immediately identified as genetic predict reading disability. Published studies analyzed one or a few risk factors at a time, with relatively inconsistent results. To assess the contribution of several putative risk factors to the development of dyslexia, we conducted…

Geothermal Target Areas in Colorado as Identified by Remote Sensing Techniques

DOE Data Explorer

Khalid Hussein

2012-02-01

This layer contains the areas identified as targets of potential geothermal activity. The Criteria used to identify the target areas include: hot/warm surface exposures modeled from ASTER/Landsat satellite imagery and geological characteristics, alteration mineral commonly associated with hot springs (clays, Si, and FeOx) modeled from ASTER and Landsat data, Colorado Geological Survey (CGS) known thermal hot springs/wells and heat-flow data points, Colorado deep-seated fault zones, weakened basement identified from isostatic gravity data, and Colorado sedimentary and topographic characteristics.

Novel β-catenin target genes identified in thalamic neurons encode modulators of neuronal excitability

PubMed Central

2012-01-01

Background LEF1/TCF transcription factors and their activator β-catenin are effectors of the canonical Wnt pathway. Although Wnt/β-catenin signaling has been implicated in neurodegenerative and psychiatric disorders, its possible role in the adult brain remains enigmatic. To address this issue, we sought to identify the genetic program activated by β-catenin in neurons. We recently showed that β-catenin accumulates specifically in thalamic neurons where it activates Cacna1g gene expression. In the present study, we combined bioinformatics and experimental approaches to find new β-catenin targets in the adult thalamus. Results We first selected the genes with at least two conserved LEF/TCF motifs within the regulatory elements. The resulting list of 428 putative LEF1/TCF targets was significantly enriched in known Wnt targets, validating our approach. Functional annotation of the presumed targets also revealed a group of 41 genes, heretofore not associated with Wnt pathway activity, that encode proteins involved in neuronal signal transmission. Using custom polymerase chain reaction arrays, we profiled the expression of these genes in the rat forebrain. We found that nine of the analyzed genes were highly expressed in the thalamus compared with the cortex and hippocampus. Removal of nuclear β-catenin from thalamic neurons in vitro by introducing its negative regulator Axin2 reduced the expression of six of the nine genes. Immunoprecipitation of chromatin from the brain tissues confirmed the interaction between β-catenin and some of the predicted LEF1/TCF motifs. The results of these experiments validated four genes as authentic and direct targets of β-catenin: Gabra3 for the receptor of GABA neurotransmitter, Calb2 for the Ca2+-binding protein calretinin, and the Cacna1g and Kcna6 genes for voltage-gated ion channels. Two other genes from the latter cluster, Cacna2d2 and Kcnh8, appeared to be regulated by β-catenin, although the binding of β-catenin to the

Primary genome scan to identify putative quantitative trait loci for feedlot growth rate, feed intake, and feed efficiency of beef cattle.

PubMed

Nkrumah, J D; Sherman, E L; Li, C; Marques, E; Crews, D H; Bartusiak, R; Murdoch, B; Wang, Z; Basarab, J A; Moore, S S

2007-12-01

Feed intake and feed efficiency of beef cattle are economically relevant traits. The study was conducted to identify QTL for feed intake and feed efficiency of beef cattle by using genotype information from 100 microsatellite markers and 355 SNP genotyped across 400 progeny of 20 Angus, Charolais, or Alberta Hybrid bulls. Traits analyzed include feedlot ADG, daily DMI, feed-to-gain ratio [F:G, which is the reciprocal of the efficiency of gain (G:F)], and residual feed intake (RFI). A mixed model with sire as random and QTL effects as fixed was used to generate an F-statistic profile across and within families for each trait along each chromosome, followed by empirical permutation tests to determine significance thresholds for QTL detection. Putative QTL for ADG (chromosome-wise P < 0.05) were detected across families on chromosomes 5 (130 cM), 6 (42 cM), 7 (84 cM), 11 (20 cM), 14 (74 cM), 16 (22 cM), 17 (9 cM), 18 (46 cM), 19 (53 cM), and 28 (23 cM). For DMI, putative QTL that exceeded the chromosome-wise P < 0.05 threshold were detected on chromosomes 1 (93 cM), 3 (123 cM), 15 (31 cM), 17 (81 cM), 18 (49 cM), 20 (56 cM), and 26 (69 cM) in the across-family analyses. Putative across-family QTL influencing F:G that exceeded the chromosome-wise P < 0.05 threshold were detected on chromosomes 3 (62 cM), 5 (129 cM), 7 (27 cM), 11 (16 cM), 16 (30 cM), 17 (81 cM), 22 (72 cM), 24 (55 cM), and 28 (24 cM). Putative QTL influencing RFI that exceeded the chromosome-wise P < 0.05 threshold were detected on chromosomes 1 (90 cM), 5 (129 cM), 7 (22 cM), 8 (80 cM), 12 (89 cM), 16 (41 cM), 17 (19 cM), and 26 (48 cM) in the across-family analyses. In addition, a total of 4, 6, 1, and 8 chromosomes showed suggestive evidence (chromosome-wise, P < 0.10) for putative ADG, DMI, F:G, and RFI QTL, respectively. Most of the QTL detected across families were also detected within families, although the locations across families were not necessarily the locations within families, which is

Random Mutagenesis of the Multidrug Transporter AcrB from Escherichia coli for Identification of Putative Target Residues of Efflux Pump Inhibitors

PubMed Central

Kohler, Samay; Buck, Annika; Dambacher, Christine; König, Armin; Bohnert, Jürgen A.; Kern, Winfried V.

2014-01-01

Efflux is an important mechanism of bacterial multidrug resistance (MDR), and the inhibition of MDR pumps by efflux pump inhibitors (EPIs) could be a promising strategy to overcome MDR. 1-(1-Naphthylmethyl)-piperazine (NMP) and phenylalanine-arginine-β-naphthylamide (PAβN) are model EPIs with activity in various Gram-negative bacteria expressing AcrB, the major efflux pump of Escherichia coli, or similar homologous pumps of the resistance-nodulation-cell division class. The aim of the present study was to generate E. coli AcrB mutants resistant to the inhibitory action of the two model EPIs and to identify putative EPI target residues in order to better understand mechanisms of pump inhibition. Using an in vitro random mutagenesis approach focusing on the periplasmic domain of AcrB, we identified the double mutation G141D N282Y, which substantially compromised the synergistic activity of NMP with linezolid, was associated with similar intracellular linezolid concentrations in the presence and absence of NMP, and did not impair the intrinsic MICs of various pump substrates and dye accumulation. We propose that these mutations near the outer face of the distal substrate binding pocket reduce NMP trapping. Other residues found to be relevant for efflux inhibition by NMP were G288 and A279, but mutations at these sites also changed the susceptibility to several pump substrates. Unlike with NMP, we were unable to generate AcrB periplasmic domain mutants with resistance or partial resistance to the EPI activity of PAβN, which is consistent with the modes of action of PAβN differing from those of NMP. PMID:25182653

Candidate adaptive genes associated with lineage divergence: identifying SNPs via next-generation targeted resequencing in mule deer (Odocoileus hemionus).

PubMed

Powell, John H; Amish, Stephen J; Haynes, Gwilym D; Luikart, Gordon; Latch, Emily K

2016-09-01

Mule deer (Odocoileus hemionus) are an excellent nonmodel species for empirically testing hypotheses in landscape and population genomics due to their large population sizes (low genetic drift), relatively continuous distribution, diversity of occupied habitats and phenotypic variation. Because few genomic resources are currently available for this species, we used exon data from a cattle (Bos taurus) reference genome to direct targeted resequencing of 5935 genes in mule deer. We sequenced approximately 3.75 Mbp at minimum 20X coverage in each of the seven mule deer, identifying 23 204 single nucleotide polymorphisms (SNPs) within, or adjacent to, 6886 exons in 3559 genes. We found 91 SNP loci (from 69 genes) with putatively fixed allele frequency differences between the two major lineages of mule deer (mule deer and black-tailed deer), and our estimate of mean genetic divergence (genome-wide FST  = 0.123) between these lineages was consistent with previous findings using microsatellite loci. We detected an over-representation of gamete generation and amino acid transport genes among the genes with SNPs exhibiting potentially fixed allele frequency differences between lineages. This targeted resequencing approach using exon capture techniques has identified a suite of loci that can be used in future research to investigate the genomic basis of adaptation and differentiation between black-tailed deer and mule deer. This study also highlights techniques (and an exon capture array) that will facilitate population genomic research in other cervids and nonmodel organisms. © 2016 John Wiley & Sons Ltd.

Emerging Putative Associations between Non-Coding RNAs and Protein-Coding Genes in Neuropathic Pain: Added Value from Reusing Microarray Data.

PubMed

Raju, Hemalatha B; Tsinoremas, Nicholas F; Capobianco, Enrico

2016-01-01

Regeneration of injured nerves is likely occurring in the peripheral nervous system, but not in the central nervous system. Although protein-coding gene expression has been assessed during nerve regeneration, little is currently known about the role of non-coding RNAs (ncRNAs). This leaves open questions about the potential effects of ncRNAs at transcriptome level. Due to the limited availability of human neuropathic pain (NP) data, we have identified the most comprehensive time-course gene expression profile referred to sciatic nerve (SN) injury and studied in a rat model using two neuronal tissues, namely dorsal root ganglion (DRG) and SN. We have developed a methodology to identify differentially expressed bioentities starting from microarray probes and repurposing them to annotate ncRNAs, while analyzing the expression profiles of protein-coding genes. The approach is designed to reuse microarray data and perform first profiling and then meta-analysis through three main steps. First, we used contextual analysis to identify what we considered putative or potential protein-coding targets for selected ncRNAs. Relevance was therefore assigned to differential expression of neighbor protein-coding genes, with neighborhood defined by a fixed genomic distance from long or antisense ncRNA loci, and of parental genes associated with pseudogenes. Second, connectivity among putative targets was used to build networks, in turn useful to conduct inference at interactomic scale. Last, network paths were annotated to assess relevance to NP. We found significant differential expression in long-intergenic ncRNAs (32 lincRNAs in SN and 8 in DRG), antisense RNA (31 asRNA in SN and 12 in DRG), and pseudogenes (456 in SN and 56 in DRG). In particular, contextual analysis centered on pseudogenes revealed some targets with known association to neurodegeneration and/or neurogenesis processes. While modules of the olfactory receptors were clearly identified in protein

A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast

DTIC Science & Technology

2004-05-01

AD Award Number: DAMD17-03-1-0232 TITLE: A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast PRINCIPAL INVESTIGATOR...Approach to Identify Novel Breast DAMD17-03-1-0232 Cancer Gene Targets in Yeast 6. A UTHOR(S) Craig Bennett, Ph.D. 7. PERFORMING ORGANIZA TION NAME(S...Unlimited 13. ABSTRACT (Maximum 200 Words) We are using the yeast Saccharomyces cerevisiae to identify new cancer gene targets that interact with the

Identification of a putative protein profile associated with tamoxifen therapy resistance in breast cancer.

PubMed

Umar, Arzu; Kang, Hyuk; Timmermans, Annemieke M; Look, Maxime P; Meijer-van Gelder, Marion E; den Bakker, Michael A; Jaitly, Navdeep; Martens, John W M; Luider, Theo M; Foekens, John A; Pasa-Tolić, Ljiljana

2009-06-01

Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on approximately 5,500 pooled tumor cells (corresponding to approximately 550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n = 24 and n = 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with > or = 2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were

Development of a Photo-Cross-Linkable Diaminoquinazoline Inhibitor for Target Identification in Plasmodium falciparum.

PubMed

Lubin, Alexandra S; Rueda-Zubiaurre, Ainoa; Matthews, Holly; Baumann, Hella; Fisher, Fabio R; Morales-Sanfrutos, Julia; Hadavizadeh, Kate S; Nardella, Flore; Tate, Edward W; Baum, Jake; Scherf, Artur; Fuchter, Matthew J

2018-04-13

Diaminoquinazolines represent a privileged scaffold for antimalarial discovery, including use as putative Plasmodium histone lysine methyltransferase inhibitors. Despite this, robust evidence for their molecular targets is lacking. Here we report the design and development of a small-molecule photo-cross-linkable probe to investigate the targets of our diaminoquinazoline series. We demonstrate the effectiveness of our designed probe for photoaffinity labeling of Plasmodium lysates and identify similarities between the target profiles of the probe and the representative diaminoquinazoline BIX-01294. Initial pull-down proteomics experiments identified 104 proteins from different classes, many of which are essential, highlighting the suitability of the developed probe as a valuable tool for target identification in Plasmodium falciparum.

Putative Porin of Bradyrhizobium sp. (Lupinus) Bacteroids Induced by Glyphosate▿

PubMed Central

de María, Nuria; Guevara, Ángeles; Serra, M. Teresa; García-Luque, Isabel; González-Sama, Alfonso; de Lacoba, Mario García; de Felipe, M. Rosario; Fernández-Pascual, Mercedes

2007-01-01

Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed. PMID:17557843

Synchrotron- and focal plane array-based Fourier-transform infrared spectroscopy differentiates the basalis and functionalis epithelial endometrial regions and identifies putative stem cell regions of human endometrial glands.

PubMed

Theophilou, Georgios; Morais, Camilo L M; Halliwell, Diane E; Lima, Kássio M G; Drury, Josephine; Martin-Hirsch, Pierre L; Stringfellow, Helen F; Hapangama, Dharani K; Martin, Francis L

2018-05-09

The cyclical process of regeneration of the endometrium suggests that it may contain a cell population that can provide daughter cells with high proliferative potential. These cell lineages are clinically significant as they may represent clonogenic cells that may also be involved in tumourigenesis as well as endometriotic lesion development. To determine whether the putative stem cell location within human uterine tissue can be derived using vibrational spectroscopy techniques, normal endometrial tissue was interrogated by two spectroscopic techniques. Paraffin-embedded uterine tissues containing endometrial glands were sectioned to 10-μm-thick parallel tissue sections and were floated onto BaF 2 slides for synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy and globar focal plane array-based FTIR spectroscopy. Different spectral characteristics were identified depending on the location of the glands examined. The resulting infrared spectra were subjected to multivariate analysis to determine associated biophysical differences along the length of longitudinal and crosscut gland sections. Comparison of the epithelial cellular layer of transverse gland sections revealed alterations indicating the presence of putative transient-amplifying-like cells in the basalis and mitotic cells in the functionalis. SR-FTIR microspectroscopy of the base of the endometrial glands identified the location where putative stem cells may reside at the same time pointing towards ν s PO 2 - in DNA and RNA, nucleic acids and amide I and II vibrations as major discriminating factors. This study supports the view that vibration spectroscopy technologies are a powerful adjunct to our understanding of the stem cell biology of endometrial tissue. Graphical abstract ᅟ.

Maternal depressive symptoms and early childhood cognitive development: a review of putative environmental mediators.

PubMed

Ahun, Marilyn N; Côté, Sylvana M

2018-06-06

Despite the abundance of research investigating the associations between maternal depressive symptoms (MDS) and children's cognitive development, little is known about the putative mechanisms through which depressive symptoms are associated with children's cognitive development. The aim of this review was to summarize the literature on family mediators (i.e., maternal parenting behaviors, mother-child interactions, and family stress) involved in this association in early childhood. The review includes seven studies, five longitudinal and two cross-sectional, which tested putative mediators of the association between MDS and children's cognitive development. Studies were selected from online databases (PubMed, PsycNet) and manual searches. Only studies which quantitatively assessed associations between MDS in the postnatal period and child cognitive development in early childhood (i.e., 0-5 years) and included mediator variables were included in the review. Six out of seven studies identified mediating variables. The mediators included maternal responsiveness, parenting style, family dysfunction, the quality of the home environment, and maternal caregiving practices. Different mediators were identified across the reviewed studies. Maternal depressive symptoms are partly associated with child cognitive development via family processes and parenting practices. Various mediating processes are at play. Further research is needed on the role of maternal and paternal mental health and gene-environment correlations in this association. A better understanding of the mediating pathways is needed for the design of preventative intervention targeting specific family processes.

Affinity resins as new tools for identifying target proteins of ascorbic acid.

PubMed

Iwaoka, Yuji; Nishino, Kohei; Ishikawa, Takahiro; Ito, Hideyuki; Sawa, Yoshihiro; Tai, Akihiro

2018-02-12

l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.

Functional kinomics identifies candidate therapeutic targets in head and neck cancer

PubMed Central

Moser, Russell; Xu, Chang; Kao, Michael; Annis, James; Lerma, Luisa Angelica; Schaupp, Christopher M.; Gurley, Kay E.; Jang, In Sock; Biktasova, Asel; Yarbrough, Wendell G.; Margolin, Adam A.; Grandori, Carla; Kemp, Christopher J.; Méndez, Eduardo

2014-01-01

Purpose To identify novel therapeutic drug targets for p53 mutant head and neck squamous cell carcinoma (HNSCC). Experimental Design RNAi kinome viability screens were performed on HNSCC cells including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19Arf. Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was utilized to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets utilizing multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition utilizing a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Results Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2/M cell cycle checkpoint, SFK, PI3K and FAK pathways. RNAi mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53 mutant HNSCC xenograft model. Conclusions WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. PMID:25125259

Functional kinomics identifies candidate therapeutic targets in head and neck cancer.

PubMed

Moser, Russell; Xu, Chang; Kao, Michael; Annis, James; Lerma, Luisa Angelica; Schaupp, Christopher M; Gurley, Kay E; Jang, In Sock; Biktasova, Asel; Yarbrough, Wendell G; Margolin, Adam A; Grandori, Carla; Kemp, Christopher J; Méndez, Eduardo

2014-08-15

To identify novel therapeutic drug targets for p53-mutant head and neck squamous cell carcinoma (HNSCC). RNAi kinome viability screens were performed on HNSCC cells, including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19(Arf). Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was used to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets using multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition using a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2-M cell-cycle checkpoint, SFK, PI3K, and FAK pathways. RNAi-mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53-mutant HNSCC xenograft model. WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. ©2014 American Association for Cancer Research.

Targeting putative mu opioid/metabotropic glutamate receptor-5 heteromers produces potent antinociception in a chronic murine bone cancer model.

PubMed

Smeester, Branden A; Lunzer, Mary M; Akgün, Eyup; Beitz, Alvin J; Portoghese, Philip S

2014-11-15

The therapeutic management of chronic pain associated with many cancers is problematic due to the development of tolerance and other adverse effects during the disease progression. Recently we reported on a bivalent ligand (MMG22) containing both mu agonist and mGluR5 antagonist pharmacophores that produced potent antinociception in mice with LPS-induced acute inflammatory pain via a putative MOR-mGluR5 heteromer. In the present study we have investigated the antinociception of MMG22 in a mouse model of bone cancer pain to determine its effectiveness in reducing this type of chronic nociception. There was a 572-fold increase in the potency of MMG22 over a period of 3-21 days that correlated with the progressive increase in hyperalgesia induced by bone tumor growth following implantation of fibrosarcoma cells in mice. The enhancement of antinociception with the progression of the cancer is possibly due to inhibition of NMDA receptor-mediated hyperalgesia via antagonism of mGluR5 and concomitant activation of MOR by the MMG22-occupied heteromer. Notably, MMG22 was 3.6-million-fold more potent than morphine at PID 21. Since MMG22 exhibited a 250,000-times greater potency than that of a mixture of the mu opioid (M19) agonist and mGluR5 antagonist (MG20) monovalent ligands, the data suggest that targeting the putative MOR-mGluR5 heteromer is far superior to univalent interaction with receptors in reducing tumor-induced nociception. In view of the high potency, long duration (>24h) of action and minimal side effects, MMG22 has the potential to be a superior pharmacological agent than morphine and other opiates in the treatment of chronic cancer pain and to serve as a novel pharmacologic tool. Copyright © 2014 Elsevier B.V. All rights reserved.

Analyses of germline variants associated with ovarian cancer survival identify functional candidates at the 1q22 and 19p12 outcome loci

PubMed Central

Glubb, Dylan M.; Johnatty, Sharon E.; Quinn, Michael C.J.; O’Mara, Tracy A.; Tyrer, Jonathan P.; Gao, Bo; Fasching, Peter A.; Beckmann, Matthias W.; Lambrechts, Diether; Vergote, Ignace; Velez Edwards, Digna R.; Beeghly-Fadiel, Alicia; Benitez, Javier; Garcia, Maria J.; Goodman, Marc T.; Thompson, Pamela J.; Dörk, Thilo; Dürst, Matthias; Modungo, Francesmary; Moysich, Kirsten; Heitz, Florian; du Bois, Andreas; Pfisterer, Jacobus; Hillemanns, Peter; Karlan, Beth Y.; Lester, Jenny; Goode, Ellen L.; Cunningham, Julie M.; Winham, Stacey J.; Larson, Melissa C.; McCauley, Bryan M.; Kjær, Susanne Krüger; Jensen, Allan; Schildkraut, Joellen M.; Berchuck, Andrew; Cramer, Daniel W.; Terry, Kathryn L.; Salvesen, Helga B.; Bjorge, Line; Webb, Penny M.; Grant, Peter; Pejovic, Tanja; Moffitt, Melissa; Hogdall, Claus K.; Hogdall, Estrid; Paul, James; Glasspool, Rosalind; Bernardini, Marcus; Tone, Alicia; Huntsman, David; Woo, Michelle; Group, AOCS; deFazio, Anna; Kennedy, Catherine J.; Pharoah, Paul D.P.; MacGregor, Stuart; Chenevix-Trench, Georgia

2017-01-01

We previously identified associations with ovarian cancer outcome at five genetic loci. To identify putatively causal genetic variants and target genes, we prioritized two ovarian outcome loci (1q22 and 19p12) for further study. Bioinformatic and functional genetic analyses indicated that MEF2D and ZNF100 are targets of candidate outcome variants at 1q22 and 19p12, respectively. At 19p12, the chromatin interaction of a putative regulatory element with the ZNF100 promoter region correlated with candidate outcome variants. At 1q22, putative regulatory elements enhanced MEF2D promoter activity and haplotypes containing candidate outcome variants modulated these effects. In a public dataset, MEF2D and ZNF100 expression were both associated with ovarian cancer progression-free or overall survival time. In an extended set of 6,162 epithelial ovarian cancer patients, we found that functional candidates at the 1q22 and 19p12 loci, as well as other regional variants, were nominally associated with patient outcome; however, no associations reached our threshold for statistical significance (p<1×10-5). Larger patient numbers will be needed to convincingly identify any true associations at these loci. PMID:29029385

SNPs in putative regulatory regions identified by human mouse comparative sequencing and transcription factor binding site data

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

Banerjee, Poulabi; Bahlo, Melanie; Schwartz, Jody R.

2002-01-01

Genome wide disease association analysis using SNPs is being explored as a method for dissecting complex genetic traits and a vast number of SNPs have been generated for this purpose. As there are cost and throughput limitations of genotyping large numbers of SNPs and statistical issues regarding the large number of dependent tests on the same data set, to make association analysis practical it has been proposed that SNPs should be prioritized based on likely functional importance. The most easily identifiable functional SNPs are coding SNPs (cSNPs) and accordingly cSNPs have been screened in a number of studies. SNPs inmore » gene regulatory sequences embedded in noncoding DNA are another class of SNPs suggested for prioritization due to their predicted quantitative impact on gene expression. The main challenge in evaluating these SNPs, in contrast to cSNPs is a lack of robust algorithms and databases for recognizing regulatory sequences in noncoding DNA. Approaches that have been previously used to delineate noncoding sequences with gene regulatory activity include cross-species sequence comparisons and the search for sequences recognized by transcription factors. We combined these two methods to sift through mouse human genomic sequences to identify putative gene regulatory elements and subsequently localized SNPs within these sequences in a 1 Megabase (Mb) region of human chromosome 5q31, orthologous to mouse chromosome 11 containing the Interleukin cluster.« less

The putative drug efflux systems of the Bacillus cereus group

PubMed Central

Elbourne, Liam D. H.; Vörös, Aniko; Kroeger, Jasmin K.; Simm, Roger; Tourasse, Nicolas J.; Finke, Sarah; Henderson, Peter J. F.; Økstad, Ole Andreas; Paulsen, Ian T.; Kolstø, Anne-Brit

2017-01-01

The Bacillus cereus group of bacteria includes seven closely related species, three of which, B. anthracis, B. cereus and B. thuringiensis, are pathogens of humans, animals and/or insects. Preliminary investigations into the transport capabilities of different bacterial lineages suggested that genes encoding putative efflux systems were unusually abundant in the B. cereus group compared to other bacteria. To explore the drug efflux potential of the B. cereus group all putative efflux systems were identified in the genomes of prototypical strains of B. cereus, B. anthracis and B. thuringiensis using our Transporter Automated Annotation Pipeline. More than 90 putative drug efflux systems were found within each of these strains, accounting for up to 2.7% of their protein coding potential. Comparative analyses demonstrated that the efflux systems are highly conserved between these species; 70–80% of the putative efflux pumps were shared between all three strains studied. Furthermore, 82% of the putative efflux system proteins encoded by the prototypical B. cereus strain ATCC 14579 (type strain) were found to be conserved in at least 80% of 169 B. cereus group strains that have high quality genome sequences available. However, only a handful of these efflux pumps have been functionally characterized. Deletion of individual efflux pump genes from B. cereus typically had little impact to drug resistance phenotypes or the general fitness of the strains, possibly because of the large numbers of alternative efflux systems that may have overlapping substrate specificities. Therefore, to gain insight into the possible transport functions of efflux systems in B. cereus, we undertook large-scale qRT-PCR analyses of efflux pump gene expression following drug shocks and other stress treatments. Clustering of gene expression changes identified several groups of similarly regulated systems that may have overlapping drug resistance functions. In this article we review current

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors

PubMed Central

Lopez, David; Ribeiro, Sébastien; Label, Philippe; Fumanal, Boris; Venisse, Jean-Stéphane; Kohler, Annegret; de Oliveira, Ricardo R.; Labutti, Kurt; Lipzen, Anna; Lail, Kathleen; Bauer, Diane; Ohm, Robin A.; Barry, Kerrie W.; Spatafora, Joseph; Grigoriev, Igor V.; Martin, Francis M.; Pujade-Renaud, Valérie

2018-01-01

Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species (Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca, and Botrosphaeria dothidea) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector-based classification

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors.

PubMed

Lopez, David; Ribeiro, Sébastien; Label, Philippe; Fumanal, Boris; Venisse, Jean-Stéphane; Kohler, Annegret; de Oliveira, Ricardo R; Labutti, Kurt; Lipzen, Anna; Lail, Kathleen; Bauer, Diane; Ohm, Robin A; Barry, Kerrie W; Spatafora, Joseph; Grigoriev, Igor V; Martin, Francis M; Pujade-Renaud, Valérie

2018-01-01

Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species ( Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca , and Botrosphaeria dothidea ) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector

Emerging Putative Associations between Non-Coding RNAs and Protein-Coding Genes in Neuropathic Pain: Added Value from Reusing Microarray Data

PubMed Central

Raju, Hemalatha B.; Tsinoremas, Nicholas F.; Capobianco, Enrico

2016-01-01

Regeneration of injured nerves is likely occurring in the peripheral nervous system, but not in the central nervous system. Although protein-coding gene expression has been assessed during nerve regeneration, little is currently known about the role of non-coding RNAs (ncRNAs). This leaves open questions about the potential effects of ncRNAs at transcriptome level. Due to the limited availability of human neuropathic pain (NP) data, we have identified the most comprehensive time-course gene expression profile referred to sciatic nerve (SN) injury and studied in a rat model using two neuronal tissues, namely dorsal root ganglion (DRG) and SN. We have developed a methodology to identify differentially expressed bioentities starting from microarray probes and repurposing them to annotate ncRNAs, while analyzing the expression profiles of protein-coding genes. The approach is designed to reuse microarray data and perform first profiling and then meta-analysis through three main steps. First, we used contextual analysis to identify what we considered putative or potential protein-coding targets for selected ncRNAs. Relevance was therefore assigned to differential expression of neighbor protein-coding genes, with neighborhood defined by a fixed genomic distance from long or antisense ncRNA loci, and of parental genes associated with pseudogenes. Second, connectivity among putative targets was used to build networks, in turn useful to conduct inference at interactomic scale. Last, network paths were annotated to assess relevance to NP. We found significant differential expression in long-intergenic ncRNAs (32 lincRNAs in SN and 8 in DRG), antisense RNA (31 asRNA in SN and 12 in DRG), and pseudogenes (456 in SN and 56 in DRG). In particular, contextual analysis centered on pseudogenes revealed some targets with known association to neurodegeneration and/or neurogenesis processes. While modules of the olfactory receptors were clearly identified in protein�

Using Click Chemistry to Identify Potential Drug Targets in Plasmodium

DTIC Science & Technology

2015-04-01

step of the Plasmodium mammalian cycle . Inhibiting this step can block malaria at an early step. However, few anti-malarials target liver infection...points in the life cycle of malaria parasites. PLoS Biol 12: e1001806. 2. Falae A, Combe A, Amaladoss A, Carvalho T, Menard R, et al. (2010) Role of...AWARD NUMBER: W81XWH-13-1-0429 TITLE: Using "Click Chemistry" to Identify Potential Drug Targets in Plasmodium PRINCIPAL INVESTIGATOR: Dr. Purnima

Putative Prostate Cancer Risk SNP in an Androgen Receptor‐Binding Site of the Melanophilin Gene Illustrates Enrichment of Risk SNPs in Androgen Receptor Target Sites

PubMed Central

Bu, Huajie; Narisu, Narisu; Schlick, Bettina; Rainer, Johannes; Manke, Thomas; Schäfer, Georg; Pasqualini, Lorenza; Chines, Peter; Schweiger, Michal R.; Fuchsberger, Christian

2015-01-01

ABSTRACT Genome‐wide association studies have identified genomic loci, whose single‐nucleotide polymorphisms (SNPs) predispose to prostate cancer (PCa). However, the mechanisms of most of these variants are largely unknown. We integrated chromatin‐immunoprecipitation‐coupled sequencing and microarray expression profiling in TMPRSS2‐ERG gene rearrangement positive DUCaP cells with the GWAS PCa risk SNPs catalog to identify disease susceptibility SNPs localized within functional androgen receptor‐binding sites (ARBSs). Among the 48 GWAS index risk SNPs and 3,917 linked SNPs, 80 were found located in ARBSs. Of these, rs11891426:T>G in an intron of the melanophilin gene (MLPH) was within a novel putative auxiliary AR‐binding motif, which is enriched in the neighborhood of canonical androgen‐responsive elements. T→G exchange attenuated the transcriptional activity of the ARBS in an AR reporter gene assay. The expression of MLPH in primary prostate tumors was significantly lower in those with the G compared with the T allele and correlated significantly with AR protein. Higher melanophilin level in prostate tissue of patients with a favorable PCa risk profile points out a tumor‐suppressive effect. These results unravel a hidden link between AR and a functional putative PCa risk SNP, whose allele alteration affects androgen regulation of its host gene MLPH. PMID:26411452

Identifying protein kinase target preferences using mass spectrometry

PubMed Central

Douglass, Jacqueline; Gunaratne, Ruwan; Bradford, Davis; Saeed, Fahad; Hoffert, Jason D.; Steinbach, Peter J.; Pisitkun, Trairak

2012-01-01

A general question in molecular physiology is how to identify candidate protein kinases corresponding to a known or hypothetical phosphorylation site in a protein of interest. It is generally recognized that the amino acid sequence surrounding the phosphorylation site provides information that is relevant to identification of the cognate protein kinase. Here, we present a mass spectrometry-based method for profiling the target specificity of a given protein kinase as well as a computational tool for the calculation and visualization of the target preferences. The mass spectrometry-based method identifies sites phosphorylated in response to in vitro incubation of protein mixtures with active recombinant protein kinases followed by standard phosphoproteomic methodologies. The computational tool, called “PhosphoLogo,” uses an information-theoretic algorithm to calculate position-specific amino acid preferences and anti-preferences from the mass-spectrometry data (http://helixweb.nih.gov/PhosphoLogo/). The method was tested using protein kinase A (catalytic subunit α), revealing the well-known preference for basic amino acids in positions −2 and −3 relative to the phosphorylated amino acid. It also provides evidence for a preference for amino acids with a branched aliphatic side chain in position +1, a finding compatible with known crystal structures of protein kinase A. The method was also employed to profile target preferences and anti-preferences for 15 additional protein kinases with potential roles in regulation of epithelial transport: CK2, p38, AKT1, SGK1, PKCδ, CaMK2δ, DAPK1, MAPKAPK2, PKD3, PIM1, OSR1, STK39/SPAK, GSK3β, Wnk1, and Wnk4. PMID:22723110

Genomic Target Database (GTD): A database of potential targets in human pathogenic bacteria

PubMed Central

Barh, Debmalya; Kumar, Anil; Misra, Amarendra Narayana

2009-01-01

A Genomic Target Database (GTD) has been developed having putative genomic drug targets for human bacterial pathogens. The selected pathogens are either drug resistant or vaccines are yet to be developed against them. The drug targets have been identified using subtractive genomics approaches and these are subsequently classified into Drug targets in pathogen specific unique metabolic pathways,Drug targets in host-pathogen common metabolic pathways, andMembrane localized drug targets. HTML code is used to link each target to its various properties and other available public resources. Essential resources and tools for subtractive genomic analysis, sub-cellular localization, vaccine and drug designing are also mentioned. To the best of authors knowledge, no such database (DB) is presently available that has listed metabolic pathways and membrane specific genomic drug targets based on subtractive genomics. Listed targets in GTD are readily available resource in developing drug and vaccine against the respective pathogen, its subtypes, and other family members. Currently GTD contains 58 drug targets for four pathogens. Shortly, drug targets for six more pathogens will be listed. Availability GTD is available at IIOAB website http://www.iioab.webs.com/GTD.htm. It can also be accessed at http://www.iioabdgd.webs.com.GTD is free for academic research and non-commercial use only. Commercial use is strictly prohibited without prior permission from IIOAB. PMID:20011153

Integrative biology approach identifies cytokine targeting strategies for psoriasis.

PubMed

Perera, Gayathri K; Ainali, Chrysanthi; Semenova, Ekaterina; Hundhausen, Christian; Barinaga, Guillermo; Kassen, Deepika; Williams, Andrew E; Mirza, Muddassar M; Balazs, Mercedesz; Wang, Xiaoting; Rodriguez, Robert Sanchez; Alendar, Andrej; Barker, Jonathan; Tsoka, Sophia; Ouyang, Wenjun; Nestle, Frank O

2014-02-12

Cytokines are critical checkpoints of inflammation. The treatment of human autoimmune disease has been revolutionized by targeting inflammatory cytokines as key drivers of disease pathogenesis. Despite this, there exist numerous pitfalls when translating preclinical data into the clinic. We developed an integrative biology approach combining human disease transcriptome data sets with clinically relevant in vivo models in an attempt to bridge this translational gap. We chose interleukin-22 (IL-22) as a model cytokine because of its potentially important proinflammatory role in epithelial tissues. Injection of IL-22 into normal human skin grafts produced marked inflammatory skin changes resembling human psoriasis. Injection of anti-IL-22 monoclonal antibody in a human xenotransplant model of psoriasis, developed specifically to test potential therapeutic candidates, efficiently blocked skin inflammation. Bioinformatic analysis integrating both the IL-22 and anti-IL-22 cytokine transcriptomes and mapping them onto a psoriasis disease gene coexpression network identified key cytokine-dependent hub genes. Using knockout mice and small-molecule blockade, we show that one of these hub genes, the so far unexplored serine/threonine kinase PIM1, is a critical checkpoint for human skin inflammation and potential future therapeutic target in psoriasis. Using in silico integration of human data sets and biological models, we were able to identify a new target in the treatment of psoriasis.

Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches

PubMed Central

Ekland, Eric H.; Schneider, Jessica; Fidock, David A.

2011-01-01

Malarial parasites have evolved resistance to all previously used therapies, and recent evidence suggests emerging resistance to the first-line artemisinins. To identify antimalarials with novel mechanisms of action, we have developed a high-throughput screen targeting the apicoplast organelle of Plasmodium falciparum. Antibiotics known to interfere with this organelle, such as azithromycin, exhibit an unusual phenotype whereby the progeny of drug-treated parasites die. Our screen exploits this phenomenon by assaying for “delayed death” compounds that exhibit a higher potency after two cycles of intraerythrocytic development compared to one. We report a primary assay employing parasites with an integrated copy of a firefly luciferase reporter gene and a secondary flow cytometry-based assay using a nucleic acid stain paired with a mitochondrial vital dye. Screening of the U.S. National Institutes of Health Clinical Collection identified known and novel antimalarials including kitasamycin. This inexpensive macrolide, used for agricultural applications, exhibited an in vitro IC50 in the 50 nM range, comparable to the 30 nM activity of our control drug, azithromycin. Imaging and pharmacologic studies confirmed kitasamycin action against the apicoplast, and in vivo activity was observed in a murine malaria model. These assays provide the foundation for high-throughput campaigns to identify novel chemotypes for combination therapies to treat multidrug-resistant malaria.—Ekland, E. H., Schneider, J., Fidock, D. A. Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches. PMID:21746861

Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms.

PubMed Central

Maeda, Kenji; Finnie, Christine; Svensson, Birte

2004-01-01

Barley thioredoxin h isoforms HvTrxh1 and HvTrxh2 differ in temporal and spatial distribution and in kinetic properties. Target proteins of HvTrxh1 and HvTrxh2 were identified in mature seeds and in seeds after 72 h of germination. Improvement of the established method for identification of thioredoxin-targeted proteins based on two-dimensional electrophoresis and fluorescence labelling of thiol groups was achieved by application of a highly sensitive Cy5 maleimide dye and large-format two-dimensional gels, resulting in a 10-fold increase in the observed number of labelled protein spots. The technique also provided information about accessible thiol groups in the proteins identified in the barley seed proteome. In total, 16 different putative target proteins were identified from 26 spots using tryptic in-gel digestion, matrix-assisted laser-desorption ionization-time-of-flight MS and database search. HvTrxh1 and HvTrxh2 were shown to have similar target specificity. Barley alpha-amylase/subtilisin inhibitor, previously demonstrated to be reduced by both HvTrxh1 and HvTrxh2, was among the identified target proteins, confirming the suitability of the method. Several alpha-amylase/trypsin inhibitors, some of which are already known as target proteins of thioredoxin h, and cyclophilin known as a target protein of m-type thioredoxin were also identified. Lipid transfer protein, embryospecific protein, three chitinase isoenzymes, a single-domain glyoxalase-like protein and superoxide dismutase were novel identifications of putative target proteins, suggesting new physiological roles of thioredoxin h in barley seeds. PMID:14636158

Identifying the cellular targets of natural products using T7 phage display.

PubMed

Piggott, Andrew M; Karuso, Peter

2016-05-04

Covering: up to the end of 2015While Nature continues to deliver a myriad of potent and structurally diverse biologically active small molecules, the cellular targets and modes of action of these natural products are rarely identified, significantly hindering their development as new chemotherapeutic agents. This article provides an introductory tutorial on the use of T7 phage display as a tool to rapidly identify the cellular targets of natural products and is aimed specifically at natural products chemists who may have only limited experience in molecular biology. A brief overview of T7 phage display is provided, including its strengths, weaknesses, and the type of problems that can and cannot be tackled with this technology. Affinity probe construction is reviewed, including linker design and natural product derivatisation strategies. A detailed description of the T7 phage biopanning procedure is provided, with valuable tips for optimising each step in the process, as well as advice for identifying and avoiding the most commonly encountered challenges and pitfalls along the way. Finally, a brief discussion is provided on techniques for validating the cellular targets identified using T7 phage display.

Genomes2Drugs: Identifies Target Proteins and Lead Drugs from Proteome Data

PubMed Central

Toomey, David; Hoppe, Heinrich C.; Brennan, Marian P.; Nolan, Kevin B.; Chubb, Anthony J.

2009-01-01

Background Genome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials. Methodology/Principal Findings To solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i) homologous to previously crystallized proteins or (ii) targets of known drugs, but are (iii) not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins. Conclusions/Significance Genomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under ‘change-of-application’ patents. PMID:19593435

Expression Profiling Identifies Klf15 as a Glucocorticoid Target That Regulates Airway Hyperresponsiveness

PubMed Central

Masuno, Kiriko; Haldar, Saptarsi M.; Jeyaraj, Darwin; Mailloux, Christina M.; Huang, Xiaozhu; Panettieri, Rey A.; Jain, Mukesh K.

2011-01-01

Glucocorticoids (GCs), which activate GC receptor (GR) signaling and thus modulate gene expression, are widely used to treat asthma. GCs exert their therapeutic effects in part through modulating airway smooth muscle (ASM) structure and function. However, the effects of genes that are regulated by GCs on airway function are not fully understood. We therefore used transcription profiling to study the effects of a potent GC, dexamethasone, on human ASM (HASM) gene expression at 4 and 24 hours. After 24 hours of dexamethasone treatment, nearly 7,500 genes had statistically distinguishable changes in expression; quantitative PCR validation of a 40-gene subset of putative GR-regulated genes in 6 HASM cell lines suggested that the early transcriptional targets of GR signaling are similar in independent HASM lines. Gene ontology analysis implicated GR targets in controlling multiple aspects of ASM function. One GR-regulated gene, the transcription factor, Kruppel-like factor 15 (Klf15), was already known to modulate vascular smooth and cardiac muscle function, but had no known role in the lung. We therefore analyzed the pulmonary phenotype of Klf15−/− mice after ovalbumin sensitization and challenge. We found diminished airway responses to acetylcholine in ovalbumin-challenged Klf15−/− mice without a significant change in the induction of asthmatic inflammation. In cultured cells, overexpression of Klf15 reduced proliferation of HASM cells, whereas apoptosis in Klf15−/− murine ASM cells was increased. Together, these results further characterize the GR-regulated gene network in ASM and establish a novel role for the GR target, Klf15, in modulating airway function. PMID:21257922

Transcriptome-Wide Survey and Expression Profile Analysis of Putative Chrysanthemum HD-Zip I and II Genes

PubMed Central

Song, Aiping; Li, Peiling; Xin, Jingjing; Chen, Sumei; Zhao, Kunkun; Wu, Dan; Fan, Qingqing; Gao, Tianwei; Chen, Fadi; Guan, Zhiyong

2016-01-01

The homeodomain-leucine zipper (HD-Zip) transcription factor family is a key transcription factor family and unique to the plant kingdom. It consists of a homeodomain and a leucine zipper that serve in combination as a dimerization motif. The family can be classified into four subfamilies, and these subfamilies participate in the development of hormones and mediation of hormone action and are involved in plant responses to environmental conditions. However, limited information on this gene family is available for the important chrysanthemum ornamental species (Chrysanthemum morifolium). Here, we characterized 17 chrysanthemum HD-Zip genes based on transcriptome sequences. Phylogenetic analyses revealed that 17 CmHB genes were distributed in the HD-Zip subfamilies I and II and identified two pairs of putative orthologous proteins in Arabidopsis and chrysanthemum and four pairs of paralogous proteins in chrysanthemum. The software MEME was used to identify 7 putative motifs with E values less than 1e-3 in the chrysanthemum HD-Zip factors, and they can be clearly classified into two groups based on the composition of the motifs. A bioinformatics analysis predicted that 8 CmHB genes could be targeted by 10 miRNA families, and the expression of these 17 genes in response to phytohormone treatments and abiotic stresses was characterized. The results presented here will promote research on the various functions of the HD-Zip gene family members in plant hormones and stress responses. PMID:27196930

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.

Comparative proteomics of cerebrospinal fluid reveals a predictive model for differential diagnosis of pneumococcal, meningococcal, and enteroviral meningitis, and novel putative therapeutic targets

PubMed Central

2015-01-01

Background Meningitis is the inflammation of the meninges in response to infection or chemical agents. While aseptic meningitis, most frequently caused by enteroviruses, is usually benign with a self-limiting course, bacterial meningitis remains associated with high morbidity and mortality rates, despite advances in antimicrobial therapy and intensive care. Fast and accurate differential diagnosis is crucial for assertive choice of the appropriate therapeutic approach for each form of meningitis. Methods We used 2D-PAGE and mass spectrometry to identify the cerebrospinal fluid proteome specifically related to the host response to pneumococcal, meningococcal, and enteroviral meningitis. The disease-specific proteome signatures were inspected by pathway analysis. Results Unique cerebrospinal fluid proteome signatures were found to the three aetiological forms of meningitis investigated, and a qualitative predictive model with four protein markers was developed for the differential diagnosis of these diseases. Nevertheless, pathway analysis of the disease-specific proteomes unveiled that Kallikrein-kinin system may play a crucial role in the pathophysiological mechanisms leading to brain damage in bacterial meningitis. Proteins taking part in this cellular process are proposed as putative targets to novel adjunctive therapies. Conclusions Comparative proteomics of cerebrospinal fluid disclosed candidate biomarkers, which were combined in a qualitative and sequential predictive model with potential to improve the differential diagnosis of pneumococcal, meningococcal and enteroviral meningitis. Moreover, we present the first evidence of the possible implication of Kallikrein-kinin system in the pathophysiology of bacterial meningitis. PMID:26040285

Comparative proteomics of cerebrospinal fluid reveals a predictive model for differential diagnosis of pneumococcal, meningococcal, and enteroviral meningitis, and novel putative therapeutic targets.

PubMed

Cordeiro, Ana Paula; Silva Pereira, Rosiane Aparecida; Chapeaurouge, Alex; Coimbra, Clarice Semião; Perales, Jonas; Oliveira, Guilherme; Sanchez Candiani, Talitah Michel; Coimbra, Roney Santos

2015-01-01

Meningitis is the inflammation of the meninges in response to infection or chemical agents. While aseptic meningitis, most frequently caused by enteroviruses, is usually benign with a self-limiting course, bacterial meningitis remains associated with high morbidity and mortality rates, despite advances in antimicrobial therapy and intensive care. Fast and accurate differential diagnosis is crucial for assertive choice of the appropriate therapeutic approach for each form of meningitis. We used 2D-PAGE and mass spectrometry to identify the cerebrospinal fluid proteome specifically related to the host response to pneumococcal, meningococcal, and enteroviral meningitis. The disease-specific proteome signatures were inspected by pathway analysis. Unique cerebrospinal fluid proteome signatures were found to the three aetiological forms of meningitis investigated, and a qualitative predictive model with four protein markers was developed for the differential diagnosis of these diseases. Nevertheless, pathway analysis of the disease-specific proteomes unveiled that Kallikrein-kinin system may play a crucial role in the pathophysiological mechanisms leading to brain damage in bacterial meningitis. Proteins taking part in this cellular process are proposed as putative targets to novel adjunctive therapies. Comparative proteomics of cerebrospinal fluid disclosed candidate biomarkers, which were combined in a qualitative and sequential predictive model with potential to improve the differential diagnosis of pneumococcal, meningococcal and enteroviral meningitis. Moreover, we present the first evidence of the possible implication of Kallikrein-kinin system in the pathophysiology of bacterial meningitis.

Combined expressional analysis, bioinformatics and targeted proteomics identify new potential therapeutic targets in glioblastoma stem cells.

PubMed

Stangeland, Biljana; Mughal, Awais A; Grieg, Zanina; Sandberg, Cecilie Jonsgar; Joel, Mrinal; Nygård, Ståle; Meling, Torstein; Murrell, Wayne; Vik Mo, Einar O; Langmoen, Iver A

2015-09-22

Glioblastoma (GBM) is both the most common and the most lethal primary brain tumor. It is thought that GBM stem cells (GSCs) are critically important in resistance to therapy. Therefore, there is a strong rationale to target these cells in order to develop new molecular therapies.To identify molecular targets in GSCs, we compared gene expression in GSCs to that in neural stem cells (NSCs) from the adult human brain, using microarrays. Bioinformatic filtering identified 20 genes (PBK/TOPK, CENPA, KIF15, DEPDC1, CDC6, DLG7/DLGAP5/HURP, KIF18A, EZH2, HMMR/RHAMM/CD168, NOL4, MPP6, MDM1, RAPGEF4, RHBDD1, FNDC3B, FILIP1L, MCC, ATXN7L4/ATXN7L1, P2RY5/LPAR6 and FAM118A) that were consistently expressed in GSC cultures and consistently not expressed in NSC cultures. The expression of these genes was confirmed in clinical samples (TCGA and REMBRANDT). The first nine genes were highly co-expressed in all GBM subtypes and were part of the same protein-protein interaction network. Furthermore, their combined up-regulation correlated negatively with patient survival in the mesenchymal GBM subtype. Using targeted proteomics and the COGNOSCENTE database we linked these genes to GBM signalling pathways.Nine genes: PBK, CENPA, KIF15, DEPDC1, CDC6, DLG7, KIF18A, EZH2 and HMMR should be further explored as targets for treatment of GBM.

Finding off-targets, biological pathways, and target diseases for chymase inhibitors via structure-based systems biology approach.

PubMed

Arooj, Mahreen; Sakkiah, Sugunadevi; Cao, Guang Ping; Kim, Songmi; Arulalapperumal, Venkatesh; Lee, Keun Woo

2015-07-01

Off-target binding connotes the binding of a small molecule of therapeutic significance to a protein target in addition to the primary target for which it was proposed. Progressively such off-targeting is emerging to be regular practice to reveal side effects. Chymase is an enzyme of hydrolase class that catalyzes hydrolysis of peptide bonds. A link between heart failure and chymase is ascribed, and a chymase inhibitor is in clinical phase II for treatment of heart failure. However, the underlying mechanisms of the off-target effects of human chymase inhibitors are still unclear. Here, we develop a robust computational strategy that is applicable to any enzyme system and that allows the prediction of drug effects on biological processes. Putative off-targets for chymase inhibitors were identified through various structural and functional similarity analyses along with molecular docking studies. Finally, literature survey was performed to incorporate these off-targets into biological pathways and to establish links between pathways and particular adverse effects. Off-targets of chymase inhibitors are linked to various biological pathways such as classical and lectin pathways of complement system, intrinsic and extrinsic pathways of coagulation cascade, and fibrinolytic system. Tissue kallikreins, granzyme M, neutrophil elastase, and mesotrypsin are also identified as off-targets. These off-targets and their associated pathways are elucidated for the effects of inflammation, cancer, hemorrhage, thrombosis, and central nervous system diseases (Alzheimer's disease). Prospectively, our approach is helpful not only to better understand the mechanisms of chymase inhibitors but also for drug repurposing exercises to find novel uses for these inhibitors. © 2014 Wiley Periodicals, Inc.

Cis-regulatory element based targeted gene finding: genome-wide identification of abscisic acid- and abiotic stress-responsive genes in Arabidopsis thaliana.

PubMed

Zhang, Weixiong; Ruan, Jianhua; Ho, Tuan-Hua David; You, Youngsook; Yu, Taotao; Quatrano, Ralph S

2005-07-15

A fundamental problem of computational genomics is identifying the genes that respond to certain endogenous cues and environmental stimuli. This problem can be referred to as targeted gene finding. Since gene regulation is mainly determined by the binding of transcription factors and cis-regulatory DNA sequences, most existing gene annotation methods, which exploit the conservation of open reading frames, are not effective in finding target genes. A viable approach to targeted gene finding is to exploit the cis-regulatory elements that are known to be responsible for the transcription of target genes. Given such cis-elements, putative target genes whose promoters contain the elements can be identified. As a case study, we apply the above approach to predict the genes in model plant Arabidopsis thaliana which are inducible by a phytohormone, abscisic acid (ABA), and abiotic stress, such as drought, cold and salinity. We first construct and analyze two ABA specific cis-elements, ABA-responsive element (ABRE) and its coupling element (CE), in A.thaliana, based on their conservation in rice and other cereal plants. We then use the ABRE-CE module to identify putative ABA-responsive genes in A.thaliana. Based on RT-PCR verification and the results from literature, this method has an accuracy rate of 67.5% for the top 40 predictions. The cis-element based targeted gene finding approach is expected to be widely applicable since a large number of cis-elements in many species are available.

Two types of putative preneoplastic lesions identified by hexosaminidase activity in whole-mounts of colons from F344 rats treated with carcinogen.

PubMed

Pretlow, T P; O'Riordan, M A; Spancake, K M; Pretlow, T G

1993-06-01

Previous studies identified as putative preneoplastic lesions 1) enzyme-altered foci in sections of methacrylate-embedded colon and 2) aberrant crypts in methylene blue-stained unembedded (whole-mount) colon and established that aberrant crypts embedded in methacrylate had enzyme alterations. We have now studied histochemically demonstrable hexosaminidase activity in unembedded or whole-mount preparations of colons from carcinogen-treated rats. These preparations have revealed two populations of crypts that are enzyme-altered: those that are morphologically altered or aberrant and those that are morphologically normal. Both populations can be quantified rigorously in less than an hour with whole-mount preparations reacted for hexosaminidase. The demonstration of phenotypic characteristics with histochemical techniques in whole-mount preparations should have wide applicability to functional studies in many normal and diseased tissues.

Two types of putative preneoplastic lesions identified by hexosaminidase activity in whole-mounts of colons from F344 rats treated with carcinogen.

PubMed Central

Pretlow, T. P.; O'Riordan, M. A.; Spancake, K. M.; Pretlow, T. G.

1993-01-01

Previous studies identified as putative preneoplastic lesions 1) enzyme-altered foci in sections of methacrylate-embedded colon and 2) aberrant crypts in methylene blue-stained unembedded (whole-mount) colon and established that aberrant crypts embedded in methacrylate had enzyme alterations. We have now studied histochemically demonstrable hexosaminidase activity in unembedded or whole-mount preparations of colons from carcinogen-treated rats. These preparations have revealed two populations of crypts that are enzyme-altered: those that are morphologically altered or aberrant and those that are morphologically normal. Both populations can be quantified rigorously in less than an hour with whole-mount preparations reacted for hexosaminidase. The demonstration of phenotypic characteristics with histochemical techniques in whole-mount preparations should have wide applicability to functional studies in many normal and diseased tissues. Images Figure 1 PMID:8506941

Evidence that the Malaria Parasite Plasmodium falciparum Putative Rhoptry Protein 2 Localizes to the Golgi Apparatus throughout the Erythrocytic Cycle.

PubMed

Hallée, Stéphanie; Richard, Dave

2015-01-01

Invasion of a red blood cell by Plasmodium falciparum merozoites is an essential step in the malaria lifecycle. Several of the proteins involved in this process are stored in the apical complex of the merozoite, a structure containing secretory organelles that are released at specific times during invasion. The molecular players involved in erythrocyte invasion thus represent potential key targets for both therapeutic and vaccine-based strategies to block parasite development. In our quest to identify and characterize new effectors of invasion, we investigated the P. falciparum homologue of a P. berghei protein putatively localized to the rhoptries, the Putative rhoptry protein 2 (PbPRP2). We show that in P. falciparum, the protein colocalizes extensively with the Golgi apparatus across the asexual erythrocytic cycle. Furthermore, imaging of merozoites caught at different times during invasion show that PfPRP2 is not secreted during the process instead staying associated with the Golgi apparatus. Our evidence therefore suggests that PfPRP2 is a Golgi protein and that it is likely not a direct effector in the process of merozoite invasion.

Known and putative mechanisms of resistance to EGFR targeted therapies in NSCLC patients with EGFR mutations—a review

PubMed Central

Stewart, Erin L.; Tan, Samuel Zhixing; Liu, Geoffrey

2015-01-01

Lung cancer is the leading cause of cancer related deaths in Canada with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Tumor characterization can identify cancer-driving mutations as treatment targets. One of the most successful examples of cancer targeted therapy is inhibition of mutated epidermal growth factor receptor (EGFR), which occurs in ~10-30% of NSCLC patients. While this treatment has benefited many patients with activating EGFR mutations, almost all who initially benefited will eventually acquire resistance. Approximately 50% of cases of acquired resistance (AR) are due to a secondary T790M mutation in exon 20 of the EGFR gene; however, many of the remaining mechanisms of resistance are still unknown. Much work has been done to elucidate the remaining mechanisms of resistance. This review aims to highlight both the mechanisms of resistance that have already been identified in patients and potential novel mechanisms identified in preclinical models which have yet to be validated in the patient settings. PMID:25806347

High-throughput identification of off-targets for the mechanistic study of severe adverse drug reactions induced by analgesics

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

Pan, Jian-Bo; Ji, Nan; Pan, Wen

2014-01-01

Drugs may induce adverse drug reactions (ADRs) when they unexpectedly bind to proteins other than their therapeutic targets. Identification of these undesired protein binding partners, called off-targets, can facilitate toxicity assessment in the early stages of drug development. In this study, a computational framework was introduced for the exploration of idiosyncratic mechanisms underlying analgesic-induced severe adverse drug reactions (SADRs). The putative analgesic-target interactions were predicted by performing reverse docking of analgesics or their active metabolites against human/mammal protein structures in a high-throughput manner. Subsequently, bioinformatics analyses were undertaken to identify ADR-associated proteins (ADRAPs) and pathways. Using the pathways and ADRAPsmore » that this analysis identified, the mechanisms of SADRs such as cardiac disorders were explored. For instance, 53 putative ADRAPs and 24 pathways were linked with cardiac disorders, of which 10 ADRAPs were confirmed by previous experiments. Moreover, it was inferred that pathways such as base excision repair, glycolysis/glyconeogenesis, ErbB signaling, calcium signaling, and phosphatidyl inositol signaling likely play pivotal roles in drug-induced cardiac disorders. In conclusion, our framework offers an opportunity to globally understand SADRs at the molecular level, which has been difficult to realize through experiments. It also provides some valuable clues for drug repurposing. - Highlights: • A novel computational framework was developed for mechanistic study of SADRs. • Off-targets of drugs were identified in large scale and in a high-throughput manner. • SADRs like cardiac disorders were systematically explored in molecular networks. • A number of ADR-associated proteins were identified.« less

The Interactions between the Long Non-coding RNA NERDL and Its Target Gene Affect Wood Formation in Populus tomentosa

PubMed Central

Shi, Wan; Quan, Mingyang; Du, Qingzhang; Zhang, Deqiang

2017-01-01

Long non-coding RNAs (lncRNAs) are important regulatory factors for plant growth and development, but little is known about the allelic interactions of lncRNAs with mRNA in perennial plants. Here, we analyzed the interaction of the NERD (Needed for RDR2-independent DNA methylation) Populus tomentosa gene PtoNERD with its putative regulator, the lncRNA NERDL (NERD-related lncRNA), which partially overlaps with the promoter region of this gene. Expression analysis in eight tissues showed a positive correlation between NERDL and PtoNERD (r = 0.62), suggesting that the interaction of NERDL with its putative target might be involved in wood formation. We conducted association mapping in a natural population of P. tomentosa (435 unrelated individuals) to evaluate genetic variation and the interaction of the lncRNA NERDL with PtoNERD. Using additive and dominant models, we identified 30 SNPs (P < 0.01) associated with five tree growth and wood property traits. Each SNP explained 3.90–8.57% of phenotypic variance, suggesting that NERDL and its putative target play a common role in wood formation. Epistasis analysis uncovered nine SNP-SNP association pairs between NERDL and PtoNERD, with an information gain of -7.55 to 2.16%, reflecting the strong interactions between NERDL and its putative target. This analysis provides a powerful method for deciphering the genetic interactions of lncRNAs with mRNA and dissecting the complex genetic network of quantitative traits in trees. PMID:28674544

Identification of novel microRNAs in Hevea brasiliensis and computational prediction of their targets

PubMed Central

2012-01-01

Background Plants respond to external stimuli through fine regulation of gene expression partially ensured by small RNAs. Of these, microRNAs (miRNAs) play a crucial role. They negatively regulate gene expression by targeting the cleavage or translational inhibition of target messenger RNAs (mRNAs). In Hevea brasiliensis, environmental and harvesting stresses are known to affect natural rubber production. This study set out to identify abiotic stress-related miRNAs in Hevea using next-generation sequencing and bioinformatic analysis. Results Deep sequencing of small RNAs was carried out on plantlets subjected to severe abiotic stress using the Solexa technique. By combining the LeARN pipeline, data from the Plant microRNA database (PMRD) and Hevea EST sequences, we identified 48 conserved miRNA families already characterized in other plant species, and 10 putatively novel miRNA families. The results showed the most abundant size for miRNAs to be 24 nucleotides, except for seven families. Several MIR genes produced both 20-22 nucleotides and 23-27 nucleotides. The two miRNA class sizes were detected for both conserved and putative novel miRNA families, suggesting their functional duality. The EST databases were scanned with conserved and novel miRNA sequences. MiRNA targets were computationally predicted and analysed. The predicted targets involved in "responses to stimuli" and to "antioxidant" and "transcription activities" are presented. Conclusions Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs when the complete genome is not yet available. Our study provided additional information for evolutionary studies and revealed potentially specific regulation of the control of redox status in Hevea. PMID:22330773

Structure, Regulation, and Putative Function of the Arginine Deiminase System of Streptococcus suis

PubMed Central

Gruening, Petra; Fulde, Marcus; Valentin-Weigand, Peter; Goethe, Ralph

2006-01-01

Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative β-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite repression

Structure, regulation, and putative function of the arginine deiminase system of Streptococcus suis.

PubMed

Gruening, Petra; Fulde, Marcus; Valentin-Weigand, Peter; Goethe, Ralph

2006-01-01

Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative beta-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite

Identifying and targeting determinants of melanoma cellular invasion.

PubMed

Jayachandran, Aparna; Prithviraj, Prashanth; Lo, Pu-Han; Walkiewicz, Marzena; Anaka, Matthew; Woods, Briannyn L; Tan, BeeShin; Behren, Andreas; Cebon, Jonathan; McKeown, Sonja J

2016-07-05

Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients.

Identifying and targeting determinants of melanoma cellular invasion

PubMed Central

Jayachandran, Aparna; Prithviraj, Prashanth; Lo, Pu-Han; Walkiewicz, Marzena; Anaka, Matthew; Woods, Briannyn L.; Tan, BeeShin

2016-01-01

Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients. PMID:27172792

Putative cognitive enhancers in preclinical models related to schizophrenia: the search for an elusive target.

PubMed

Barak, Segev; Weiner, Ina

2011-08-01

Several developments have converged to drive what may be called "the cognitive revolution" in drug discovery in schizophrenia (SCZ), including the emphasis on cognitive deficits as a core disabling aspect of SCZ, the increasing consensus that cognitive deficits are not treated satisfactorily by the available antipsychotic drugs (APDs), and the failure of animal models to predict drug efficacy for cognitive deficits in clinical trials. Consequently, in recent years, a paradigm shift has been encouraged in animal modeling, triggered by the NIMH sponsored Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) initiative, and intended to promote the development and use of behavioral measures in animals that can generate valid (clinically relevant) measures of cognition and thus promote the identification of cognition enhancers for SCZ. Here, we provide a non-exhaustive survey of the effects of putative cognition enhancers (PCEs) representing 10 pharmacological targets as well as antipsychotic drugs (APDs), on SCZ-mimetic drugs (NMDA antagonists, muscarinic antagonist scopolamine and dopaminergic agonist amphetamine), in several tasks considered to measure cognitive processes/domains that are disrupted in SCZ (the five choice serial reaction time task, sustain attention task, working and/or recognition memory (delayed (non)matching to sample, delayed alternation task, radial arm maze, novel object recognition), reversal learning, attentional set shifting, latent inhibition and spatial learning and memory). We conclude that most of the available models have no capacity to distinguish between PCEs and APDs and that there is a need to establish models based on tasks whose perturbations lead to performance impairments that are resistant to APDs, and/or to accept APDs as a "weak gold standard". Several directions derived from the surveyed data are suggested. Copyright © 2011 Elsevier Inc. All rights reserved.

Coupling bimolecular PARylation biosensors with genetic screens to identify PARylation targets.

PubMed

Krastev, Dragomir B; Pettitt, Stephen J; Campbell, James; Song, Feifei; Tanos, Barbara E; Stoynov, Stoyno S; Ashworth, Alan; Lord, Christopher J

2018-05-22

Poly (ADP-ribose)ylation is a dynamic protein modification that regulates multiple cellular processes. Here, we describe a system for identifying and characterizing PARylation events that exploits the ability of a PBZ (PAR-binding zinc finger) protein domain to bind PAR with high-affinity. By linking PBZ domains to bimolecular fluorescent complementation biosensors, we developed fluorescent PAR biosensors that allow the detection of temporal and spatial PARylation events in live cells. Exploiting transposon-mediated recombination, we integrate the PAR biosensor en masse into thousands of protein coding genes in living cells. Using these PAR-biosensor "tagged" cells in a genetic screen we carry out a large-scale identification of PARylation targets. This identifies CTIF (CBP80/CBP20-dependent translation initiation factor) as a novel PARylation target of the tankyrase enzymes in the centrosomal region of cells, which plays a role in the distribution of the centrosomal satellites.

Characterization of putative toxin/antitoxin systems in Vibrio parahaemolyticus.

PubMed

Hino, M; Zhang, J; Takagi, H; Miyoshi, T; Uchiumi, T; Nakashima, T; Kakuta, Y; Kimura, M

2014-07-01

To obtain more information about the toxin/antitoxin (TA) systems in the Vibrio genus and also to examine their involvement in the induction of a viable but nonculturable (VBNC) state, we searched homologues of the Escherichia coli TA systems in the Vibrio parahaemolyticus genome. We found that a gene cluster, vp1842/vp1843, in the V. parahaemolyticus genome database has homology to that encoding the E. coli TA proteins, DinJ/YafQ. Expression of the putative toxin gene vp1843 in E. coli cells strongly inhibited the cell growth, while coexpression with the putative antitoxin gene vp1842 neutralized this effect. Mutational analysis identified Lys37 and Pro45 in the gene product VP1843 of vp1843 as crucial residues for the growth retardation of E. coli cells. VP1843, unlike the E. coli toxin YafQ, has no protein synthesis inhibitory activity, and that instead the expression of vp1843 in E. coli caused morphological change of the cells. The gene cluster vp1842/vp1843 encodes the V. parahaemolyticus TA system; VP1843 inhibits cell growth, whereas VP1842 serves as an antitoxin by forming a stable complex with VP1843. The putative toxin, VP1843, may be involved in the induction of the VBNC state in V. parahaemolyticus by inhibiting cell division. © 2014 The Society for Applied Microbiology.

A Strategy for Identifying Quantitative Trait Genes Using Gene Expression Analysis and Causal Analysis.

PubMed

Ishikawa, Akira

2017-11-27

Large numbers of quantitative trait loci (QTL) affecting complex diseases and other quantitative traits have been reported in humans and model animals. However, the genetic architecture of these traits remains elusive due to the difficulty in identifying causal quantitative trait genes (QTGs) for common QTL with relatively small phenotypic effects. A traditional strategy based on techniques such as positional cloning does not always enable identification of a single candidate gene for a QTL of interest because it is difficult to narrow down a target genomic interval of the QTL to a very small interval harboring only one gene. A combination of gene expression analysis and statistical causal analysis can greatly reduce the number of candidate genes. This integrated approach provides causal evidence that one of the candidate genes is a putative QTG for the QTL. Using this approach, I have recently succeeded in identifying a single putative QTG for resistance to obesity in mice. Here, I outline the integration approach and discuss its usefulness using my studies as an example.

Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues

PubMed Central

Tulloch, Lindsay B.; Menzies, Stefanie K.; Fraser, Andrew L.; Gould, Eoin R.; King, Elizabeth F.; Zacharova, Marija K.; Florence, Gordon J.

2017-01-01

Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or

Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability.

PubMed

Nilsson, Martin; Chiang, Wen-Chi; Fazli, Mustafa; Gjermansen, Morten; Givskov, Michael; Tolker-Nielsen, Tim

2011-05-01

We report a study of the role of putative exopolysaccharide gene clusters in the formation and stability of Pseudomonas putida KT2440 biofilm. Two novel putative exopolysaccharide gene clusters, pea and peb, were identified, and evidence is provided that they encode products that stabilize P. putida KT2440 biofilm. The gene clusters alg and bcs, which code for proteins mediating alginate and cellulose biosynthesis, were found to play minor roles in P. putida KT2440 biofilm formation and stability under the conditions tested. A P. putida KT2440 derivative devoid of any identifiable exopolysaccharide genes was found to form biofilm with a structure similar to wild-type biofilm, but with a stability lower than that of wild-type biofilm. Based on our data, we suggest that the formation of structured P. putida KT2440 biofilm can occur in the absence of exopolysaccharides; however, exopolysaccharides play a role as structural stabilizers. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

PUTATIVE GENE PROMOTER SEQUENCES IN THE CHLORELLA VIRUSES

PubMed Central

Fitzgerald, Lisa A.; Boucher, Philip T.; Yanai-Balser, Giane; Suhre, Karsten; Graves, Michael V.; Van Etten, James L.

2008-01-01

Three short (7 to 9 nucleotides) highly conserved nucleotide sequences were identified in the putative promoter regions (150 bp upstream and 50 bp downstream of the ATG translation start site) of three members of the genus Chlorovirus, family Phycodnaviridae. Most of these sequences occurred in similar locations within the defined promoter regions. The sequence and location of the motifs were often conserved among homologous ORFs within the Chlorovirus family. One of these conserved sequences (AATGACA) is predominately associated with genes expressed early in virus replication. PMID:18768195

Identifying MicroRNAs and Transcript Targets in Jatropha Seeds

PubMed Central

Galli, Vanessa; Guzman, Frank; de Oliveira, Luiz F. V.; Loss-Morais, Guilherme; Körbes, Ana P.; Silva, Sérgio D. A.; Margis-Pinheiro, Márcia M. A. N.; Margis, Rogério

2014-01-01

MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs) as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs. PMID:24551031

Identifying Drug-Target Interactions with Decision Templates.

PubMed

Yan, Xiao-Ying; Zhang, Shao-Wu

2018-01-01

During the development process of new drugs, identification of the drug-target interactions wins primary concerns. However, the chemical or biological experiments bear the limitation in coverage as well as the huge cost of both time and money. Based on drug similarity and target similarity, chemogenomic methods can be able to predict potential drug-target interactions (DTIs) on a large scale and have no luxurious need about target structures or ligand entries. In order to reflect the cases that the drugs having variant structures interact with common targets and the targets having dissimilar sequences interact with same drugs. In addition, though several other similarity metrics have been developed to predict DTIs, the combination of multiple similarity metrics (especially heterogeneous similarities) is too naïve to sufficiently explore the multiple similarities. In this paper, based on Gene Ontology and pathway annotation, we introduce two novel target similarity metrics to address above issues. More importantly, we propose a more effective strategy via decision template to integrate multiple classifiers designed with multiple similarity metrics. In the scenarios that predict existing targets for new drugs and predict approved drugs for new protein targets, the results on the DTI benchmark datasets show that our target similarity metrics are able to enhance the predictive accuracies in two scenarios. And the elaborate fusion strategy of multiple classifiers has better predictive power than the naïve combination of multiple similarity metrics. Compared with other two state-of-the-art approaches on the four popular benchmark datasets of binary drug-target interactions, our method achieves the best results in terms of AUC and AUPR for predicting available targets for new drugs (S2), and predicting approved drugs for new protein targets (S3).These results demonstrate that our method can effectively predict the drug-target interactions. The software package can

New generic primer system targeting mucosal/genital and cutaneous human papillomaviruses leads to the characterization of HPV 115, a novel Beta-papillomavirus species 3

PubMed Central

Chouhy, Diego; Gorosito, Mario; Sánchez, Adriana; Serra, Esteban C; Bergero, Adriana; Bussy, Ramón Fernandez; Giri, Adriana A

2009-01-01

We explored the cutaneotropic HPV genetic diversity in 71 subjects from Argentina. New generic primers (CUT) targeting 88 mucosal/cutaneous HPV were designed and compared to FAP primers. Overall, 69 different HPV types/putative types were identified, being 17 of them novel putative types. Phylogenetic analysis of partial L1 sequences grouped 2 novel putative types in the Beta-PV, 14 in the Gamma-PV and 1 in the Mu-PV genera. CUT primers showed broader capacity than FAP primers in detecting different genera/species and novel putative types (p<0.01). Using overlapping PCR, the full-length genome of a Beta-PV putative type was amplified and cloned. The new virus, designated HPV 115, encodes 5 early genes and 2 late genes. Phylogenetic analysis indicated HPV 115 as the most divergent type within the genus Beta-PV species 3. This report is the first providing data on cutaneous HPVs circulating in South America and expands our knowledge of the Papillomaviridae family. PMID:19948351

Developing putative AOPs from high content dataDeveloping putative AOPs from high content dataDeveloping putative AOPs from high content dataDeveloping putative AOPs from high content data

EPA Science Inventory

Developing putative AOPs from high content data Shannon M. Bell1,2, Stephen W. Edwards2 1 Oak Ridge Institute for Science and Education 2 Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development,...

Identifying relationships between unrelated pharmaceutical target proteins on the basis of shared active compounds.

PubMed

Miljković, Filip; Kunimoto, Ryo; Bajorath, Jürgen

2017-08-01

Computational exploration of small-molecule-based relationships between target proteins from different families. Target annotations of drugs and other bioactive compounds were systematically analyzed on the basis of high-confidence activity data. A total of 286 novel chemical links were established between distantly related or unrelated target proteins. These relationships involved a total of 1859 bioactive compounds including 147 drugs and 141 targets. Computational analysis of large amounts of compounds and activity data has revealed unexpected relationships between diverse target proteins on the basis of compounds they share. These relationships are relevant for drug discovery efforts. Target pairs that we have identified and associated compound information are made freely available.

Fibroblast growth factor receptor (FGFR) alterations in squamous differentiated bladder cancer: a putative therapeutic target for a small subgroup.

PubMed

Baldia, Philipp H; Maurer, Angela; Heide, Timon; Rose, Michael; Stoehr, Robert; Hartmann, Arndt; Williams, Sarah V; Knowles, Margaret A; Knuechel, Ruth; Gaisa, Nadine T

2016-11-01

Although drugable fibroblast growth factor receptor (FGFR) alterations in squamous cell carcinomas (SCC) of various entities are well known, little is known about FGFR modifications in squamous differentiated bladder cancer. Therefore, our study evaluated FGFR1-3 alterations as a putative therapeutic target in this subgroup. We analyzed 73 squamous differentiated bladder cancers (n = 10 pT2, n = 55 pT3, n = 8 pT4) for FGFR1-3 protein expression, FGFR1-3 copy number variations, FGFR3 chromosomal rearrangements (fluorescence in situ hybridization (FISH)) and FGFR3 mutations (SNapShot analysis). Only single cases displayed enhanced protein expression, most frequently FGFR3 overexpression (9.4% (6/64)). FISH showed no amplifications of FGFR1, 2 or 3. Break apart events were only slightly above the cut off in 12.1% (8/66) of cases and no FGFR3-TACC3 rearrangements could be proven by qPCR. FGFR3 mutations (p.S249C) were found in 8.5% (6/71) of tumors and were significantly associated with FGFR3 protein overexpression (p < 0.001), and unfavourable clinical outcome (p = 0.001). Our findings are consistent with the results of the TCGA data set for the "squamous-like" subtype of bladder cancer (n = 85), which revealed reduced overall expression of FGFR1 and FGFR2 in tumors compared to normal tissue, while expression of FGFR3 remained high. In the TCGA "squamous-like" subtype FGFR3 mutations were found in 4.9% and correlated with high FGFR3 RNA expression. Mutations of FGFR1 and FGFR2 were less frequent (2.4% and 1.2%). Hence, our comprehensive study provides novel insights into a subgroup of squamous differentiated bladder tumors that hold clues for novel therapeutic regimens and may benefit from FGFR3-targeted therapies.

Using Functional Signature Ontology (FUSION) to Identify Mechanisms of Action for Natural Products

PubMed Central

Potts, Malia B.; Kim, Hyun Seok; Fisher, Kurt W.; Hu, Youcai; Carrasco, Yazmin P.; Bulut, Gamze Betul; Ou, Yi-Hung; Herrera-Herrera, Mireya L.; Cubillos, Federico; Mendiratta, Saurabh; Xiao, Guanghua; Hofree, Matan; Ideker, Trey; Xie, Yang; Huang, Lily Jun-shen; Lewis, Robert E.; MacMillan, John B.; White, Michael A.

2014-01-01

A challenge for biomedical research is the development of pharmaceuticals that appropriately target disease mechanisms. Natural products can be a rich source of bioactive chemicals for medicinal applications but can act through unknown mechanisms and can be difficult to produce or obtain. To address these challenges, we developed a new marine-derived, renewable natural products resource and a method for linking bioactive derivatives of this library to the proteins and biological processes that they target in cells. We used cell-based screening and computational analysis to match gene expression signatures produced by natural products to those produced by siRNA and synthetic microRNA libraries. With this strategy, we matched proteins and microRNAs with diverse biological processes and also identified putative protein targets and mechanisms of action for several previously undescribed marine-derived natural products. We confirmed mechanistic relationships for selected short-interfering RNAs, microRNAs, and compounds with functional roles in autophagy, chemotaxis mediated by discoidin domain receptor 2, or activation of the kinase AKT. Thus, this approach may be an effective method for screening new drugs while simultaneously identifying their targets. PMID:24129700

Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

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

Whitley, Dexter S.; Yu, Kwang; Sample, Robert C.

2010-09-30

Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granularmore » bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.« less

Genome-wide identification of Hami melon miRNAs with putative roles during fruit development

PubMed Central

Wang, Guangzhi; Ma, Xinli; Li, Meihua; Wu, Haibo; Fu, Qiushi; Zhang, Yi; Yi, Hongping

2017-01-01

MicroRNAs represent a family of small endogenous, non-coding RNAs that play critical regulatory roles in plant growth, development, and environmental stress responses. Hami melon is famous for its attractive flavor and excellent nutritional value, however, the mechanisms underlying the fruit development and ripening remains largely unknown. Here, we performed small RNA sequencing to investigate the roles of miRNAs during Hami melon fruit development. Two batches of flesh samples were collected at four fruit development stages. Small RNA sequencing yielded a total of 54,553,424 raw reads from eight libraries. 113 conserved miRNAs belonging to 30 miRNA families and nine novel miRNAs comprising nine miRNA families were identified. The expression of 42 conserved miRNAs and three Hami melon-specific miRNAs significantly changed during fruit development. Furthermore, 484 and 124 melon genes were predicted as putative targets of 29 conserved and nine Hami melon-specific miRNA families, respectively. GO enrichment analysis were performed on target genes, “transcription, DNA-dependent”, “rRNA processing”, “oxidation reduction”, “signal transduction”, “regulation of transcription, DNA-dependent”, and “metabolic process” were the over-represented biological process terms. Cleavage sites of six target genes were validated using 5’ RACE. Our results present a comprehensive set of identification and characterization of Hami melon fruit miRNAs and their potential targets, which provide valuable basis towards understanding the regulatory mechanisms in programmed process of normal Hami fruit development and ripening. Specific miRNAs could be selected for further research and applications in breeding practices. PMID:28742088

Putative golden proportions as predictors of facial esthetics in adolescents.

PubMed

Kiekens, Rosemie M A; Kuijpers-Jagtman, Anne Marie; van 't Hof, Martin A; van 't Hof, Bep E; Maltha, Jaap C

2008-10-01

In orthodontics, facial esthetics is assumed to be related to golden proportions apparent in the ideal human face. The aim of the study was to analyze the putative relationship between facial esthetics and golden proportions in white adolescents. Seventy-six adult laypeople evaluated sets of photographs of 64 adolescents on a visual analog scale (VAS) from 0 to 100. The facial esthetic value of each subject was calculated as a mean VAS score. Three observers recorded the position of 13 facial landmarks included in 19 putative golden proportions, based on the golden proportions as defined by Ricketts. The proportions and each proportion's deviation from the golden target (1.618) were calculated. This deviation was then related to the VAS scores. Only 4 of the 19 proportions had a significant negative correlation with the VAS scores, indicating that beautiful faces showed less deviation from the golden standard than less beautiful faces. Together, these variables explained only 16% of the variance. Few golden proportions have a significant relationship with facial esthetics in adolescents. The explained variance of these variables is too small to be of clinical importance.

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.

A vector space model approach to identify genetically related diseases.

PubMed

Sarkar, Indra Neil

2012-01-01

The relationship between diseases and their causative genes can be complex, especially in the case of polygenic diseases. Further exacerbating the challenges in their study is that many genes may be causally related to multiple diseases. This study explored the relationship between diseases through the adaptation of an approach pioneered in the context of information retrieval: vector space models. A vector space model approach was developed that bridges gene disease knowledge inferred across three knowledge bases: Online Mendelian Inheritance in Man, GenBank, and Medline. The approach was then used to identify potentially related diseases for two target diseases: Alzheimer disease and Prader-Willi Syndrome. In the case of both Alzheimer Disease and Prader-Willi Syndrome, a set of plausible diseases were identified that may warrant further exploration. This study furthers seminal work by Swanson, et al. that demonstrated the potential for mining literature for putative correlations. Using a vector space modeling approach, information from both biomedical literature and genomic resources (like GenBank) can be combined towards identification of putative correlations of interest. To this end, the relevance of the predicted diseases of interest in this study using the vector space modeling approach were validated based on supporting literature. The results of this study suggest that a vector space model approach may be a useful means to identify potential relationships between complex diseases, and thereby enable the coordination of gene-based findings across multiple complex diseases.

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

Whole-Genome Survey of the Putative ATP-Binding Cassette Transporter Family Genes in Vitis vinifera

PubMed Central

Çakır, Birsen; Kılıçkaya, Ozan

2013-01-01

The ATP-binding cassette (ABC) protein superfamily constitutes one of the largest protein families known in plants. In this report, we performed a complete inventory of ABC protein genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with ABC protein members of Arabidopsis thaliana, we identified 135 putative ABC proteins with 1 or 2 NBDs in V. vinifera. Of these, 120 encode intrinsic membrane proteins, and 15 encode proteins missing TMDs. V. vinifera ABC proteins can be divided into 13 subfamilies with 79 “full-size,” 41 “half-size,” and 15 “soluble” putative ABC proteins. The main feature of the Vitis ABC superfamily is the presence of 2 large subfamilies, ABCG (pleiotropic drug resistance and white-brown complex homolog) and ABCC (multidrug resistance-associated protein). We identified orthologs of V. vinifera putative ABC transporters in different species. This work represents the first complete inventory of ABC transporters in V. vinifera. The identification of Vitis ABC transporters and their comparative analysis with the Arabidopsis counterparts revealed a strong conservation between the 2 species. This inventory could help elucidate the biological and physiological functions of these transporters in V. vinifera. PMID:24244377

Genomic identification of direct target genes of LEAFY

PubMed Central

William, Dilusha A.; Su, Yanhui; Smith, Michael R.; Lu, Meina; Baldwin, Don A.; Wagner, Doris

2004-01-01

The switch from vegetative to reproductive development in plants necessitates a switch in the developmental program of the descendents of the stem cells in the shoot apical meristem. Genetic and molecular investigations have demonstrated that the plant-specific transcription factor and meristem identity regulator LEAFY (LFY) controls this developmental transition by inducing expression of a second transcription factor, APETALA1, and by regulating the expression of additional, as yet unknown, genes. Here we show that the additional LFY targets include the APETALA1-related factor, CAULI-FLOWER, as well as three transcription factors and two putative signal transduction pathway components. These genes are up-regulated by LFY even when protein synthesis is inhibited and, hence, appear to be direct targets of LFY. Supporting this conclusion, cis-regulatory regions upstream of these genes are bound by LFY in vivo. The newly identified LFY targets likely initiate the transcriptional changes that are required for the switch from vegetative to reproductive development in Arabidopsis. PMID:14736918

Factor Analysis of Therapist-Identified Treatment Targets in Community-Based Children's Mental Health.

PubMed

Love, Allison R; Okado, Izumi; Orimoto, Trina E; Mueller, Charles W

2018-01-01

The present study used exploratory and confirmatory factor analyses to identify underlying latent factors affecting variation in community therapists' endorsement of treatment targets. As part of a statewide practice management program, therapist completed monthly reports of treatment targets (up to 10 per month) for a sample of youth (n = 790) receiving intensive in-home therapy. Nearly 75 % of youth were diagnosed with multiple co-occurring disorders. Five factors emerged: Disinhibition, Societal Rules Evasion, Social Engagement Deficits, Emotional Distress, and Management of Biodevelopmental Outcomes. Using logistic regression, primary diagnosis predicted therapist selection of Disinhibition and Emotional Distress targets. Client age predicted endorsement of Societal Rules Evasion targets. Practice-to-research implications are discussed.

Identifying Belief-Based Targets for the Promotion of Leisure-Time Walking

ERIC Educational Resources Information Center

Rhodes, Ryan E.; Blanchard, Chris M.; Courneya, Kerry S.; Plotnikoff, Ronald C.

2009-01-01

Walking is the most common type of physical activity (PA) and the likely target of efforts to increase PA. No studies, however, have identified the belief-level correlates for walking using the theory of planned behavior. This study elicits salient beliefs about walking and evaluates beliefs that may be most important for walking-promotion…

In silico molecular comparisons of C. elegans and mammalian pharmacology identify distinct targets that regulate feeding.

PubMed

Lemieux, George A; Keiser, Michael J; Sassano, Maria F; Laggner, Christian; Mayer, Fahima; Bainton, Roland J; Werb, Zena; Roth, Bryan L; Shoichet, Brian K; Ashrafi, Kaveh

2013-11-01

Phenotypic screens can identify molecules that are at once penetrant and active on the integrated circuitry of a whole cell or organism. These advantages are offset by the need to identify the targets underlying the phenotypes. Additionally, logistical considerations limit screening for certain physiological and behavioral phenotypes to organisms such as zebrafish and C. elegans. This further raises the challenge of elucidating whether compound-target relationships found in model organisms are preserved in humans. To address these challenges we searched for compounds that affect feeding behavior in C. elegans and sought to identify their molecular mechanisms of action. Here, we applied predictive chemoinformatics to small molecules previously identified in a C. elegans phenotypic screen likely to be enriched for feeding regulatory compounds. Based on the predictions, 16 of these compounds were tested in vitro against 20 mammalian targets. Of these, nine were active, with affinities ranging from 9 nM to 10 µM. Four of these nine compounds were found to alter feeding. We then verified the in vitro findings in vivo through genetic knockdowns, the use of previously characterized compounds with high affinity for the four targets, and chemical genetic epistasis, which is the effect of combined chemical and genetic perturbations on a phenotype relative to that of each perturbation in isolation. Our findings reveal four previously unrecognized pathways that regulate feeding in C. elegans with strong parallels in mammals. Together, our study addresses three inherent challenges in phenotypic screening: the identification of the molecular targets from a phenotypic screen, the confirmation of the in vivo relevance of these targets, and the evolutionary conservation and relevance of these targets to their human orthologs.

Novel target for high-risk neuroblastoma identified in pre-clinical research | Center for Cancer Research

Cancer.gov

Pre-clinical research by investigators at the Center for Cancer Research and their colleagues have identified a number of novel epigenetic targets for high-risk neuroblastoma and validated a promising new targeted inhibitor in pre-clinical models.  Read more...

GRIL-Seq, a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation

PubMed Central

Han, Kook; Tjaden, Brian; Lory, Stephen

2017-01-01

The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base-pairing with partially complementary sequences of targeted transcripts. We present a simple method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique (referred to as GRIL-Seq) is based on preferential ligation of sRNAs to ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimeras. In addition to the RNA chaperone Hfq, the GRIL-Seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, we demonstrate that direct regulatory targets of this sRNA can be readily identified. Therefore, GRIL-Seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but can also result in uncovering novel roles for sRNAs and their targets in complex regulatory networks. PMID:28005055

Genetic toxicology of putative nongenotoxic carcinogens

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

Jackson, M.A.; Stack, H.F.; Waters, M.D.

1993-01-01

The report examines a group of putative nongenotoxic carcinogens that have been cited in the published literature. Using short-term test data from the US Environmental Protection Agency/International Agency for Research on Cancer genetic activity profile (EPA/IARC GAP) database, these agents are classified on the basis of their mutagenicity emphasizing three genetic endpoints: gene mutation, chromosomal aberration and aneuploidy. On the basis of results of short-term tests for these effects, criteria was defined for evidence of mutagenicity (and nonmutagenicity) these criteria were applied in classifying the group of putative nongenotoxic carcinogens. The results from this evaluation based on the EPA/IARC GAPmore » database are presented along with a summary of the short-term test data for each chemical and the relevant carcinogenicity results from the NTP, Gene-Tox and IARC databases. The data clearly demonstrate that many of the putative nongenotoxic carcinogens that have been adequately tested in short-term bioassays induce gene or chromosomal mutations or aneuploidy.« less

Genome-wide analysis of putative peroxiredoxin in unicellular and filamentous cyanobacteria.

PubMed

Cui, Hongli; Wang, Yipeng; Wang, Yinchu; Qin, Song

2012-11-16

Cyanobacteria are photoautotrophic prokaryotes with wide variations in genome sizes and ecological habitats. Peroxiredoxin (PRX) is an important protein that plays essential roles in protecting own cells against reactive oxygen species (ROS). PRXs have been identified from mammals, fungi and higher plants. However, knowledge on cyanobacterial PRXs still remains obscure. With the availability of 37 sequenced cyanobacterial genomes, we performed a comprehensive comparative analysis of PRXs and explored their diversity, distribution, domain structure and evolution. Overall 244 putative prx genes were identified, which were abundant in filamentous diazotrophic cyanobacteria, Acaryochloris marina MBIC 11017, and unicellular cyanobacteria inhabiting freshwater and hot-springs, while poor in all Prochlorococcus and marine Synechococcus strains. Among these putative genes, 25 open reading frames (ORFs) encoding hypothetical proteins were identified as prx gene family members and the others were already annotated as prx genes. All 244 putative PRXs were classified into five major subfamilies (1-Cys, 2-Cys, BCP, PRX5_like, and PRX-like) according to their domain structures. The catalytic motifs of the cyanobacterial PRXs were similar to those of eukaryotic PRXs and highly conserved in all but the PRX-like subfamily. Classical motif (CXXC) of thioredoxin was detected in protein sequences from the PRX-like subfamily. Phylogenetic tree constructed of catalytic domains coincided well with the domain structures of PRXs and the phylogenies based on 16s rRNA. The distribution of genes encoding PRXs in different unicellular and filamentous cyanobacteria especially those sub-families like PRX-like or 1-Cys PRX correlate with the genome size, eco-physiology, and physiological properties of the organisms. Cyanobacterial and eukaryotic PRXs share similar conserved motifs, indicating that cyanobacteria adopt similar catalytic mechanisms as eukaryotes. All cyanobacterial PRX proteins

Genome-wide analysis of putative peroxiredoxin in unicellular and filamentous cyanobacteria

PubMed Central

2012-01-01

Background Cyanobacteria are photoautotrophic prokaryotes with wide variations in genome sizes and ecological habitats. Peroxiredoxin (PRX) is an important protein that plays essential roles in protecting own cells against reactive oxygen species (ROS). PRXs have been identified from mammals, fungi and higher plants. However, knowledge on cyanobacterial PRXs still remains obscure. With the availability of 37 sequenced cyanobacterial genomes, we performed a comprehensive comparative analysis of PRXs and explored their diversity, distribution, domain structure and evolution. Results Overall 244 putative prx genes were identified, which were abundant in filamentous diazotrophic cyanobacteria, Acaryochloris marina MBIC 11017, and unicellular cyanobacteria inhabiting freshwater and hot-springs, while poor in all Prochlorococcus and marine Synechococcus strains. Among these putative genes, 25 open reading frames (ORFs) encoding hypothetical proteins were identified as prx gene family members and the others were already annotated as prx genes. All 244 putative PRXs were classified into five major subfamilies (1-Cys, 2-Cys, BCP, PRX5_like, and PRX-like) according to their domain structures. The catalytic motifs of the cyanobacterial PRXs were similar to those of eukaryotic PRXs and highly conserved in all but the PRX-like subfamily. Classical motif (CXXC) of thioredoxin was detected in protein sequences from the PRX-like subfamily. Phylogenetic tree constructed of catalytic domains coincided well with the domain structures of PRXs and the phylogenies based on 16s rRNA. Conclusions The distribution of genes encoding PRXs in different unicellular and filamentous cyanobacteria especially those sub-families like PRX-like or 1-Cys PRX correlate with the genome size, eco-physiology, and physiological properties of the organisms. Cyanobacterial and eukaryotic PRXs share similar conserved motifs, indicating that cyanobacteria adopt similar catalytic mechanisms as eukaryotes. All

Using Bioinformatic Approaches to Identify Pathways Targeted by Human Leukemogens

PubMed Central

Thomas, Reuben; Phuong, Jimmy; McHale, Cliona M.; Zhang, Luoping

2012-01-01

We have applied bioinformatic approaches to identify pathways common to chemical leukemogens and to determine whether leukemogens could be distinguished from non-leukemogenic carcinogens. From all known and probable carcinogens classified by IARC and NTP, we identified 35 carcinogens that were associated with leukemia risk in human studies and 16 non-leukemogenic carcinogens. Using data on gene/protein targets available in the Comparative Toxicogenomics Database (CTD) for 29 of the leukemogens and 11 of the non-leukemogenic carcinogens, we analyzed for enrichment of all 250 human biochemical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The top pathways targeted by the leukemogens included metabolism of xenobiotics by cytochrome P450, glutathione metabolism, neurotrophin signaling pathway, apoptosis, MAPK signaling, Toll-like receptor signaling and various cancer pathways. The 29 leukemogens formed 18 distinct clusters comprising 1 to 3 chemicals that did not correlate with known mechanism of action or with structural similarity as determined by 2D Tanimoto coefficients in the PubChem database. Unsupervised clustering and one-class support vector machines, based on the pathway data, were unable to distinguish the 29 leukemogens from 11 non-leukemogenic known and probable IARC carcinogens. However, using two-class random forests to estimate leukemogen and non-leukemogen patterns, we estimated a 76% chance of distinguishing a random leukemogen/non-leukemogen pair from each other. PMID:22851955

Combining functional genomics and chemical biology to identify targets of bioactive compounds.

PubMed

Ho, Cheuk Hei; Piotrowski, Jeff; Dixon, Scott J; Baryshnikova, Anastasia; Costanzo, Michael; Boone, Charles

2011-02-01

Genome sequencing projects have revealed thousands of suspected genes, challenging researchers to develop efficient large-scale functional analysis methodologies. Determining the function of a gene product generally requires a means to alter its function. Genetically tractable model organisms have been widely exploited for the isolation and characterization of activating and inactivating mutations in genes encoding proteins of interest. Chemical genetics represents a complementary approach involving the use of small molecules capable of either inactivating or activating their targets. Saccharomyces cerevisiae has been an important test bed for the development and application of chemical genomic assays aimed at identifying targets and modes of action of known and uncharacterized compounds. Here we review yeast chemical genomic assays strategies for drug target identification. Copyright © 2010 Elsevier Ltd. All rights reserved.

Preferential Representation of Past Outcome Information and Future Choice Behavior by Putative Inhibitory Interneurons Rather Than Putative Pyramidal Neurons in the Primate Dorsal Anterior Cingulate Cortex.

PubMed

Kawai, Takashi; Yamada, Hiroshi; Sato, Nobuya; Takada, Masahiko; Matsumoto, Masayuki

2018-05-02

The dorsal anterior cingulate cortex (dACC) plays crucial roles in monitoring the outcome of a choice and adjusting a subsequent choice behavior based on the outcome information. In the present study, we investigated how different types of dACC neurons, that is, putative pyramidal neurons and putative inhibitory interneurons, contribute to these processes. We analyzed single-unit database obtained from the dACC in monkeys performing a reversal learning task. The monkey was required to adjust choice behavior from past outcome experiences. Depending on their action potential waveforms, the recorded neurons were classified into putative pyramidal neurons and putative inhibitory interneurons. We found that these neurons do not equally contribute to outcome monitoring and behavioral adjustment. Although both neuron types evenly responded to the current outcome, a larger proportion of putative inhibitory interneurons than putative pyramidal neurons stored the information about the past outcome. The putative inhibitory interneurons further represented choice-related signals more frequently, such as whether the monkey would shift the last choice to an alternative at the next choice opportunity. Our findings suggest that putative inhibitory interneurons, which are thought not to project to brain areas outside the dACC, preferentially transmit signals that would adjust choice behavior based on past outcome experiences.

Beyond the binding site: in vivo identification of tbx2, smarca5 and wnt5b as molecular targets of CNBP during embryonic development.

PubMed

Armas, Pablo; Margarit, Ezequiel; Mouguelar, Valeria S; Allende, Miguel L; Calcaterra, Nora B

2013-01-01

CNBP is a nucleic acid chaperone implicated in vertebrate craniofacial development, as well as in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human muscle diseases. CNBP is highly conserved among vertebrates and has been implicated in transcriptional regulation; however, its DNA binding sites and molecular targets remain elusive. The main goal of this work was to identify CNBP DNA binding sites that might reveal target genes involved in vertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound in vivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequency of putative G-quadruplex DNA secondary structure. Moreover, an in silico approach enabled us to establish the CNBP DNA-binding site and to predict CNBP putative targets based on gene ontology terms and synexpression with CNBP. The direct interaction between CNBP and candidate genes was proved by EMSA and ChIP assays. Besides, the role of CNBP upon the identified genes was validated in loss-of-function experiments in developing zebrafish. We successfully confirmed that CNBP up-regulates tbx2b and smarca5, and down-regulates wnt5b gene expression. The highly stringent strategy used in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonic development. Furthermore, it represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates.

Beyond the Binding Site: In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development

PubMed Central

Mouguelar, Valeria S.; Allende, Miguel L.; Calcaterra, Nora B.

2013-01-01

CNBP is a nucleic acid chaperone implicated in vertebrate craniofacial development, as well as in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human muscle diseases. CNBP is highly conserved among vertebrates and has been implicated in transcriptional regulation; however, its DNA binding sites and molecular targets remain elusive. The main goal of this work was to identify CNBP DNA binding sites that might reveal target genes involved in vertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound in vivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequency of putative G-quadruplex DNA secondary structure. Moreover, an in silico approach enabled us to establish the CNBP DNA-binding site and to predict CNBP putative targets based on gene ontology terms and synexpression with CNBP. The direct interaction between CNBP and candidate genes was proved by EMSA and ChIP assays. Besides, the role of CNBP upon the identified genes was validated in loss-of-function experiments in developing zebrafish. We successfully confirmed that CNBP up-regulates tbx2b and smarca5, and down-regulates wnt5b gene expression. The highly stringent strategy used in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonic development. Furthermore, it represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates. PMID:23667590

Virus-encoded chemokine receptors--putative novel antiviral drug targets.

PubMed

Rosenkilde, Mette M

2005-01-01

Large DNA viruses, in particular herpes- and poxviruses, have evolved proteins that serve as mimics or decoys for endogenous proteins in the host. The chemokines and their receptors serve key functions in both innate and adaptive immunity through control of leukocyte trafficking, and have as such a paramount role in the antiviral immune responses. It is therefore not surprising that viruses have found ways to exploit and subvert the chemokine system by means of molecular mimicry. By ancient acts of molecular piracy and by induction and suppression of endogenous genes, viruses have utilized chemokines and their receptors to serve a variety of roles in viral life-cycle. This review focuses on the pharmacology of virus-encoded chemokine receptors, yet also the family of virus-encoded chemokines and chemokine-binding proteins will be touched upon. Key properties of the virus-encoded receptors, compared to their closest endogenous homologs, are interactions with a wider range of chemokines, which can act as agonists, antagonists and inverse agonists, and the exploitation of many signal transduction pathways. High constitutive activity is another key property of some--but not all--of these receptors. The chemokine receptors belong to the superfamily of G-protein coupled 7TM receptors that per se are excellent drug targets. At present, non-peptide antagonists have been developed against many chemokine receptors. The potentials of the virus-encoded chemokine receptors as drug targets--ie. as novel antiviral strategies--will be highlighted here together with the potentials of the virus-encoded chemokines and chemokine-binding proteins as novel anti-inflammatory biopharmaceutical strategies.

GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation.

PubMed

Han, Kook; Tjaden, Brian; Lory, Stephen

2016-12-22

The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base pairing with partially complementary sequences of targeted transcripts. We present a simple method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique, referred to as global small non-coding RNA target identification by ligation and sequencing (GRIL-seq), is based on preferential ligation of sRNAs to the ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimaeras. In addition to the RNA chaperone Hfq, the GRIL-seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, we demonstrated that direct regulatory targets of this sRNA can readily be identified. Therefore, GRIL-seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but also for uncovering novel roles for sRNAs and their targets in complex regulatory networks.

Uridine monophosphate kinase as potential target for tuberculosis: from target to lead identification.

PubMed

Arvind, Akanksha; Jain, Vaibhav; Saravanan, Parameswaran; Mohan, C Gopi

2013-12-01

Mycobacterium tuberculosis (Mtb) is a causative agent of tuberculosis (TB) disease, which has affected approximately 2 billion people worldwide. Due to the emergence of resistance towards the existing drugs, discovery of new anti-TB drugs is an important global healthcare challenge. To address this problem, there is an urgent need to identify new drug targets in Mtb. In the present study, the subtractive genomics approach has been employed for the identification of new drug targets against TB. Screening the Mtb proteome using the Database of Essential Genes (DEG) and human proteome resulted in the identification of 60 key proteins which have no eukaryotic counterparts. Critical analysis of these proteins using Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways database revealed uridine monophosphate kinase (UMPK) enzyme as a potential drug target for developing novel anti-TB drugs. Homology model of Mtb-UMPK was constructed for the first time on the basis of the crystal structure of E. coli-UMPK, in order to understand its structure-function relationships, and which would in turn facilitate to perform structure-based inhibitor design. Furthermore, the structural similarity search was carried out using physiological inhibitor UTP of Mtb-UMPK to virtually screen ZINC database. Retrieved hits were further screened by implementing several filters like ADME and toxicity followed by molecular docking. Finally, on the basis of the Glide docking score and the mode of binding, 6 putative leads were identified as inhibitors of this enzyme which can potentially emerge as future drugs for the treatment of TB.

Differential Expression Patterns in Chemosensory and Non-Chemosensory Tissues of Putative Chemosensory Genes Identified by Transcriptome Analysis of Insect Pest the Purple Stem Borer Sesamia inferens (Walker)

PubMed Central

Zhang, Ya-Nan; Jin, Jun-Yan; Jin, Rong; Xia, Yi-Han; Zhou, Jing-Jiang; Deng, Jian-Yu; Dong, Shuang-Lin

2013-01-01

Background A large number of insect chemosensory genes from different gene subfamilies have been identified and annotated, but their functional diversity and complexity are largely unknown. A systemic examination of expression patterns in chemosensory organs could provide important information. Methodology/Principal Findings We identified 92 putative chemosensory genes by analysing the transcriptome of the antennae and female sex pheromone gland of the purple stem borer Sesamia inferens, among them 87 are novel in this species, including 24 transcripts encoding for odorant binding proteins (OBPs), 24 for chemosensory proteins (CSPs), 2 for sensory neuron membrane proteins (SNMPs), 39 for odorant receptors (ORs) and 3 for ionotropic receptors (IRs). The transcriptome analyses were validated and quantified with a detailed global expression profiling by Reverse Transcription-PCR for all 92 transcripts and by Quantitative Real Time RT-PCR for selected 16 ones. Among the chemosensory gene subfamilies, CSP transcripts are most widely and evenly expressed in different tissues and stages, OBP transcripts showed a clear antenna bias and most of OR transcripts are only detected in adult antennae. Our results also revealed that some OR transcripts, such as the transcripts of SNMP2 and 2 IRs were expressed in non-chemosensory tissues, and some CSP transcripts were antenna-biased expression. Furthermore, no chemosensory transcript is specific to female sex pheromone gland and very few are found in the heads. Conclusion Our study revealed that there are a large number of chemosensory genes expressed in S. inferens, and some of them displayed unusual expression profile in non-chemosensory tissues. The identification of a large set of putative chemosensory genes of each subfamily from a single insect species, together with their different expression profiles provide further information in understanding the functions of these chemosensory genes in S. inferens as well as other

Differential expression patterns in chemosensory and non-chemosensory tissues of putative chemosensory genes identified by transcriptome analysis of insect pest the purple stem borer Sesamia inferens (Walker).

PubMed

Zhang, Ya-Nan; Jin, Jun-Yan; Jin, Rong; Xia, Yi-Han; Zhou, Jing-Jiang; Deng, Jian-Yu; Dong, Shuang-Lin

2013-01-01

A large number of insect chemosensory genes from different gene subfamilies have been identified and annotated, but their functional diversity and complexity are largely unknown. A systemic examination of expression patterns in chemosensory organs could provide important information. We identified 92 putative chemosensory genes by analysing the transcriptome of the antennae and female sex pheromone gland of the purple stem borer Sesamia inferens, among them 87 are novel in this species, including 24 transcripts encoding for odorant binding proteins (OBPs), 24 for chemosensory proteins (CSPs), 2 for sensory neuron membrane proteins (SNMPs), 39 for odorant receptors (ORs) and 3 for ionotropic receptors (IRs). The transcriptome analyses were validated and quantified with a detailed global expression profiling by Reverse Transcription-PCR for all 92 transcripts and by Quantitative Real Time RT-PCR for selected 16 ones. Among the chemosensory gene subfamilies, CSP transcripts are most widely and evenly expressed in different tissues and stages, OBP transcripts showed a clear antenna bias and most of OR transcripts are only detected in adult antennae. Our results also revealed that some OR transcripts, such as the transcripts of SNMP2 and 2 IRs were expressed in non-chemosensory tissues, and some CSP transcripts were antenna-biased expression. Furthermore, no chemosensory transcript is specific to female sex pheromone gland and very few are found in the heads. Our study revealed that there are a large number of chemosensory genes expressed in S. inferens, and some of them displayed unusual expression profile in non-chemosensory tissues. The identification of a large set of putative chemosensory genes of each subfamily from a single insect species, together with their different expression profiles provide further information in understanding the functions of these chemosensory genes in S. inferens as well as other insects.

Omen: identifying potential spear-phishing targets before the email is sent.

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

Wendt, Jeremy Daniel.

2013-07-01

We present the results of a two year project focused on a common social engineering attack method called "spear phishing". In a spear phishing attack, the user receives an email with information specifically focused on the user. This email contains either a malware-laced attachment or a link to download the malware that has been disguised as a useful program. Spear phishing attacks have been one of the most effective avenues for attackers to gain initial entry into a target network. This project focused on a proactive approach to spear phishing. To create an effective, user-specific spear phishing email, the attackermore » must research the intended recipient. We believe that much of the information used by the attacker is provided by the target organization's own external website. Thus when researching potential targets, the attacker leaves signs of his research in the webserver's logs. We created tools and visualizations to improve cybersecurity analysts' abilities to quickly understand a visitor's visit patterns and interests. Given these suspicious visitors and log-parsing tools, analysts can more quickly identify truly suspicious visitors, search for potential spear-phishing targeted users, and improve security around those users before the spear phishing email is sent.« less

Targeted plasma proteomics identifies a novel, robust association between cornulin and Swedish moist snuff.

PubMed

Sundkvist, Anneli; Myte, Robin; Bodén, Stina; Enroth, Stefan; Gyllensten, Ulf; Harlid, Sophia; van Guelpen, Bethany

2018-02-02

Lifestyle behaviors are believed to influence the body's inflammatory state. Chronic low-grade inflammation contributes to the development of major non-communicable diseases such as diabetes, cardiovascular disease and cancer. Inflammation may thus be an important link between lifestyle and disease. We evaluated self-reported physical activity, tobacco use and alcohol consumption in relation to plasma levels of 160 validated inflammatory and cancer biomarkers. The study included 138 participants from a population-based cohort, all with repeated sampling of plasma and data ten years apart, allowing consideration of both intra- and inter-individual variation. Of 17 relationships identified, the strongest was an independent, positive association between cornulin (CRNN) and Swedish moist snuff (snus) use. We replicated the finding in a second cohort of 501 individuals, in which a dose-response relationship was also observed. Snus explained approximately one fifth of the variance in CRNN levels in both sample sets (18% and 23%). In conclusion, we identified a novel, independent, dose-dependent association between CRNN and snus use. Further study is warranted, to evaluate the performance of CRNN as a potential snus biomarker. The putative importance of lifestyle behaviors on a wide range of protein biomarkers illustrates the need for more personalized biomarker cut-offs.

Large-Scale Chemical Similarity Networks for Target Profiling of Compounds Identified in Cell-Based Chemical Screens

PubMed Central

Lo, Yu-Chen; Senese, Silvia; Li, Chien-Ming; Hu, Qiyang; Huang, Yong; Damoiseaux, Robert; Torres, Jorge Z.

2015-01-01

Target identification is one of the most critical steps following cell-based phenotypic chemical screens aimed at identifying compounds with potential uses in cell biology and for developing novel disease therapies. Current in silico target identification methods, including chemical similarity database searches, are limited to single or sequential ligand analysis that have limited capabilities for accurate deconvolution of a large number of compounds with diverse chemical structures. Here, we present CSNAP (Chemical Similarity Network Analysis Pulldown), a new computational target identification method that utilizes chemical similarity networks for large-scale chemotype (consensus chemical pattern) recognition and drug target profiling. Our benchmark study showed that CSNAP can achieve an overall higher accuracy (>80%) of target prediction with respect to representative chemotypes in large (>200) compound sets, in comparison to the SEA approach (60–70%). Additionally, CSNAP is capable of integrating with biological knowledge-based databases (Uniprot, GO) and high-throughput biology platforms (proteomic, genetic, etc) for system-wise drug target validation. To demonstrate the utility of the CSNAP approach, we combined CSNAP's target prediction with experimental ligand evaluation to identify the major mitotic targets of hit compounds from a cell-based chemical screen and we highlight novel compounds targeting microtubules, an important cancer therapeutic target. The CSNAP method is freely available and can be accessed from the CSNAP web server (http://services.mbi.ucla.edu/CSNAP/). PMID:25826798

Targeted Approach to Identify Genetic Loci Associated with ...

EPA Pesticide Factsheets

Extreme tolerance to highly toxic dioxin-like contaminants (DLCs) has evolved independently and contemporaneously in (at least) four populations of Atlantic killifish (Fundulus heteroclitus). Surprisingly, the magnitude and phenotype of DLC tolerance is similar among these killifish populations that have adapted to varied, but highly contaminated urban/industrialized estuaries of the US Atlantic coast. We hypothesized that comparisons among tolerant populations and in contrast to their sensitive neighboring killifish might reveal genetic loci associated with DLC tolerance. Since the aryl hydrocarbon receptor (AHR) pathway partly or fully mediates DLC toxicity in vertebrates, we identified single nucleotide polymorphisms (SNPs) from 43 genes associated with the AHR to serve as targeted markers. Wild fish from the four highly tolerant killifish populations and four nearby sensitive populations were genotyped using 59 SNP markers. Consistent with other killifish population genetic analyses, our results revealed strong genetic differentiation among populations, consistent with isolation by distance models. Pairwise comparisons of nearby tolerant and sensitive populations revealed differentiation among these loci: AHR 1 and 2, cathepsin Z, the cytochrome P450s (CYP) 1A and 3A30, and the NADH ubiquinone oxidoreductase MLRQ subunit. By grouping tolerant versus sensitive populations, we also identified cytochrome P450 1A and the AHR2 loci as under selection, lend

Targeted proteomics identifies liquid-biopsy signatures for extracapsular prostate cancer

PubMed Central

Kim, Yunee; Jeon, Jouhyun; Mejia, Salvador; Yao, Cindy Q; Ignatchenko, Vladimir; Nyalwidhe, Julius O; Gramolini, Anthony O; Lance, Raymond S; Troyer, Dean A; Drake, Richard R; Boutros, Paul C; Semmes, O. John; Kislinger, Thomas

2016-01-01

Biomarkers are rapidly gaining importance in personalized medicine. Although numerous molecular signatures have been developed over the past decade, there is a lack of overlap and many biomarkers fail to validate in independent patient cohorts and hence are not useful for clinical application. For these reasons, identification of novel and robust biomarkers remains a formidable challenge. We combine targeted proteomics with computational biology to discover robust proteomic signatures for prostate cancer. Quantitative proteomics conducted in expressed prostatic secretions from men with extraprostatic and organ-confined prostate cancers identified 133 differentially expressed proteins. Using synthetic peptides, we evaluate them by targeted proteomics in a 74-patient cohort of expressed prostatic secretions in urine. We quantify a panel of 34 candidates in an independent 207-patient cohort. We apply machine-learning approaches to develop clinical predictive models for prostate cancer diagnosis and prognosis. Our results demonstrate that computationally guided proteomics can discover highly accurate non-invasive biomarkers. PMID:27350604

Identifying antimalarial compounds targeting dihydrofolate reductase-thymidylate synthase (DHFR-TS) by chemogenomic profiling.

PubMed

Aroonsri, Aiyada; Akinola, Olugbenga; Posayapisit, Navaporn; Songsungthong, Warangkhana; Uthaipibull, Chairat; Kamchonwongpaisan, Sumalee; Gbotosho, Grace O; Yuthavong, Yongyuth; Shaw, Philip J

2016-07-01

The mode of action of many antimalarial drugs is unknown. Chemogenomic profiling is a powerful method to address this issue. This experimental approach entails disruption of gene function and phenotypic screening for changes in sensitivity to bioactive compounds. Here, we describe the application of reverse genetics for chemogenomic profiling in Plasmodium. Plasmodium falciparum parasites harbouring a transgenic insertion of the glmS ribozyme downstream of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene were used for chemogenomic profiling of antimalarial compounds to identify those which target DHFR-TS. DHFR-TS expression can be attenuated by exposing parasites to glucosamine. Parasites with attenuated DHFR-TS expression were significantly more sensitive to antifolate drugs known to target DHFR-TS. In contrast, no change in sensitivity to other antimalarial drugs with different modes of action was observed. Chemogenomic profiling was performed using the Medicines for Malaria Venture (Switzerland) Malaria Box compound library, and two compounds were identified as novel DHFR-TS inhibitors. We also tested the glmS ribozyme in Plasmodium berghei, a rodent malaria parasite. The expression of reporter genes with downstream glmS ribozyme could be attenuated in transgenic parasites comparable with that obtained in P. falciparum. The chemogenomic profiling method was applied in a P. berghei line expressing a pyrimethamine-resistant Toxoplasma gondii DHFR-TS reporter gene under glmS ribozyme control. Parasites with attenuated expression of this gene were significantly sensitised to antifolates targeting DHFR-TS, but not other drugs with different modes of action. In conclusion, these data show that the glmS ribozyme reverse genetic tool can be applied for identifying primary targets of antimalarial compounds in human and rodent malaria parasites. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Proteome-wide prediction of targets for aspirin: new insight into the molecular mechanism of aspirin

PubMed Central

Dai, Shao-Xing; Li, Wen-Xing

2016-01-01

Besides its anti-inflammatory, analgesic and anti-pyretic properties, aspirin is used for the prevention of cardiovascular disease and various types of cancer. The multiple activities of aspirin likely involve several molecular targets and pathways rather than a single target. Therefore, systematic identification of these targets of aspirin can help us understand the underlying mechanisms of the activities. In this study, we identified 23 putative targets of aspirin in the human proteome by using binding pocket similarity detecting tool combination with molecular docking, free energy calculation and pathway analysis. These targets have diverse folds and are derived from different protein family. However, they have similar aspirin-binding pockets. The binding free energy with aspirin for newly identified targets is comparable to that for the primary targets. Pathway analysis revealed that the targets were enriched in several pathways such as vascular endothelial growth factor (VEGF) signaling, Fc epsilon RI signaling and arachidonic acid metabolism, which are strongly involved in inflammation, cardiovascular disease and cancer. Therefore, the predicted target profile of aspirin suggests a new explanation for the disease prevention ability of aspirin. Our findings provide a new insight of aspirin and its efficacy of disease prevention in a systematic and global view. PMID:26989626

Proteome-wide prediction of targets for aspirin: new insight into the molecular mechanism of aspirin.

PubMed

Dai, Shao-Xing; Li, Wen-Xing; Li, Gong-Hua; Huang, Jing-Fei

2016-01-01

Besides its anti-inflammatory, analgesic and anti-pyretic properties, aspirin is used for the prevention of cardiovascular disease and various types of cancer. The multiple activities of aspirin likely involve several molecular targets and pathways rather than a single target. Therefore, systematic identification of these targets of aspirin can help us understand the underlying mechanisms of the activities. In this study, we identified 23 putative targets of aspirin in the human proteome by using binding pocket similarity detecting tool combination with molecular docking, free energy calculation and pathway analysis. These targets have diverse folds and are derived from different protein family. However, they have similar aspirin-binding pockets. The binding free energy with aspirin for newly identified targets is comparable to that for the primary targets. Pathway analysis revealed that the targets were enriched in several pathways such as vascular endothelial growth factor (VEGF) signaling, Fc epsilon RI signaling and arachidonic acid metabolism, which are strongly involved in inflammation, cardiovascular disease and cancer. Therefore, the predicted target profile of aspirin suggests a new explanation for the disease prevention ability of aspirin. Our findings provide a new insight of aspirin and its efficacy of disease prevention in a systematic and global view.

The Biogeography of Putative Microbial Antibiotic Production

PubMed Central

Bryant, Jessica A.; Charkoudian, Louise K.; Docherty, Kathryn M.; Jones, Evan; Kembel, Steven W.; Green, Jessica L.; Bohannan, Brendan J. M.

2015-01-01

Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics. PMID:26102275

RNAi phenotype profiling of kinases identifies potential therapeutic targets in Ewing's sarcoma.

PubMed

Arora, Shilpi; Gonzales, Irma M; Hagelstrom, R Tanner; Beaudry, Christian; Choudhary, Ashish; Sima, Chao; Tibes, Raoul; Mousses, Spyro; Azorsa, David O

2010-08-18

Ewing's sarcomas are aggressive musculoskeletal tumors occurring most frequently in the long and flat bones as a solitary lesion mostly during the teen-age years of life. With current treatments, significant number of patients relapse and survival is poor for those with metastatic disease. As part of novel target discovery in Ewing's sarcoma, we applied RNAi mediated phenotypic profiling to identify kinase targets involved in growth and survival of Ewing's sarcoma cells. Four Ewing's sarcoma cell lines TC-32, TC-71, SK-ES-1 and RD-ES were tested in high throughput-RNAi screens using a siRNA library targeting 572 kinases. Knockdown of 25 siRNAs reduced the growth of all four Ewing's sarcoma cell lines in replicate screens. Of these, 16 siRNA were specific and reduced proliferation of Ewing's sarcoma cells as compared to normal fibroblasts. Secondary validation and preliminary mechanistic studies highlighted the kinases STK10 and TNK2 as having important roles in growth and survival of Ewing's sarcoma cells. Furthermore, knockdown of STK10 and TNK2 by siRNA showed increased apoptosis. In summary, RNAi-based phenotypic profiling proved to be a powerful gene target discovery strategy, leading to successful identification and validation of STK10 and TNK2 as two novel potential therapeutic targets for Ewing's sarcoma.

A screen to identify drug resistant variants to target-directed anti-cancer agents

PubMed Central

Azam, Mohammad; Raz, Tal; Nardi, Valentina; Opitz, Sarah L.

2003-01-01

The discovery of oncogenes and signal transduction pathways important for mitogenesis has triggered the development of target-specific small molecule anti-cancer compounds. As exemplified by imatinib (Gleevec), a specific inhibitor of the Chronic Myeloid Leukemia (CML)-associated Bcr-Abl kinase, these agents promise impressive activity in clinical trials, with low levels of clinical toxicity. However, such therapy is susceptible to the emergence of drug resistance due to amino acid substitutions in the target protein. Defining the spectrum of such mutations is important for patient monitoring and the design of next-generation inhibitors. Using imatinib and BCR/ABL as a paradigm for a drug-target pair, we recently reported a retroviral vector-based screening strategy to identify the spectrum of resistance-conferring mutations. Here we provide a detailed methodology for the screen, which can be generally applied to any drug-target pair. PMID:14615817

Identification of distant drug off-targets by direct superposition of binding pocket surfaces.

PubMed

Schumann, Marcel; Armen, Roger S

2013-01-01

Correctly predicting off-targets for a given molecular structure, which would have the ability to bind a large range of ligands, is both particularly difficult and important if they share no significant sequence or fold similarity with the respective molecular target ("distant off-targets"). A novel approach for identification of off-targets by direct superposition of protein binding pocket surfaces is presented and applied to a set of well-studied and highly relevant drug targets, including representative kinases and nuclear hormone receptors. The entire Protein Data Bank is searched for similar binding pockets and convincing distant off-target candidates were identified that share no significant sequence or fold similarity with the respective target structure. These putative target off-target pairs are further supported by the existence of compounds that bind strongly to both with high topological similarity, and in some cases, literature examples of individual compounds that bind to both. Also, our results clearly show that it is possible for binding pockets to exhibit a striking surface similarity, while the respective off-target shares neither significant sequence nor significant fold similarity with the respective molecular target ("distant off-target").

Unbiased Combinatorial Genomic Approaches to Identify Alternative Therapeutic Targets within the TSC Signaling Network

DTIC Science & Technology

2015-09-01

assessed the specificity of mutation in Drosophila S2R+ cells. We generated a quantitative mutation reporter vector in which an sgRNA target sequence ...phosphatases (563 genes) in the Drosophila genome (Figure 4). 65 samples that displayed synthetic lethality (15 genes) or synthetic increases in viability...targeting all kinases and phosphatases (563 genes) in the Drosophila genome . . Identified three hits (mRNA-Cap, Pitslre and CycT) that scored as

Acyl-Homoserine Lactone Production in Nitrifying Bacteria of the Genera Nitrosospira, Nitrobacter, and Nitrospira Identified via a Survey of Putative Quorum-Sensing Genes.

PubMed

Mellbye, Brett L; Spieck, Eva; Bottomley, Peter J; Sayavedra-Soto, Luis A

2017-11-15

The genomes of many bacteria that participate in nitrogen cycling through the process of nitrification contain putative genes associated with acyl-homoserine lactone (AHL) quorum sensing (QS). AHL QS or bacterial cell-cell signaling is a method of bacterial communication and gene regulation and may be involved in nitrogen oxide fluxes or other important phenotypes in nitrifying bacteria. Here, we carried out a broad survey of AHL production in nitrifying bacteria in three steps. First, we analyzed the evolutionary history of AHL synthase and AHL receptor homologs in sequenced genomes and metagenomes of nitrifying bacteria to identify AHL synthase homologs in ammonia-oxidizing bacteria (AOB) of the genus Nitrosospira and nitrite-oxidizing bacteria (NOB) of the genera Nitrococcus , Nitrobacter , and Nitrospira Next, we screened cultures of both AOB and NOB with uncharacterized AHL synthase genes and AHL synthase-negative nitrifiers by a bioassay. Our results suggest that an AHL synthase gene is required for, but does not guarantee, cell density-dependent AHL production under the conditions tested. Finally, we utilized mass spectrometry to identify the AHLs produced by the AOB Nitrosospira multiformis and Nitrosospira briensis and the NOB Nitrobacter vulgaris and Nitrospira moscoviensis as N -decanoyl-l-homoserine lactone (C 10 -HSL), N -3-hydroxy-tetradecanoyl-l-homoserine lactone (3-OH-C 14 -HSL), a monounsaturated AHL (C 10:1 -HSL), and N -octanoyl-l-homoserine lactone (C 8 -HSL), respectively. Our survey expands the list of AHL-producing nitrifiers to include a representative of Nitrospira lineage II and suggests that AHL production is widespread in nitrifying bacteria. IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite by nitrifying microorganisms, plays an important role in environmental nitrogen cycling from agricultural fertilization to wastewater treatment. The genomes of many nitrifying bacteria contain genes associated with

Fragment-Based Screening of a Natural Product Library against 62 Potential Malaria Drug Targets Employing Native Mass Spectrometry

PubMed Central

2018-01-01

Natural products are well known for their biological relevance, high degree of three-dimensionality, and access to areas of largely unexplored chemical space. To shape our understanding of the interaction between natural products and protein targets in the postgenomic era, we have used native mass spectrometry to investigate 62 potential protein targets for malaria using a natural-product-based fragment library. We reveal here 96 low-molecular-weight natural products identified as binding partners of 32 of the putative malarial targets. Seventy-nine (79) fragments have direct growth inhibition on Plasmodium falciparum at concentrations that are promising for the development of fragment hits against these protein targets. This adds a fragment library to the published HTS active libraries in the public domain. PMID:29436819

Fragment-Based Screening of a Natural Product Library against 62 Potential Malaria Drug Targets Employing Native Mass Spectrometry.

PubMed

Vu, Hoan; Pedro, Liliana; Mak, Tin; McCormick, Brendan; Rowley, Jessica; Liu, Miaomiao; Di Capua, Angela; Williams-Noonan, Billy; Pham, Ngoc B; Pouwer, Rebecca; Nguyen, Bao; Andrews, Katherine T; Skinner-Adams, Tina; Kim, Jessica; Hol, Wim G J; Hui, Raymond; Crowther, Gregory J; Van Voorhis, Wesley C; Quinn, Ronald J

2018-04-13

Natural products are well known for their biological relevance, high degree of three-dimensionality, and access to areas of largely unexplored chemical space. To shape our understanding of the interaction between natural products and protein targets in the postgenomic era, we have used native mass spectrometry to investigate 62 potential protein targets for malaria using a natural-product-based fragment library. We reveal here 96 low-molecular-weight natural products identified as binding partners of 32 of the putative malarial targets. Seventy-nine (79) fragments have direct growth inhibition on Plasmodium falciparum at concentrations that are promising for the development of fragment hits against these protein targets. This adds a fragment library to the published HTS active libraries in the public domain.

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target.

PubMed

Manguso, Robert T; Pope, Hans W; Zimmer, Margaret D; Brown, Flavian D; Yates, Kathleen B; Miller, Brian C; Collins, Natalie B; Bi, Kevin; LaFleur, Martin W; Juneja, Vikram R; Weiss, Sarah A; Lo, Jennifer; Fisher, David E; Miao, Diana; Van Allen, Eliezer; Root, David E; Sharpe, Arlene H; Doench, John G; Haining, W Nicholas

2017-07-27

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

Identification of plant glutaredoxin targets.

PubMed

Rouhier, Nicolas; Villarejo, Arsenio; Srivastava, Manoj; Gelhaye, Eric; Keech, Olivier; Droux, Michel; Finkemeier, Iris; Samuelsson, Göran; Dietz, Karl Josef; Jacquot, Jean-Pierre; Wingsle, Gunnar

2005-01-01

Glutaredoxins (Grxs) are small ubiquitous proteins of the thioredoxin (Trx) family, which catalyze dithiol-disulfide exchange reactions or reduce protein-mixed glutathione disulfides. In plants, several Trx-interacting proteins have been isolated from different compartments, whereas very few Grx-interacting proteins are known. We describe here the determination of Grx target proteins using a mutated poplar Grx, various tissular and subcellular plant extracts, and liquid chromatography coupled to tandem mass spectrometry detection. We have identified 94 putative targets, involved in many processes, including oxidative stress response [peroxiredoxins (Prxs), ascorbate peroxidase, catalase], nitrogen, sulfur, and carbon metabolisms (methionine synthase, alanine aminotransferase, phosphoglycerate kinase), translation (elongation factors E and Tu), or protein folding (heat shock protein 70). Some of these proteins were previously found to interact with Trx or to be glutathiolated in other organisms, but others could be more specific partners of Grx. To substantiate further these data, Grx was shown to support catalysis of the stroma beta-type carbonic anhydrase and Prx IIF of Arabidopsis thaliana, but not of poplar 2-Cys Prx. Overall, these data suggest that the interaction could occur randomly either with exposed cysteinyl disulfide bonds formed within or between target proteins or with mixed disulfides between a protein thiol and glutathione.

Complementary Approaches to Existing Target Based Drug Discovery for Identifying Novel Drug Targets.

PubMed

Vasaikar, Suhas; Bhatia, Pooja; Bhatia, Partap G; Chu Yaiw, Koon

2016-11-21

In the past decade, it was observed that the relationship between the emerging New Molecular Entities and the quantum of R&D investment has not been favorable. There might be numerous reasons but few studies stress the introduction of target based drug discovery approach as one of the factors. Although a number of drugs have been developed with an emphasis on a single protein target, yet identification of valid target is complex. The approach focuses on an in vitro single target, which overlooks the complexity of cell and makes process of validation drug targets uncertain. Thus, it is imperative to search for alternatives rather than looking at success stories of target-based drug discovery. It would be beneficial if the drugs were developed to target multiple components. New approaches like reverse engineering and translational research need to take into account both system and target-based approach. This review evaluates the strengths and limitations of known drug discovery approaches and proposes alternative approaches for increasing efficiency against treatment.

Researchers identify potential therapeutic targets for a rare childhood cancer | Center for Cancer Research

Cancer.gov

CCR researchers have identified the mechanism behind a rare but extremely aggressive childhood cancer called alveolar rhabdomyosarcoma (ARMS) and have pinpointed a potential drug target for its treatment. Learn more...

Pan-genome analysis of human gastric pathogen H. pylori: comparative genomics and pathogenomics approaches to identify regions associated with pathogenicity and prediction of potential core therapeutic targets.

PubMed

Ali, Amjad; Naz, Anam; Soares, Siomar C; Bakhtiar, Marriam; Tiwari, Sandeep; Hassan, Syed S; Hanan, Fazal; Ramos, Rommel; Pereira, Ulisses; Barh, Debmalya; Figueiredo, Henrique César Pereira; Ussery, David W; Miyoshi, Anderson; Silva, Artur; Azevedo, Vasco

2015-01-01

Helicobacter pylori is a human gastric pathogen implicated as the major cause of peptic ulcer and second leading cause of gastric cancer (~70%) around the world. Conversely, an increased resistance to antibiotics and hindrances in the development of vaccines against H. pylori are observed. Pan-genome analyses of the global representative H. pylori isolates consisting of 39 complete genomes are presented in this paper. Phylogenetic analyses have revealed close relationships among geographically diverse strains of H. pylori. The conservation among these genomes was further analyzed by pan-genome approach; the predicted conserved gene families (1,193) constitute ~77% of the average H. pylori genome and 45% of the global gene repertoire of the species. Reverse vaccinology strategies have been adopted to identify and narrow down the potential core-immunogenic candidates. Total of 28 nonhost homolog proteins were characterized as universal therapeutic targets against H. pylori based on their functional annotation and protein-protein interaction. Finally, pathogenomics and genome plasticity analysis revealed 3 highly conserved and 2 highly variable putative pathogenicity islands in all of the H. pylori genomes been analyzed.

The LIM-homeodomain transcription factor LMX1B regulates expression of NF-kappa B target genes

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

Rascle, Anne; Neumann, Tanja; Raschta, Anne-Sarah

2009-01-01

LMX1B is a LIM-homeodomain transcription factor essential for development. Putative LMX1B target genes have been identified through mouse gene targeting studies, but their identity as direct LMX1B targets remains hypothetical. We describe here the first molecular characterization of LMX1B target gene regulation. Microarray analysis using a tetracycline-inducible LMX1B expression system in HeLa cells revealed that a subset of NF-{kappa}B target genes, including IL-6 and IL-8, are upregulated in LMX1B-expressing cells. Inhibition of NF-{kappa}B activity by short interfering RNA-mediated knock-down of p65 impairs, while activation of NF-{kappa}B activity by TNF-{alpha} synergizes induction of NF-{kappa}B target genes by LMX1B. Chromatin immunoprecipitation demonstratedmore » that LMX1B binds to the proximal promoter of IL-6 and IL-8 in vivo, in the vicinity of the characterized {kappa}B site, and that LMX1B recruitment correlates with increased NF-{kappa}B DNA association. IL-6 promoter-reporter assays showed that the {kappa}B site and an adjacent putative LMX1B binding motif are both involved in LMX1B-mediated transcription. Expression of NF-{kappa}B target genes is affected in the kidney of Lmx1b{sup -/-} knock-out mice, thus supporting the biological relevance of our findings. Together, these data demonstrate for the first time that LMX1B directly regulates transcription of a subset of NF-{kappa}B target genes in cooperation with nuclear p50/p65 NF-{kappa}B.« less

Maize OXIDATIVE STRESS2 Homologs Enhance Cadmium Tolerance in Arabidopsis through Activation of a Putative SAM-Dependent Methyltransferase Gene.

PubMed

He, Lilong; Ma, Xiaoling; Li, Zhenzhen; Jiao, Zhengli; Li, Yongqing; Ow, David W

2016-07-01

Previously the Arabidopsis (Arabidopsis thaliana) zinc finger protein OXIDATIVE STRESS2 (AtOXS2) and four OXS2-like (AtO2L) family members were described to play a role in stress tolerance and stress escape. For stress escape, SOC1 was a target of AtOXS2. However, for stress tolerance, the downstream targets were not identified. We cloned two OXS2 homolog genes from sweet corn, ZmOXS2b and ZmO2L1 Both genes are transiently inducible by Cd treatment. When expressed in Arabidopsis, each enhances tolerance against cadmium. Further analysis showed that ZmOXS2b and ZmO2L1 proteins enhance Cd tolerance in Arabidopsis by activating at least one target gene, that encoding a putative S-adenosyl-l-Met-dependent methyltransferase superfamily protein (AT5G37990), which we named CIMT1 This activation involves the in vivo interaction with a segment of the CIMT1 promoter that contains a BOXS2 motif previously identified as the binding element for AtOXS2. More importantly, CIMT1 is induced by Cd treatment, and overexpression of this gene alone was sufficient to enhance Cd tolerance in Arabidopsis. The connection of ZmOXS2b and ZmO2L1 to Arabidopsis CIMT1 suggests a similar network may exist in maize (Zea mays) and may provide a clue to possibly using a CIMT1 maize homolog to engineer stress tolerance in a major crop. © 2016 American Society of Plant Biologists. All Rights Reserved.

Identification of Distant Drug Off-Targets by Direct Superposition of Binding Pocket Surfaces

PubMed Central

Schumann, Marcel; Armen, Roger S.

2013-01-01

Correctly predicting off-targets for a given molecular structure, which would have the ability to bind a large range of ligands, is both particularly difficult and important if they share no significant sequence or fold similarity with the respective molecular target (“distant off-targets”). A novel approach for identification of off-targets by direct superposition of protein binding pocket surfaces is presented and applied to a set of well-studied and highly relevant drug targets, including representative kinases and nuclear hormone receptors. The entire Protein Data Bank is searched for similar binding pockets and convincing distant off-target candidates were identified that share no significant sequence or fold similarity with the respective target structure. These putative target off-target pairs are further supported by the existence of compounds that bind strongly to both with high topological similarity, and in some cases, literature examples of individual compounds that bind to both. Also, our results clearly show that it is possible for binding pockets to exhibit a striking surface similarity, while the respective off-target shares neither significant sequence nor significant fold similarity with the respective molecular target (“distant off-target”). PMID:24391782

Chemical proteomics approaches for identifying the cellular targets of natural products

PubMed Central

Sieber, S. A.

2016-01-01

Covering: 2010 up to 2016 Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied “in situ” – in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide–alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss ‘competitive mode’ approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed. PMID:27098809

Chemical proteomics approaches for identifying the cellular targets of natural products.

PubMed

Wright, M H; Sieber, S A

2016-05-04

Covering: 2010 up to 2016Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied "in situ" - in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide-alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss 'competitive mode' approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed.

Prospects for detection of target-dependent annual modulation in direct dark matter searches

DOE PAGES

Nobile, Eugenio Del; Gelmini, Graciela B.; Witte, Samuel J.

2016-02-03

Earth's rotation about the Sun produces an annual modulation in the expected scattering rate at direct dark matter detection experiments. The annual modulation as a function of the recoil energy E R imparted by the dark matter particle to a target nucleus is expected to vary depending on the detector material. However, for most interactions a change of variables from E R to v min, the minimum speed a dark matter particle must have to impart a fixed E R to a target nucleus, produces an annual modulation independent of the target element. We recently showed that if the darkmore » matter-nucleus cross section contains a non-factorizable target and dark matter velocity dependence, the annual modulation as a function of v min can be target dependent. Here we examine more extensively the necessary conditions for target-dependent modulation, its observability in present-day experiments, and the extent to which putative signals could identify a dark matter-nucleus differential cross section with a non-factorizable dependence on the dark matter velocity.« less

Endothelial cell palmitoylproteomics identifies novel lipid modified targets and potential substrates for protein acyl transferases

PubMed Central

Marin, Ethan P.; Derakhshan, Behrad; Lam, TuKiet T.; Davalos, Alberto; Sessa, William C.

2012-01-01

Rationale Protein S-palmitoylation is the post-translational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well understood, in part due to technological limits on palmitoylprotein detection. Objective To develop a method using acyl-biotinyl exchange (ABE) technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in EC. Methods and Results More than 150 putative palmitoyl proteins were identified in EC using ABE and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase 1 (SOD1), an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine 6 prevents palmitoylation, leads to reduction in SOD1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for SOD1 palmitoylation. Moreover, we used ABE to search for substrates of particular protein acyl transferases in EC. We found that palmitoylation of the cell adhesion protein PECAM1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of PECAM1 at the cell surface. Conclusions Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important post-translational lipid modification in EC biology. PMID:22496122

Emissions of putative isoprene oxidation products from mango branches under abiotic stress

PubMed Central

Jardine, Kolby J.; Meyers, Kimberly; Abrell, Leif; Alves, Eliane G.; Yanez Serrano, Ana Maria; Kesselmeier, Jürgen; Karl, Thomas; Guenther, Alex; Vickers, Claudia; Chambers, Jeffrey Q.

2013-01-01

Although several per cent of net carbon assimilation can be re-released as isoprene emissions to the atmosphere by many tropical plants, much uncertainty remains regarding its biological significance. In a previous study, we detected emissions of isoprene and its oxidation products methyl vinyl ketone (MVK) and methacrolein (MACR) from tropical plants under high temperature/light stress, suggesting that isoprene is oxidized not only in the atmosphere but also within plants. However, a comprehensive analysis of the suite of isoprene oxidation products in plants has not been performed and production relationships with environmental stress have not been described. In this study, putative isoprene oxidation products from mango (Mangifera indica) branches under abiotic stress were first identified. High temperature/light and freeze–thaw treatments verified direct emissions of the isoprene oxidation products MVK and MACR together with the first observations of 3-methyl furan (3-MF) and 2-methyl-3-buten-2-ol (MBO) as putative novel isoprene oxidation products. Mechanical wounding also stimulated emissions of MVK and MACR. Photosynthesis under 13CO2 resulted in rapid (<30min) labelling of up to five carbon atoms of isoprene, with a similar labelling pattern observed in the putative oxidation products. These observations highlight the need to investigate further the mechanisms of isoprene oxidation within plants under stress and its biological and atmospheric significance. PMID:23881400

Emissions of putative isoprene oxidation products from mango branches under abiotic stress.

PubMed

Jardine, Kolby J; Meyers, Kimberly; Abrell, Leif; Alves, Eliane G; Yanez Serrano, Ana Maria; Kesselmeier, Jürgen; Karl, Thomas; Guenther, Alex; Chambers, Jeffrey Q; Vickers, Claudia

2013-09-01

Although several per cent of net carbon assimilation can be re-released as isoprene emissions to the atmosphere by many tropical plants, much uncertainty remains regarding its biological significance. In a previous study, we detected emissions of isoprene and its oxidation products methyl vinyl ketone (MVK) and methacrolein (MACR) from tropical plants under high temperature/light stress, suggesting that isoprene is oxidized not only in the atmosphere but also within plants. However, a comprehensive analysis of the suite of isoprene oxidation products in plants has not been performed and production relationships with environmental stress have not been described. In this study, putative isoprene oxidation products from mango (Mangifera indica) branches under abiotic stress were first identified. High temperature/light and freeze-thaw treatments verified direct emissions of the isoprene oxidation products MVK and MACR together with the first observations of 3-methyl furan (3-MF) and 2-methyl-3-buten-2-ol (MBO) as putative novel isoprene oxidation products. Mechanical wounding also stimulated emissions of MVK and MACR. Photosynthesis under (13)CO2 resulted in rapid (<30 min) labelling of up to five carbon atoms of isoprene, with a similar labelling pattern observed in the putative oxidation products. These observations highlight the need to investigate further the mechanisms of isoprene oxidation within plants under stress and its biological and atmospheric significance.

Characterisation of putative oxygen chemoreceptors in bowfin (Amia calva).

PubMed

Porteus, Cosima S; Wright, Patricia A; Milsom, William K

2014-04-15

Serotonin containing neuroepithelial cells (NECs) are putative oxygen sensing cells found in different locations within the gills of fish. In this study we wished to determine the effect of sustained internal (blood) hypoxaemia versus external (aquatic) hypoxia on the size and density of NECs in the first gill arch of bowfin (Amia calva), a facultative air breather. We identified five different populations of serotonergic NECs in this species (Types I-V) based on location, presence of synaptic vesicles (SV) that stain for the antibody SV2, innervation and labelling with the neural crest marker HNK-1. Cell Types I-III were innervated, and these cells, which participate in central O2 chemoreflexes, were studied further. Although there was no change in the density of any cell type in bowfin after exposure to sustained hypoxia (6.0 kPa for 7 days) without access to air, all three of these cell types increased in size. In contrast, only Type II and III cells increased in size in bowfin exposed to sustained hypoxia with access to air. These data support the suggestion that NECs are putative oxygen-sensing cells, that they occur in several locations, and that Type I cells monitor only hypoxaemia, whereas both other cell types monitor hypoxia and hypoxaemia.

Duplications and losses in gene families of rust pathogens highlight putative effectors.

PubMed

Pendleton, Amanda L; Smith, Katherine E; Feau, Nicolas; Martin, Francis M; Grigoriev, Igor V; Hamelin, Richard; Nelson, C Dana; Burleigh, J Gordon; Davis, John M

2014-01-01

Rust fungi are a group of fungal pathogens that cause some of the world's most destructive diseases of trees and crops. A shared characteristic among rust fungi is obligate biotrophy, the inability to complete a lifecycle without a host. This dependence on a host species likely affects patterns of gene expansion, contraction, and innovation within rust pathogen genomes. The establishment of disease by biotrophic pathogens is reliant upon effector proteins that are encoded in the fungal genome and secreted from the pathogen into the host's cell apoplast or within the cells. This study uses a comparative genomic approach to elucidate putative effectors and determine their evolutionary histories. We used OrthoMCL to identify nearly 20,000 gene families in proteomes of 16 diverse fungal species, which include 15 basidiomycetes and one ascomycete. We inferred patterns of duplication and loss for each gene family and identified families with distinctive patterns of expansion/contraction associated with the evolution of rust fungal genomes. To recognize potential contributors for the unique features of rust pathogens, we identified families harboring secreted proteins that: (i) arose or expanded in rust pathogens relative to other fungi, or (ii) contracted or were lost in rust fungal genomes. While the origin of rust fungi appears to be associated with considerable gene loss, there are many gene duplications associated with each sampled rust fungal genome. We also highlight two putative effector gene families that have expanded in Cqf that we hypothesize have roles in pathogenicity.

Complete Genome Sequence of a Putative Densovirus of the Asian Citrus Psyllid, Diaphorina citri.

PubMed

Nigg, Jared C; Nouri, Shahideh; Falk, Bryce W

2016-07-28

Here, we report the complete genome sequence of a putative densovirus of the Asian citrus psyllid, Diaphorina citri Diaphorina citri densovirus (DcDNV) was originally identified through metagenomics, and here, we obtained the complete nucleotide sequence using PCR-based approaches. Phylogenetic analysis places DcDNV between viruses of the Ambidensovirus and Iteradensovirus genera. Copyright © 2016 Nigg et al.

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.

Putative human sperm Interactome: a networks study.

PubMed

Ordinelli, Alessandra; Bernabò, Nicola; Orsini, Massimiliano; Mattioli, Mauro; Barboni, Barbara

2018-04-11

For over sixty years, it has been known that mammalian spermatozoa immediately after ejaculation are virtually infertile. They became able to fertilize only after they reside for long time (hours to days) within female genital tract where they complete their functional maturation, the capacitation. This process is finely regulated by the interaction with the female environment and involves, in spermatozoa, a myriad of molecules as messengers and target of signals. Since, to date, a model able to represent the molecular interaction that characterize sperm physiology does not exist, we realized the Human Sperm Interactme Network3.0 (HSIN3.0) and its main component (HSNI3.0_MC), starting from the pathway active in male germ cells. HSIN3.0 and HSIN3.0_MC are scale free networks, adherent to the Barabasi-Albert model, and are characterised by an ultra-small world topology. We found that they are resistant to random attacks and that are designed to respond quickly and specifically to external inputs. In addition, it has been possible to identify the most connected nodes (the hubs) and the bottlenecks nodes. This result allowed us to explore the control mechanisms active in driving sperm biochemical machinery and to verify the different levels of controls: party vs. date hubs and hubs vs. bottlenecks, thanks the availability of data from KO mice. Finally, we found that several key nodes represent molecules specifically involved in function that are thought to be not present or not active in sperm cells, such as control of cell cycle, proteins synthesis, nuclear trafficking, and immune response, thus potentially open new perspectives on the study of sperm biology. For the first time we present a network representing putative human sperm interactome. This result gives very intriguing biological information and could contribute to the knowledge of spermatozoa, either in physiological or pathological conditions.

Threading the Needle: Small-Molecule Targeting of a Xenobiotic Receptor to Ablate Escherichia coli Polysaccharide Capsule Expression Without Altering Antibiotic Resistance.

PubMed

Arshad, Mehreen; Goller, Carlos C; Pilla, Danielle; Schoenen, Frank J; Seed, Patrick C

2016-04-15

Uropathogenic Escherichia coli (UPEC), a leading cause of urinary tract and invasive infections worldwide, is rapidly acquiring multidrug resistance, hastening the need for selective new anti-infective agents. Here we demonstrate the molecular target of DU011, our previously discovered potent, nontoxic, small-molecule inhibitor of UPEC polysaccharide capsule biogenesis and virulence. Real-time polymerase chain reaction analysis and a target-overexpression drug-suppressor screen were used to localize the putative inhibitor target. A thermal shift assay quantified interactions between the target protein and the inhibitor, and a novel DNase protection assay measured chemical inhibition of protein-DNA interactions. Virulence of a regulatory target mutant was assessed in a murine sepsis model. MprA, a MarR family transcriptional repressor, was identified as the putative target of the DU011 inhibitor. Thermal shift measurements indicated the formation of a stable DU011-MprA complex, and DU011 abrogated MprA binding to its DNA promoter site. Knockout of mprA had effects similar to that of DU011 treatment of wild-type bacteria: a loss of encapsulation and complete attenuation in a murine sepsis model, without any negative change in antibiotic resistance. MprA regulates UPEC polysaccharide encapsulation, is essential for UPEC virulence, and can be targeted without inducing antibiotic resistance. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

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.

Systematic Identification, Characterization and Target Gene Analysis of microRNAs Involved in Osteoarthritis Subchondral Bone Pathogenesis.

PubMed

Prasadam, Indira; Batra, Jyotsna; Perry, Samuel; Gu, Wenyi; Crawford, Ross; Xiao, Yin

2016-07-01

This study aimed to identify the microRNAs associated with sclerotic status of subchondral bone in the pathogenesis of osteoarthritis (OA). Total RNA was extracted from non-sclerotic and sclerotic OA subchondral bone from patients undergoing knee replacement surgeries. miRCURY™ LNA miRNA chip and qRT-PCR were used to profile and validate differential microRNA expression. In addition, we further confirmed profiles of altered miRNAs in an OA rat meniscectomy animal model and their putative targets of the miRNAs were predicted using ingenuity (IPA) software. Finally, five short-listed miRNAs were reactivated by transient in vitro overexpression (miRNA mimics) in subchondral bone osteoblasts and their phenotypes were assessed. Functional screening identified 30 differentiated miRNAs in sclerotic subchondral bone compared to non-sclerotic bone of OA patients. Data integration resulted in confirmation of the eight miRNAs, with aberrant expression in independent human OA bone sample set. In silico analysis (IPA) identified 732 mRNA transcripts as putative targets of the eight altered miRNAs, of which twenty genes were validated to be differentially expressed in sclerotic compared to non-sclerotic bone samples. Out of eight dysregulated miRNA's, five of them showed consistent time-dependent downregulation in a rat OA model. Furthermore, synthetic miR-199a-3p, miR-199a-5p, miR-590-5p, and miR-211-5p mimics rescued the abnormal osteoarthritic subchondral bone osteoblast gene expression and mineralization. We have identified four novel miRNAs that play important roles in subchondral bone pathogenesis in OA. Additional studies are required to develop these miRNAs into therapeutic modalities for OA.

Application of Targeted Functional Assays to Assess a Putative Vascular Disruption Developmental Toxicity Pathway Informed By ToxCast High-Throughput Screening Data

EPA Science Inventory

Chemical perturbation of vascular development is a putative toxicity pathway which may result in developmental toxicity. EPA’s high-throughput screening (HTS) ToxCast program contains assays which measure cellular signals and biological processes critical for blood vessel develop...

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.

Actin dynamics at focal adhesions: a common endpoint and putative therapeutic target for proteinuric kidney diseases.

PubMed

Sever, Sanja; Schiffer, Mario

2018-06-01

Proteinuria encompasses diverse causes including both genetic diseases and acquired forms such as diabetic and hypertensive nephropathy. The basis of proteinuria is a disturbance in size selectivity of the glomerular filtration barrier, which largely depends on the podocyte: a terminally differentiated epithelial cell type covering the outer surface of the glomerulus. Compromised podocyte structure is one of the earliest signs of glomerular injury. The phenotype of diverse animal models and podocyte cell culture firmly established the essential role of the actin cytoskeleton in maintaining functional podocyte structure. Podocyte foot processes, actin-based membrane extensions, contain 2 molecularly distinct "hubs" that control actin dynamics: a slit diaphragm and focal adhesions. Although loss of foot processes encompasses disassembly of slit diaphragm multiprotein complexes, as long as cells are attached to the glomerular basement membrane, focal adhesions will be the sites in which stress due to filtration flow is counteracted by forces generated by the actin network in foot processes. Numerous studies within last 20 years have identified actin binding and regulatory proteins as well as integrins as essential components of signaling and actin dynamics at focal adhesions in podocytes, suggesting that some of them may become novel, druggable targets for proteinuric kidney diseases. Here we review evidence supporting the idea that current treatments for chronic kidney diseases beneficially and directly target the podocyte actin cytoskeleton associated with focal adhesions and suggest that therapeutic reagents that target the focal adhesion-regulated actin cytoskeleton in foot processes have potential to modernize treatments for chronic kidney diseases. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Vitiligo blood transcriptomics provides new insights into disease mechanisms and identifies potential novel therapeutic targets.

PubMed

Dey-Rao, Rama; Sinha, Animesh A

2017-01-28

Significant gaps remain regarding the pathomechanisms underlying the autoimmune response in vitiligo (VL), where the loss of self-tolerance leads to the targeted killing of melanocytes. Specifically, there is incomplete information regarding alterations in the systemic environment that are relevant to the disease state. We undertook a genome-wide profiling approach to examine gene expression in the peripheral blood of VL patients and healthy controls in the context of our previously published VL-skin gene expression profile. We used several in silico bioinformatics-based analyses to provide new insights into disease mechanisms and suggest novel targets for future therapy. Unsupervised clustering methods of the VL-blood dataset demonstrate a "disease-state"-specific set of co-expressed genes. Ontology enrichment analysis of 99 differentially expressed genes (DEGs) uncovers a down-regulated immune/inflammatory response, B-Cell antigen receptor (BCR) pathways, apoptosis and catabolic processes in VL-blood. There is evidence for both type I and II interferon (IFN) playing a role in VL pathogenesis. We used interactome analysis to identify several key blood associated transcriptional factors (TFs) from within (STAT1, STAT6 and NF-kB), as well as "hidden" (CREB1, MYC, IRF4, IRF1, and TP53) from the dataset that potentially affect disease pathogenesis. The TFs overlap with our reported lesional-skin transcriptional circuitry, underscoring their potential importance to the disease. We also identify a shared VL-blood and -skin transcriptional "hot spot" that maps to chromosome 6, and includes three VL-blood dysregulated genes (PSMB8, PSMB9 and TAP1) described as potential VL-associated genetic susceptibility loci. Finally, we provide bioinformatics-based support for prioritizing dysregulated genes in VL-blood or skin as potential therapeutic targets. We examined the VL-blood transcriptome in context with our (previously published) VL-skin transcriptional profile to address

Identification of the receptor tyrosine kinase AXL in breast cancer as a target for the human miR-34a microRNA

PubMed Central

Mackiewicz, Mark; Huppi, Konrad; Pitt, Jason J.; Dorsey, Tiffany H.; Ambs, Stefan

2012-01-01

The identification of molecular features that contribute to the progression of breast cancer can provide valuable insight into the pathogenesis of this disease. Deregulated microRNA expression represents one type of molecular event that has been associated with many different human cancers. In order to identify a miRNA/mRNA regulatory interaction that is biologically relevant to the triple-negative breast cancer genotype/phenotype, we initially conducted a miRNA profiling experiment to detect differentially expressed miRNAs in cell line models representing triple-negative (MDA-MB-231), ER+ (MCF7), and HER-2 over expressed (SK-BR-3) histotypes. We identified human miR-34a expression as being >3-fold down (from its median expression value across all cell lines) in MDA-MB-231 cells, and identified AXL as a putative mRNA target using multiple miRNA/target prediction algorithms. The miR-34a/AXL interaction was functionally characterized through ectopic over expression experiments with a miR-34a mimic in two independent triple-negative breast cancer cell lines. In reporter assays, miR-34a binds to its putative target site within the AXL 3′UTR to inhibit luciferase expression. We also observed degradation of AXL mRNA and decreased AXL protein levels, as well as cell signaling effects on AKT phosphorylation and phenotypic effects on cell migration. Finally, we present an inverse correlative trend in miR-34a and AXL expression for both cell line and patient tumor samples. PMID:21814748

Integrative screening approach identifies regulators of polyploidization and targets for acute megakaryocytic leukemia

PubMed Central

Wen, Qiang; Goldenson, Benjamin; Silver, Serena J.; Schenone, Monica; Dancik, Vladimir; Huang, Zan; Wang, Ling-Zhi; Lewis, Timothy; An, W. Frank; Li, Xiaoyu; Bray, Mark-Anthony; Thiollier, Clarisse; Diebold, Lauren; Gilles, Laure; Vokes, Martha S.; Moore, Christopher B.; Bliss-Moreau, Meghan; VerPlank, Lynn; Tolliday, Nicola J.; Mishra, Rama; Vemula, Sasidhar; Shi, Jianjian; Wei, Lei; Kapur, Reuben; Lopez, Cécile K.; Gerby, Bastien; Ballerini, Paola; Pflumio, Francoise; Gilliland, D. Gary; Goldberg, Liat; Birger, Yehudit; Izraeli, Shai; Gamis, Alan S.; Smith, Franklin O.; Woods, William G.; Taub, Jeffrey; Scherer, Christina A.; Bradner, James; Goh, Boon-Cher; Mercher, Thomas; Carpenter, Anne E.; Gould, Robert J.; Clemons, Paul A.; Carr, Steven A.; Root, David E.; Schreiber, Stuart L.; Stern, Andrew M.; Crispino, John D.

2012-01-01

Summary The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. We found that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. A broadly applicable, highly integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora A kinase (AURKA), which has not been studied extensively in megakaryocytes. Moreover, we discovered that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in AMKL blasts and displayed potent anti-AMKL activity in vivo. This research provides the rationale to support clinical trials of MLN8237 and other inducers of polyploidization in AMKL. Finally, we have identified five networks of kinases that regulate the switch to polyploidy. PMID:22863010

The Near-Earth Object Human Space Flight Accessible Targets Study (NHATS) List of Near-Earth Asteroids: Identifying Potential Targets for Future Exploration

NASA Technical Reports Server (NTRS)

Abell, Paul A.; Barbee, B. W.; Mink, R. G.; Alberding, C. M.; Adamo, D. R.; Mazanek, D. D.; Johnson, L. N.; Yeomans, D. K.; Chodas, P. W.; Chamberlin, A. B.;

A putative hybrid swarm within Oonopsis foliosa (Asteraceae: Astereae)

USGS Publications Warehouse

Hughes, J.F.; Brown, G.K.

2004-01-01

Oo??nopsis foliosa var. foliosa and var. monocephala are endemic to short-grass steppe of southeastern Colorado and until recently were considered geographically disjunct. The only known qualitative feature separating these 2 varieties is floral head type; var. foliosa has radiate heads, whereas var. monocephala heads are discoid. Sympatry between these varieties is restricted to a small area in which a range of parental types and intermediate head morphologies is observed. We used distribution mapping, morphometric analyses, chromosome cytology, and pollen stainability to characterize the sympatric zone. Morphometrics confirms that the only discrete difference between var. foliosa and var. monocephala is radiate versus discoid heads, respectively. The outer florets of putative hybrid individuals ranged from conspicuously elongated yet radially symmetric disc-floret corollas, to elongated radially asymmetric bilabiate- or deeply cleft corollas, to stunted ray florets with appendages remnant of corolla lobes. Chromosome cytology of pollen mother cells from both putative parental varieties and a series of intermediate morphological types collected at the sympatric zone reveal evidence of translocation heterozygosity. Pollen stainability shows no significant differences in viability between the parental varieties and putative hybrids. The restricted distribution of putative hybrids to a narrow zone of sympatry between the parental types and the presence of meiotic chromosome-pairing anomalies in these intermediate plants are consistent with a hybrid origin. The high stainability of putative-hybrid pollen adds to a growing body of evidence that hybrids are not universally unfit.

Network Understanding of Herb Medicine via Rapid Identification of Ingredient-Target Interactions

NASA Astrophysics Data System (ADS)

Zhang, Hai-Ping; Pan, Jian-Bo; Zhang, Chi; Ji, Nan; Wang, Hao; Ji, Zhi-Liang

2014-01-01

Today, herb medicines have become the major source for discovery of novel agents in countermining diseases. However, many of them are largely under-explored in pharmacology due to the limitation of current experimental approaches. Therefore, we proposed a computational framework in this study for network understanding of herb pharmacology via rapid identification of putative ingredient-target interactions in human structural proteome level. A marketing anti-cancer herb medicine in China, Yadanzi (Brucea javanica), was chosen for mechanistic study. Total 7,119 ingredient-target interactions were identified for thirteen Yadanzi active ingredients. Among them, about 29.5% were estimated to have better binding affinity than their corresponding marketing drug-target interactions. Further Bioinformatics analyses suggest that simultaneous manipulation of multiple proteins in the MAPK signaling pathway and the phosphorylation process of anti-apoptosis may largely answer for Yadanzi against non-small cell lung cancers. In summary, our strategy provides an efficient however economic solution for systematic understanding of herbs' power.

Network understanding of herb medicine via rapid identification of ingredient-target interactions.

PubMed

Zhang, Hai-Ping; Pan, Jian-Bo; Zhang, Chi; Ji, Nan; Wang, Hao; Ji, Zhi-Liang

2014-01-16

Today, herb medicines have become the major source for discovery of novel agents in countermining diseases. However, many of them are largely under-explored in pharmacology due to the limitation of current experimental approaches. Therefore, we proposed a computational framework in this study for network understanding of herb pharmacology via rapid identification of putative ingredient-target interactions in human structural proteome level. A marketing anti-cancer herb medicine in China, Yadanzi (Brucea javanica), was chosen for mechanistic study. Total 7,119 ingredient-target interactions were identified for thirteen Yadanzi active ingredients. Among them, about 29.5% were estimated to have better binding affinity than their corresponding marketing drug-target interactions. Further Bioinformatics analyses suggest that simultaneous manipulation of multiple proteins in the MAPK signaling pathway and the phosphorylation process of anti-apoptosis may largely answer for Yadanzi against non-small cell lung cancers. In summary, our strategy provides an efficient however economic solution for systematic understanding of herbs' power.

Identifying biomarkers for asthma diagnosis using targeted metabolomics approaches.

PubMed

Checkley, William; Deza, Maria P; Klawitter, Jost; Romero, Karina M; Klawitter, Jelena; Pollard, Suzanne L; Wise, Robert A; Christians, Uwe; Hansel, Nadia N

2016-12-01

The diagnosis of asthma in children is challenging and relies on a combination of clinical factors and biomarkers including methacholine challenge, lung function, bronchodilator responsiveness, and presence of airway inflammation. No single test is diagnostic. We sought to identify a pattern of inflammatory biomarkers that was unique to asthma using a targeted metabolomics approach combined with data science methods. We conducted a nested case-control study of 100 children living in a peri-urban community in Lima, Peru. We defined cases as children with current asthma, and controls as children with no prior history of asthma and normal lung function. We further categorized enrollment following a factorial design to enroll equal numbers of children as either overweight or not. We obtained a fasting venous blood sample to characterize a comprehensive panel of targeted markers using a metabolomics approach based on high performance liquid chromatography-mass spectrometry. A statistical comparison of targeted metabolites between children with asthma (n = 50) and healthy controls (n = 49) revealed distinct patterns in relative concentrations of several metabolites: children with asthma had approximately 40-50% lower relative concentrations of ascorbic acid, 2-isopropylmalic acid, shikimate-3-phosphate, and 6-phospho-d-gluconate when compared to children without asthma, and 70% lower relative concentrations of reduced glutathione (all p < 0.001 after Bonferroni correction). Moreover, a combination of 2-isopropylmalic acid and betaine strongly discriminated between children with asthma (2-isopropylmalic acid ≤ 13 077 normalized counts/second) and controls (2-isopropylmalic acid > 13 077 normalized counts/second and betaine ≤ 16 47 121 normalized counts/second). By using a metabolomics approach applied to serum, we were able to discriminate between children with and without asthma by revealing different metabolic patterns. These results suggest that

Dihydroartemisinin as a Putative STAT3 Inhibitor, Suppresses the Growth of Head and Neck Squamous Cell Carcinoma by Targeting Jak2/STAT3 Signaling

PubMed Central

Jia, Lifeng; Song, Qi; Zhou, Chenyang; Li, Xiaoming; Pi, Lihong; Ma, Xiuru; Li, Hui; Lu, Xiuying; Shen, Yupeng

2016-01-01

Developing drugs that can effectively block STAT3 activation may serve as one of the most promising strategy for cancer treatment. Currently, there is no putative STAT3 inhibitor that can be safely and effectively used in clinic. In the present study, we investigated the potential of dihydroartemisinin (DHA) as a putative STAT3 inhibitor and its antitumor activities in head and neck squamous cell carcinoma (HNSCC). The inhibitory effects of DHA on STAT3 activation along with its underlying mechanisms were studied in HNSCC cells. The antitumor effects of DHA against HNSCC cells were explored both in vitro and in vivo. An investigation on cooperative effects of DHA with cisplatin in killing HNSCC cells was also implemented. DHA exhibited remarkable and specific inhibitory effects on STAT3 activation via selectively blocking Jak2/STAT3 signaling. Besides, DHA significantly inhibited HNSCC growth both in vitro and in vivo possibly through induction of apoptosis and attenuation of cell migration. DHA also synergized with cisplatin in tumor inhibition in HNSCC cells. Our findings demonstrate that DHA is a putative STAT3 inhibitor that may represent a new and effective drug for cancer treatment and therapeutic sensitization in HNSCC patients. PMID:26784960

Differential microRNA Analysis of Glandular Trichomes and Young Leaves in Xanthium strumarium L. Reveals Their Putative Roles in Regulating Terpenoid Biosynthesis.

PubMed

Fan, Rongyan; Li, Yuanjun; Li, Changfu; Zhang, Yansheng

2015-01-01

The medicinal plant Xanthium strumarium L. (X. strumarium) is covered with glandular trichomes, which are the sites for synthesizing pharmacologically active terpenoids such as xanthatin. MicroRNAs (miRNAs) are a class of 21-24 nucleotide (nt) non-coding RNAs, most of which are identified as regulators of plant growth development. Identification of miRNAs involved in the biosynthesis of plant secondary metabolites remains limited. In this study, high-throughput Illumina sequencing, combined with target gene prediction, was performed to discover novel and conserved miRNAs with potential roles in regulating terpenoid biosynthesis in X. strumarium glandular trichomes. Two small RNA libraries from leaves and glandular trichomes of X. strumarium were established. In total, 1,185 conserved miRNAs and 37 novel miRNAs were identified, with 494 conserved miRNAs and 18 novel miRNAs being differentially expressed between the two tissue sources. Based on the X. strumarium transcriptome data that we recently constructed, 3,307 annotated mRNA transcripts were identified as putative targets of the differentially expressed miRNAs. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that some of the differentially expressed miRNAs, including miR6435, miR5021 and miR1134, might be involved in terpenoid biosynthesis in the X. strumarium glandular trichomes. This study provides the first comprehensive analysis of miRNAs in X. strumarium, which forms the basis for further understanding of miRNA-based regulation on terpenoid biosynthesis.

Differential microRNA Analysis of Glandular Trichomes and Young Leaves in Xanthium strumarium L. Reveals Their Putative Roles in Regulating Terpenoid Biosynthesis

PubMed Central

Fan, Rongyan; Li, Yuanjun; Li, Changfu; Zhang, Yansheng

2015-01-01

The medicinal plant Xanthium strumarium L. (X. strumarium) is covered with glandular trichomes, which are the sites for synthesizing pharmacologically active terpenoids such as xanthatin. MicroRNAs (miRNAs) are a class of 21–24 nucleotide (nt) non-coding RNAs, most of which are identified as regulators of plant growth development. Identification of miRNAs involved in the biosynthesis of plant secondary metabolites remains limited. In this study, high-throughput Illumina sequencing, combined with target gene prediction, was performed to discover novel and conserved miRNAs with potential roles in regulating terpenoid biosynthesis in X. strumarium glandular trichomes. Two small RNA libraries from leaves and glandular trichomes of X. strumarium were established. In total, 1,185 conserved miRNAs and 37 novel miRNAs were identified, with 494 conserved miRNAs and 18 novel miRNAs being differentially expressed between the two tissue sources. Based on the X. strumarium transcriptome data that we recently constructed, 3,307 annotated mRNA transcripts were identified as putative targets of the differentially expressed miRNAs. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that some of the differentially expressed miRNAs, including miR6435, miR5021 and miR1134, might be involved in terpenoid biosynthesis in the X. strumarium glandular trichomes. This study provides the first comprehensive analysis of miRNAs in X. strumarium, which forms the basis for further understanding of miRNA-based regulation on terpenoid biosynthesis. PMID:26406988

Promoter-enhancer interactions identified from Hi-C data using probabilistic models and hierarchical topological domains.

PubMed

Ron, Gil; Globerson, Yuval; Moran, Dror; Kaplan, Tommy

2017-12-21

Proximity-ligation methods such as Hi-C allow us to map physical DNA-DNA interactions along the genome, and reveal its organization into topologically associating domains (TADs). As the Hi-C data accumulate, computational methods were developed for identifying domain borders in multiple cell types and organisms. Here, we present PSYCHIC, a computational approach for analyzing Hi-C data and identifying promoter-enhancer interactions. We use a unified probabilistic model to segment the genome into domains, which we then merge hierarchically and fit using a local background model, allowing us to identify over-represented DNA-DNA interactions across the genome. By analyzing the published Hi-C data sets in human and mouse, we identify hundreds of thousands of putative enhancers and their target genes, and compile an extensive genome-wide catalog of gene regulation in human and mouse. As we show, our predictions are highly enriched for ChIP-seq and DNA accessibility data, evolutionary conservation, eQTLs and other DNA-DNA interaction data.

Identifying mechanism-of-action targets for drugs and probes

PubMed Central

Gregori-Puigjané, Elisabet; Setola, Vincent; Hert, Jérôme; Crews, Brenda A.; Irwin, John J.; Lounkine, Eugen; Marnett, Lawrence; Roth, Bryan L.; Shoichet, Brian K.

2012-01-01

Notwithstanding their key roles in therapy and as biological probes, 7% of approved drugs are purported to have no known primary target, and up to 18% lack a well-defined mechanism of action. Using a chemoinformatics approach, we sought to “de-orphanize” drugs that lack primary targets. Surprisingly, targets could be easily predicted for many: Whereas these targets were not known to us nor to the common databases, most could be confirmed by literature search, leaving only 13 Food and Drug Administration—approved drugs with unknown targets; the number of drugs without molecular targets likely is far fewer than reported. The number of worldwide drugs without reasonable molecular targets similarly dropped, from 352 (25%) to 44 (4%). Nevertheless, there remained at least seven drugs for which reasonable mechanism-of-action targets were unknown but could be predicted, including the antitussives clemastine, cloperastine, and nepinalone; the antiemetic benzquinamide; the muscle relaxant cyclobenzaprine; the analgesic nefopam; and the immunomodulator lobenzarit. For each, predicted targets were confirmed experimentally, with affinities within their physiological concentration ranges. Turning this question on its head, we next asked which drugs were specific enough to act as chemical probes. Over 100 drugs met the standard criteria for probes, and 40 did so by more stringent criteria. A chemical information approach to drug-target association can guide therapeutic development and reveal applications to probe biology, a focus of much current interest. PMID:22711801

Aberrant TGFβ/SMAD4 signaling contributes to epigenetic silencing of a putative tumor suppressor, RunX1T1, in ovarian cancer

PubMed Central

Yang, Hui-Wen; Chou, Jian-Liang; Chen, Lin-Yu; Yeh, Chia-Ming; Chen, Yu-Hsin; Lin, Ru-Inn; Su, Her-Young; Chen, Gary CW; Deatherage, Daniel E; Huang, Yi-Wen; Yan, Pearlly S; Lin, Huey-Jen; Nephew, Kenneth P; Huang, Tim H-M; Lai, Hung-Cheng

2011-01-01

Aberrant TGFβ signaling pathway may alter the expression of down-stream targets and promotes ovarian carcinogenesis. However, the mechanism of this impairment is not fully understood. Our previous study identified RunX1T1 as a putative SMAD4 target in an immortalized ovarian surface epithelial cell line, IOSE. In this study, we report that transcription of RunX1T1 was confirmed to be positively regulated by SMAD4 in IOSE cells and epigenetically silenced in a panel of ovarian cancer cell lines by promoter hypermethylation and histone methylation at H3 lysine 9. SMAD4 depletion increased repressive histone modifications of RunX1T1 promoter without affecting promoter methylation in IOSE cells. Epigenetic treatment can restore RunX1T1 expression by reversing its epigenetic status in MCP 3 ovarian cancer cells. When transiently treated with a demethylating agent, the expression of RunX1T1 was partially restored in MCP 3 cells, but gradual re-silencing through promoter re-methylation was observed after the treatment. Interestingly, SMAD4 knockdown accelerated this re-silencing process, suggesting that normal TGFβ signaling is essential for the maintenance of RunX1T1 expression. In vivo analysis confirmed that hypermethylation of RunX1T1 was detected in 35.7% (34/95) of ovarian tumors with high clinical stages (p = 0.035) and in 83% (5/6) of primary ovarian cancer-initiating cells. Additionally, concurrent methylation of RunX1T1 and another SMAD4 target, FBXO32 which was previously found to be hypermethylated in ovarian cancer was observed in this same sample cohort (p < 0.05). Restoration of RunX1T1 inhibited cancer cell growth. Taken together, dysregulated TGFβ/SMAD4 signaling may lead to epigenetic silencing of a putative tumor suppressor, RunX1T1, during ovarian carcinogenesis. PMID:21540640

Omics of Brucella: Species-Specific sRNA-Mediated Gene Ontology Regulatory Networks Identified by Computational Biology.

PubMed

Vishnu, Udayakumar S; Sankarasubramanian, Jagadesan; Gunasekaran, Paramasamy; Sridhar, Jayavel; Rajendhran, Jeyaprakash

2016-06-01

Brucella is an intracellular bacterium that causes the zoonotic infectious disease, brucellosis. Brucella species are currently intensively studied with a view to developing novel global health diagnostics and therapeutics. In this context, small RNAs (sRNAs) are one of the emerging topical areas; they play significant roles in regulating gene expression and cellular processes in bacteria. In the present study, we forecast sRNAs in three Brucella species that infect humans, namely Brucella melitensis, Brucella abortus, and Brucella suis, using a computational biology analysis. We combined two bioinformatic algorithms, SIPHT and sRNAscanner. In B. melitensis 16M, 21 sRNA candidates were identified, of which 14 were novel. Similarly, 14 sRNAs were identified in B. abortus, of which four were novel. In B. suis, 16 sRNAs were identified, and five of them were novel. TargetRNA2 software predicted the putative target genes that could be regulated by the identified sRNAs. The identified mRNA targets are involved in carbohydrate, amino acid, lipid, nucleotide, and coenzyme metabolism and transport, energy production and conversion, replication, recombination, repair, and transcription. Additionally, the Gene Ontology (GO) network analysis revealed the species-specific, sRNA-based regulatory networks in B. melitensis, B. abortus, and B. suis. Taken together, although sRNAs are veritable modulators of gene expression in prokaryotes, there are few reports on the significance of sRNAs in Brucella. This report begins to address this literature gap by offering a series of initial observations based on computational biology to pave the way for future experimental analysis of sRNAs and their targets to explain the complex pathogenesis of Brucella.

Precambrian animal diversity: putative phosphatized embryos from the Doushantuo Formation of China

NASA Technical Reports Server (NTRS)

Chen, J. Y.; Oliveri, P.; Li, C. W.; Zhou, G. Q.; Gao, F.; Hagadorn, J. W.; Peterson, K. J.; Davidson, E. H.

2000-01-01

Putative fossil embryos and larvae from the Precambrian phosphorite rocks of the Doushantuo Formation in Southwest China have been examined in thin section by bright field and polarized light microscopy. Although we cannot completely exclude a nonbiological or nonmetazoan origin, we identified what appear to be modern cnidarian developmental stages, including both anthozoan planula larvae and hydrozoan embryos. Most importantly, the sections contain a variety of small (

Proteomics as a Tool to Identify New Targets Against Aspergillus and Scedosporium in the Context of Cystic Fibrosis.

PubMed

Ramirez-Garcia, Andoni; Pellon, Aize; Buldain, Idoia; Antoran, Aitziber; Arbizu-Delgado, Aitana; Guruceaga, Xabier; Rementeria, Aitor; Hernando, Fernando L

2018-02-01

Cystic fibrosis (CF) is a genetic disorder that increases the risk of suffering microbial, including fungal, infections. In this paper, proteomics-based information was collated relating to secreted and cell wall proteins with potential medical applications from the most common filamentous fungi in CF, i.e., Aspergillus and Scedosporium/Lomentospora species. Among the Aspergillus fumigatus secreted allergens, β-1,3-endoglucanase, the alkaline protease 1 (Alp1/oryzin), Asp f 2, Asp f 13/15, chitinase, chitosanase, dipeptidyl-peptidase V (DppV), the metalloprotease Asp f 5, mitogillin/Asp f 1, and thioredoxin reductase receive a special mention. In addition, the antigens β-glucosidase 1, catalase, glucan endo-1,3-β-glucosidase EglC, β-1,3-glucanosyltransferases Gel1 and Gel2, and glutaminase A were also identified in secretomes of other Aspergillus species associated with CF: Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, and Aspergillus terreus. Regarding cell wall proteins, cytochrome P450 and eEF-3 were proposed as diagnostic targets, and alkaline protease 2 (Alp2), Asp f 3 (putative peroxiredoxin pmp20), probable glycosidases Asp f 9/Crf1 and Crf2, GPI-anchored protein Ecm33, β-1,3-glucanosyltransferase Gel4, conidial hydrophobin Hyp1/RodA, and secreted aspartyl protease Pep2 as protective vaccines in A. fumigatus. On the other hand, for Scedosporium/Lomentospora species, the heat shock protein Hsp70 stands out as a relevant secreted and cell wall antigen. Additionally, the secreted aspartyl proteinase and an ortholog of Asp f 13, as well as the cell wall endo-1,3-β-D-glucosidase and 1,3-β-glucanosyl transferase, were also found to be significant proteins. In conclusion, proteins mentioned in this review may be promising candidates for developing innovative diagnostic and therapeutic tools for fungal infections in CF patients.

Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters

PubMed Central

Baseler, Laura; Scott, Dana P.; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz

2016-01-01

Background Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Methodology/Principal Findings Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Conclusions/Significance Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central

Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters.

PubMed

Baseler, Laura; Scott, Dana P; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz; de Wit, Emmie

2016-11-01

Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central nervous system pathology.

CRISPR-Cas systems target a diverse collection of invasive mobile genetic elements in human microbiomes

PubMed Central

2013-01-01

Background Bacteria and archaea develop immunity against invading genomes by incorporating pieces of the invaders' sequences, called spacers, into a clustered regularly interspaced short palindromic repeats (CRISPR) locus between repeats, forming arrays of repeat-spacer units. When spacers are expressed, they direct CRISPR-associated (Cas) proteins to silence complementary invading DNA. In order to characterize the invaders of human microbiomes, we use spacers from CRISPR arrays that we had previously assembled from shotgun metagenomic datasets, and identify contigs that contain these spacers' targets. Results We discover 95,000 contigs that are putative invasive mobile genetic elements, some targeted by hundreds of CRISPR spacers. We find that oral sites in healthy human populations have a much greater variety of mobile genetic elements than stool samples. Mobile genetic elements carry genes encoding diverse functions: only 7% of the mobile genetic elements are similar to known phages or plasmids, although a much greater proportion contain phage- or plasmid-related genes. A small number of contigs share similarity with known integrative and conjugative elements, providing the first examples of CRISPR defenses against this class of element. We provide detailed analyses of a few large mobile genetic elements of various types, and a relative abundance analysis of mobile genetic elements and putative hosts, exploring the dynamic activities of mobile genetic elements in human microbiomes. A joint analysis of mobile genetic elements and CRISPRs shows that protospacer-adjacent motifs drive their interaction network; however, some CRISPR-Cas systems target mobile genetic elements lacking motifs. Conclusions We identify a large collection of invasive mobile genetic elements in human microbiomes, an important resource for further study of the interaction between the CRISPR-Cas immune system and invaders. PMID:23628424

Identification of a RAC/AKT-like gene in Leishmania parasites as a putative therapeutic target in leishmaniasis.

PubMed

Varela-M, Rubén E; Ochoa, Rodrigo; Muskus, Carlos E; Muro, Antonio; Mollinedo, Faustino

2017-10-10

Leishmaniasis is one of the world's most neglected diseases caused by at least 20 different species of the protozoan parasite Leishmania. Although new drugs have become recently available, current therapy for leishmaniasis is still unsatisfactory. A subgroup of serine/threonine protein kinases named as related to A and C protein kinases (RAC), or protein kinase B (PKB)/AKT, has been identified in several organisms including Trypanosoma cruzi parasites. PKB/AKT plays a critical role in mammalian cell signaling promoting cell survival and is a major drug target in cancer therapy. However, the role of protozoan parasitic PKB/AKT remains to be elucidated. We have found that anti-human AKT antibodies recognized a protein of about 57 kDa in Leishmania spp. parasites. Anti-human phospho-AKT(Thr308) antibodies identified a protein in extracts from Leishmania spp. that was upregulated following parasite exposure to stressful conditions, such as nutrient deprivation or heat shock. Incubation of AKT inhibitor X with Leishmania spp. promastigotes under stressful conditions or with Leishmania-infected macrophages led to parasite cell death. We have identified and cloned a novel gene from Leishmania donovani named Ld-RAC/AKT-like gene, encoding a 510-amino acid protein of approximately 57.6 kDa that shows a 26.5% identity with mammalian AKT1. Ld-RAC/AKT-like protein contains major mammalian PKB/AKT hallmarks, including the typical pleckstrin, protein kinase and AGC kinase domains. Unlike mammalian AKT that contains key phosphorylation sites at Thr308 and Ser473 in the activation loop and hydrophobic motif, respectively, Ld-RAC/AKT-like protein has a Thr residue in both motifs. By domain sequence comparison, we classified AKT proteins from different origins in four major subcategories that included different parasites. Our data suggest that Ld-RAC/AKT-like protein represents a Leishmania orthologue of mammalian AKT involved in parasite stress response and survival, and

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

Predicting essential genes for identifying potential drug targets in Aspergillus fumigatus.

PubMed

Lu, Yao; Deng, Jingyuan; Rhodes, Judith C; Lu, Hui; Lu, Long Jason

2014-06-01

Aspergillus fumigatus (Af) is a ubiquitous and opportunistic pathogen capable of causing acute, invasive pulmonary disease in susceptible hosts. Despite current therapeutic options, mortality associated with invasive Af infections remains unacceptably high, increasing 357% since 1980. Therefore, there is an urgent need for the development of novel therapeutic strategies, including more efficacious drugs acting on new targets. Thus, as noted in a recent review, "the identification of essential genes in fungi represents a crucial step in the development of new antifungal drugs". Expanding the target space by rapidly identifying new essential genes has thus been described as "the most important task of genomics-based target validation". In previous research, we were the first to show that essential gene annotation can be reliably transferred between distantly related four Prokaryotic species. In this study, we extend our machine learning approach to the much more complex Eukaryotic fungal species. A compendium of essential genes is predicted in Af by transferring known essential gene annotations from another filamentous fungus Neurospora crassa. This approach predicts essential genes by integrating diverse types of intrinsic and context-dependent genomic features encoded in microbial genomes. The predicted essential datasets contained 1674 genes. We validated our results by comparing our predictions with known essential genes in Af, comparing our predictions with those predicted by homology mapping, and conducting conditional expressed alleles. We applied several layers of filters and selected a set of potential drug targets from the predicted essential genes. Finally, we have conducted wet lab knockout experiments to verify our predictions, which further validates the accuracy and wide applicability of the machine learning approach. The approach presented here significantly extended our ability to predict essential genes beyond orthologs and made it possible to

Identifying Minefields and Verifying Clearance: Adapting Statistical Methods for UXO Target Detection

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

Gilbert, Richard O.; O'Brien, Robert F.; Wilson, John E.

2003-09-01

It may not be feasible to completely survey large tracts of land suspected of containing minefields. It is desirable to develop a characterization protocol that will confidently identify minefields within these large land tracts if they exist. Naturally, surveying areas of greatest concern and most likely locations would be necessary but will not provide the needed confidence that an unknown minefield had not eluded detection. Once minefields are detected, methods are needed to bound the area that will require detailed mine detection surveys. The US Department of Defense Strategic Environmental Research and Development Program (SERDP) is sponsoring the development ofmore » statistical survey methods and tools for detecting potential UXO targets. These methods may be directly applicable to demining efforts. Statistical methods are employed to determine the optimal geophysical survey transect spacing to have confidence of detecting target areas of a critical size, shape, and anomaly density. Other methods under development determine the proportion of a land area that must be surveyed to confidently conclude that there are no UXO present. Adaptive sampling schemes are also being developed as an approach for bounding the target areas. These methods and tools will be presented and the status of relevant research in this area will be discussed.« less

Identification of aryl hydrocarbon receptor binding targets in mouse hepatic tissue treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

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

Lo, Raymond; Celius, Trine; Forgacs, Agnes L.

2011-11-15

Genome-wide, promoter-focused ChIP-chip analysis of hepatic aryl hydrocarbon receptor (AHR) binding sites was conducted in 8-week old female C57BL/6 treated with 30 {mu}g/kg/body weight 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 2 h and 24 h. These studies identified 1642 and 508 AHR-bound regions at 2 h and 24 h, respectively. A total of 430 AHR-bound regions were common between the two time points, corresponding to 403 unique genes. Comparison with previous AHR ChIP-chip studies in mouse hepatoma cells revealed that only 62 of the putative target genes overlapped with the 2 h AHR-bound regions in vivo. Transcription factor binding site analysis revealed anmore » over-representation of aryl hydrocarbon response elements (AHREs) in AHR-bound regions with 53% (2 h) and 68% (24 h) of them containing at least one AHRE. In addition to AHREs, E2f-Myc activator motifs previously implicated in AHR function, as well as a number of other motifs, including Sp1, nuclear receptor subfamily 2 factor, and early growth response factor motifs were also identified. Expression microarray studies identified 133 unique genes differentially regulated after 4 h treatment with TCDD. Of which, 39 were identified as AHR-bound genes at 2 h. Ingenuity Pathway Analysis on the 39 AHR-bound TCDD responsive genes identified potential perturbation in biological processes such as lipid metabolism, drug metabolism, and endocrine system development as a result of TCDD-mediated AHR activation. Our findings identify direct AHR target genes in vivo, highlight in vitro and in vivo differences in AHR signaling and show that AHR recruitment does not necessarily result in changes in target gene expression. -- Highlights: Black-Right-Pointing-Pointer ChIP-chip analysis of hepatic AHR binding after 2 h and 24 h of TCDD. Black-Right-Pointing-Pointer We identified 1642 and 508 AHR-bound regions at 2 h and 24 h. Black-Right-Pointing-Pointer 430 regions were common to both time points and highly enriched

Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics.

PubMed

Dhouib, R; Laroche-Traineau, J; Shaha, R; Lapaillerie, D; Solier, E; Rualès, J; Pina, M; Villeneuve, P; Carrière, F; Bonneu, M; Arondel, V

2011-01-01

Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 μmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 μmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 μmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 μmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex. © 2010 The Authors Journal compilation © 2010 FEBS.

Structural modelling and comparative analysis of homologous, analogous and specific proteins from Trypanosoma cruzi versus Homo sapiens: putative drug targets for chagas' disease treatment.

PubMed

Capriles, Priscila V S Z; Guimarães, Ana C R; Otto, Thomas D; Miranda, Antonio B; Dardenne, Laurent E; Degrave, Wim M

2010-10-29

Trypanosoma cruzi is the etiological agent of Chagas' disease, an endemic infection that causes thousands of deaths every year in Latin America. Therapeutic options remain inefficient, demanding the search for new drugs and/or new molecular targets. Such efforts can focus on proteins that are specific to the parasite, but analogous enzymes and enzymes with a three-dimensional (3D) structure sufficiently different from the corresponding host proteins may represent equally interesting targets. In order to find these targets we used the workflows MHOLline and AnEnΠ obtaining 3D models from homologous, analogous and specific proteins of Trypanosoma cruzi versus Homo sapiens. We applied genome wide comparative modelling techniques to obtain 3D models for 3,286 predicted proteins of T. cruzi. In combination with comparative genome analysis to Homo sapiens, we were able to identify a subset of 397 enzyme sequences, of which 356 are homologous, 3 analogous and 38 specific to the parasite. In this work, we present a set of 397 enzyme models of T. cruzi that can constitute potential structure-based drug targets to be investigated for the development of new strategies to fight Chagas' disease. The strategies presented here support the concept of structural analysis in conjunction with protein functional analysis as an interesting computational methodology to detect potential targets for structure-based rational drug design. For example, 2,4-dienoyl-CoA reductase (EC 1.3.1.34) and triacylglycerol lipase (EC 3.1.1.3), classified as analogous proteins in relation to H. sapiens enzymes, were identified as new potential molecular targets.

Systems pharmacology identifies drug targets for Stargardt disease–associated retinal degeneration

PubMed Central

Chen, Yu; Palczewska, Grazyna; Mustafi, Debarshi; Golczak, Marcin; Dong, Zhiqian; Sawada, Osamu; Maeda, Tadao; Maeda, Akiko; Palczewski, Krzysztof

2013-01-01

A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases. PMID:24231350

Putative SF2 helicases of the early-branching eukaryote Giardia lamblia are involved in antigenic variation and parasite differentiation into cysts

PubMed Central

2012-01-01

Background Regulation of surface antigenic variation in Giardia lamblia is controlled post-transcriptionally by an RNA-interference (RNAi) pathway that includes a Dicer-like bidentate RNase III (gDicer). This enzyme, however, lacks the RNA helicase domain present in Dicer enzymes from higher eukaryotes. The participation of several RNA helicases in practically all organisms in which RNAi was studied suggests that RNA helicases are potentially involved in antigenic variation, as well as during Giardia differentiation into cysts. Results An extensive in silico analysis of the Giardia genome identified 32 putative Super Family 2 RNA helicases that contain almost all the conserved RNA helicase motifs. Phylogenetic studies and sequence analysis separated them into 22 DEAD-box, 6 DEAH-box and 4 Ski2p-box RNA helicases, some of which are homologs of well-characterized helicases from higher organisms. No Giardia putative helicase was found to have significant homology to the RNA helicase domain of Dicer enzymes. Additionally a series of up- and down-regulated putative RNA helicases were found during encystation and antigenic variation by qPCR experiments. Finally, we were able to recognize 14 additional putative helicases from three different families (RecQ family, Swi2/Snf2 and Rad3 family) that could be considered DNA helicases. Conclusions This is the first comprehensive analysis of the Super Family 2 helicases from the human intestinal parasite G. lamblia. The relative and variable expression of particular RNA helicases during both antigenic variation and encystation agrees with the proposed participation of these enzymes during both adaptive processes. The putatives RNA and DNA helicases identified in this early-branching eukaryote provide initial information regarding the biological role of these enzymes in cell adaptation and differentiation. PMID:23190735

Methylation of DNA and chromatin as a mechanism of oncogenesis and therapeutic target in neuroblastoma.

PubMed

Ram Kumar, Ram Mohan; Schor, Nina Felice

2018-04-24

Neuroblastoma (NB), a developmental cancer, is often fatal, emphasizing the need to understand its pathogenesis and identify new therapeutic targets. The heterogeneous pathological and clinical phenotype of NB underscores the cryptic biological and genetic features of this tumor that result in outcomes ranging from rapid progression to spontaneous regression. Despite recent genome-wide mutation analyses, most primary NBs do not harbor driver mutations, implicating epigenetically-mediated gene regulatory mechanisms in the initiation and maintenance of NB. Aberrant epigenomic mechanisms, as demonstrated by global changes in DNA methylation signatures, acetylation, re-distribution of histone marks, and change in the chromatin architecture, are hypothesized to play a role in NB oncogenesis. This paper reviews the evidence for, putative mechanisms underlying, and prospects for therapeutic targeting of NB oncogenesis related to DNA methylation.

Differential splicing of human androgen receptor pre-mRNA in X-linked reifenstein syndrome, because of a deletion involving a putative branch site

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

Ris-Stalpers, C.; Verleun-Mooijman, M.C.T.; Blaeij, T.J.P. de

1994-04-01

The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of >6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-pointmore » sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS. 42 refs., 6 figs., 1 tab.« less

Identify mutation in amyotrophic lateral sclerosis cases using HaloPlex target enrichment system.

PubMed

Liu, Zhi-Jun; Li, Hong-Fu; Tan, Guo-He; Tao, Qing-Qing; Ni, Wang; Cheng, Xue-Wen; Xiong, Zhi-Qi; Wu, Zhi-Ying

2014-12-01

To date, at least 18 causative genes have been identified in amyotrophic lateral sclerosis (ALS). Because of the clinical and genetic heterogeneity, molecular diagnosis for ALS faces great challenges. HaloPlex target enrichment system is a new targeted sequencing approach, which can detect already known mutations or candidate genes. We performed this approach to screen 18 causative genes of ALS, including SOD1, SETX, FUS, ANG, TARDBP, ALS2, FIG4, VAPB, OPTN, DAO, VCP, UBQLN2, SPG11, SIGMAR1, DCTN1, SQSTM1, PFN1, and CHMP2B in 8 ALS probands. Using this approach, we got an average of 9.5 synonymous or missense mutations per sample. After validation by Sanger sequencing, we identified 3 documented SOD1 mutations (p.F21C, p.G148D, and p.C147R) and 1 novel DCTN1 p.G59R mutation in 4 probands. The novel DCTN1 mutation appeared to segregate with the disease in the pedigree and was absent in 200 control subjects. The high throughput and efficiency of this approach indicated that it could be applied to diagnose ALS and other inherited diseases with multiple causative genes in clinical practice. Copyright © 2014 Elsevier Inc. All rights reserved.

FOXP2 Targets Show Evidence of Positive Selection in European Populations

PubMed Central

Ayub, Qasim; Yngvadottir, Bryndis; Chen, Yuan; Xue, Yali; Hu, Min; Vernes, Sonja C.; Fisher, Simon E.; Tyler-Smith, Chris

2013-01-01

Forkhead box P2 (FOXP2) is a highly conserved transcription factor that has been implicated in human speech and language disorders and plays important roles in the plasticity of the developing brain. The pattern of nucleotide polymorphisms in FOXP2 in modern populations suggests that it has been the target of positive (Darwinian) selection during recent human evolution. In our study, we searched for evidence of selection that might have followed FOXP2 adaptations in modern humans. We examined whether or not putative FOXP2 targets identified by chromatin-immunoprecipitation genomic screening show evidence of positive selection. We developed an algorithm that, for any given gene list, systematically generates matched lists of control genes from the Ensembl database, collates summary statistics for three frequency-spectrum-based neutrality tests from the low-coverage resequencing data of the 1000 Genomes Project, and determines whether these statistics are significantly different between the given gene targets and the set of controls. Overall, there was strong evidence of selection of FOXP2 targets in Europeans, but not in the Han Chinese, Japanese, or Yoruba populations. Significant outliers included several genes linked to cellular movement, reproduction, development, and immune cell trafficking, and 13 of these constituted a significant network associated with cardiac arteriopathy. Strong signals of selection were observed for CNTNAP2 and RBFOX1, key neurally expressed genes that have been consistently identified as direct FOXP2 targets in multiple studies and that have themselves been associated with neurodevelopmental disorders involving language dysfunction. PMID:23602712

Targeted next generation sequencing identifies functionally deleterious germline mutations in novel genes in early-onset/familial prostate cancer.

PubMed

Paulo, Paula; Maia, Sofia; Pinto, Carla; Pinto, Pedro; Monteiro, Augusta; Peixoto, Ana; Teixeira, Manuel R

2018-04-01

Considering that mutations in known prostate cancer (PrCa) predisposition genes, including those responsible for hereditary breast/ovarian cancer and Lynch syndromes, explain less than 5% of early-onset/familial PrCa, we have sequenced 94 genes associated with cancer predisposition using next generation sequencing (NGS) in a series of 121 PrCa patients. We found monoallelic truncating/functionally deleterious mutations in seven genes, including ATM and CHEK2, which have previously been associated with PrCa predisposition, and five new candidate PrCa associated genes involved in cancer predisposing recessive disorders, namely RAD51C, FANCD2, FANCI, CEP57 and RECQL4. Furthermore, using in silico pathogenicity prediction of missense variants among 18 genes associated with breast/ovarian cancer and/or Lynch syndrome, followed by KASP genotyping in 710 healthy controls, we identified "likely pathogenic" missense variants in ATM, BRIP1, CHEK2 and TP53. In conclusion, this study has identified putative PrCa predisposing germline mutations in 14.9% of early-onset/familial PrCa patients. Further data will be necessary to confirm the genetic heterogeneity of inherited PrCa predisposition hinted in this study.

CRISPR-mediated HDAC2 disruption identifies two distinct classes of target genes in human cells.

PubMed

Somanath, Priyanka; Herndon Klein, Rachel; Knoepfler, Paul S

2017-01-01

The transcriptional functions of the class I histone deacetylases (HDACs) HDAC1 and HDAC2 are mainly viewed as both repressive and redundant based on murine knockout studies, but they may have additional independent roles and their physiological functions in human cells are not as clearly defined. To address the individual epigenomic functions of HDAC2, here we utilized CRISPR-Cas9 to disrupt HDAC2 in human cells. We find that while HDAC2 null cells exhibited signs of cross-regulation between HDAC1 and HDAC2, specific epigenomic phenotypes were still apparent using RNA-seq and ChIP assays. We identified specific targets of HDAC2 repression, and defined a novel class of genes that are actively expressed in a partially HDAC2-dependent manner. While HDAC2 was required for the recruitment of HDAC1 to repressed HDAC2-gene targets, HDAC2 was dispensable for HDAC1 binding to HDAC2-activated targets, supporting the notion of distinct classes of targets. Both active and repressed classes of gene targets demonstrated enhanced histone acetylation and methylation in HDAC2-null cells. Binding of the HDAC1/2-associated SIN3A corepressor was altered at most HDAC2-targets, but without a clear pattern. Overall, our study defines two classes of HDAC2 targets in human cells, with a dependence of HDAC1 on HDAC2 at one class of targets, and distinguishes unique functions for HDAC2.

Comparative analyses of putative toxin gene homologs from an Old World viper, Daboia russelii

PubMed Central

Krishnan, Neeraja M.

2017-01-01

Availability of snake genome sequences has opened up exciting areas of research on comparative genomics and gene diversity. One of the challenges in studying snake genomes is the acquisition of biological material from live animals, especially from the venomous ones, making the process cumbersome and time-consuming. Here, we report comparative sequence analyses of putative toxin gene homologs from Russell’s viper (Daboia russelii) using whole-genome sequencing data obtained from shed skin. When compared with the major venom proteins in Russell’s viper studied previously, we found 45–100% sequence similarity between the venom proteins and their putative homologs in the skin. Additionally, comparative analyses of 20 putative toxin gene family homologs provided evidence of unique sequence motifs in nerve growth factor (NGF), platelet derived growth factor (PDGF), Kunitz/Bovine pancreatic trypsin inhibitor (Kunitz BPTI), cysteine-rich secretory proteins, antigen 5, andpathogenesis-related1 proteins (CAP) and cysteine-rich secretory protein (CRISP). In those derived proteins, we identified V11 and T35 in the NGF domain; F23 and A29 in the PDGF domain; N69, K2 and A5 in the CAP domain; and Q17 in the CRISP domain to be responsible for differences in the largest pockets across the protein domain structures in crotalines, viperines and elapids from the in silico structure-based analysis. Similarly, residues F10, Y11 and E20 appear to play an important role in the protein structures across the kunitz protein domain of viperids and elapids. Our study highlights the usefulness of shed skin in obtaining good quality high-molecular weight DNA for comparative genomic studies, and provides evidence towards the unique features and evolution of putative venom gene homologs in vipers. PMID:29230357

Secretome Characterization and Correlation Analysis Reveal Putative Pathogenicity Mechanisms and Identify Candidate Avirulence Genes in the Wheat Stripe Rust Fungus Puccinia striiformis f. sp. tritici.

PubMed

Xia, Chongjing; Wang, Meinan; Cornejo, Omar E; Jiwan, Derick A; See, Deven R; Chen, Xianming

2017-01-01

Stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici ( Pst ), is one of the most destructive diseases of wheat worldwide. Planting resistant cultivars is an effective way to control this disease, but race-specific resistance can be overcome quickly due to the rapid evolving Pst population. Studying the pathogenicity mechanisms is critical for understanding how Pst virulence changes and how to develop wheat cultivars with durable resistance to stripe rust. We re-sequenced 7 Pst isolates and included additional 7 previously sequenced isolates to represent balanced virulence/avirulence profiles for several avirulence loci in seretome analyses. We observed an uneven distribution of heterozygosity among the isolates. Secretome comparison of Pst with other rust fungi identified a large portion of species-specific secreted proteins, suggesting that they may have specific roles when interacting with the wheat host. Thirty-two effectors of Pst were identified from its secretome. We identified candidates for Avr genes corresponding to six Yr genes by correlating polymorphisms for effector genes to the virulence/avirulence profiles of the 14 Pst isolates. The putative AvYr76 was present in the avirulent isolates, but absent in the virulent isolates, suggesting that deleting the coding region of the candidate avirulence gene has produced races virulent to resistance gene Yr76 . We conclude that incorporating avirulence/virulence phenotypes into correlation analysis with variations in genomic structure and secretome, particularly presence/absence polymorphisms of effectors, is an efficient way to identify candidate Avr genes in Pst . The candidate effector genes provide a rich resource for further studies to determine the evolutionary history of Pst populations and the co-evolutionary arms race between Pst and wheat. The Avr candidates identified in this study will lead to cloning avirulence genes in Pst , which will enable us to understand molecular mechanisms

Putative therapeutic targets for symptom subtypes of adult ADHD: D4 receptor agonism and COMT inhibition improve attention and response inhibition in a novel translational animal model.

PubMed

Tomlinson, Anneka; Grayson, Ben; Marsh, Samuel; Hayward, Andrew; Marshall, Kay M; Neill, Joanna C

2015-04-01

Prefrontal cortical dopamine plays an important role in cognitive control, specifically in attention and response inhibition; the core deficits in ADHD. We have previously shown that methylphenidate and atomoxetine differentially improve these deficits dependent on baseline performance. The present study extends this work to investigate the effects of putative therapeutic targets in our model. A selective dopamine D4 receptor agonist (A-412997) and the catechol-O-methyl-transferase (COMT) inhibitor; tolcapone, were investigated in the combined subtype of adult ADHD (ADHD-C). Adult female rats were trained to criterion in the 5C-CPT (5-Choice Continuous Performance Task) and then separated into subgroups according to baseline levels of sustained attention, vigilance, and response disinhibition. The subgroups included: high-attentive (HA) and low-attentive with high response disinhibition (ADHD-C). The ADHD-C subgroup was selected to represent the combined subtype of adult ADHD. Effects of tolcapone (3.0, 10.0, 15.0mg/kg) and A-412997 (0.1, 0.3, 1.0µmol/kg) were tested by increasing the variable inter-trial-interval (ITI) duration in the 5C-CPT. Tolcapone (15mg/kg) significantly increased sustained attention, vigilance and response inhibition in ADHD-C animals, and impaired attention in HA animals. A-412997 (1.0µmol/kg) significantly increased vigilance and response inhibition in ADHD-C animals only, with no effect in HA animals. This is the first study to use the translational 5C-CPT to model the adult ADHD-C subtype in rats and to study new targets in this model. Both tolcapone and A-412997 increased vigilance and response inhibition in the ADHD-C subgroup. D4 and COMT are emerging as important potential therapeutic targets in adult ADHD that warrant further investigation. Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.

A meta-analysis of multiple myeloma risk regions in African and European ancestry populations identifies putatively functional loci

PubMed Central

Rand, Kristin A.; Song, Chi; Dean, Eric; Serie, Daniel J.; Curtin, Karen; Sheng, Xin; Hu, Donglei; Huff, Carol Ann; Bernal-Mizrachi, Leon; Tomasson, Michael H.; Ailawadhi, Sikander; Singhal, Seema; Pawlish, Karen; Peters, Edward S.; Bock, Cathryn H.; Stram, Alex; Van Den Berg, David J; Edlund, Christopher K.; V.Conti, David; Zimmerman, Todd; Hwang, Amie E.; Huntsman, Scott; Graff, John; Nooka, Ajay; Kong, Yinfei; Pregja, Silvana L.; Berndt, Sonja I.; Blot, William J.; Carpten, John; Casey, Graham; Chu, Lisa; Diver, W. Ryan; Stevens, Victoria L.; Lieber, Michael R.; Goodman, Phyllis J.; Hennis, Anselm J.M.; Hsing, Ann W.; Mehta, Jayesh; Kittles, Rick A.; Kolb, Suzanne; Klein, Eric A.; Leske, Cristina; Murphy, Adam B.; Nemesure, Barbara; Neslund-Dudas, Christine; Strom, Sara S.; Vij, Ravi; Rybicki, Benjamin A.; Stanford, Janet L.; Signorello, Lisa B.; Witte, John S.; Ambrosone, Christine B.; Bhatti, Parveen; John, Esther M.; Bernstein, Leslie; Zheng, Wei; Olshan, Andrew F.; Hu, Jennifer J.; Ziegler, Regina G.; Nyante, Sarah J.; Bandera, Elisa V.; Birmann, Brenda M.; Ingles, Sue A.; Press, Michael F.; Atanackovic, Djordje; Glenn, Martha J.; Cannon-Albright, Lisa A.; Jones, Brandt; Tricot, Guido; Martin, Thomas G.; Kumar, Shaji K.; Wolf, Jeffrey L.; Deming, Sandra L.; Rothman, Nathaniel; Brooks-Wilson, Angela R.; Rajkumar, S. Vincent; Kolonel, Laurence N.; Chanock, Stephen J.; Slager, Susan L.; Severson, Richard K.; Janakiraman, Nalini; Terebelo, Howard R.; Brown, Elizabeth E.; De Roos, Anneclaire J.; Mohrbacher, Ann F.; Colditz, Graham A.; Giles, Graham G.; Spinelli, John J.; Chiu, Brian C.; Munshi, Nikhil C.; Anderson, Kenneth C.; Levy, Joan; Zonder, Jeffrey A.; Orlowski, Robert Z.; Lonial, Sagar; Camp, Nicola J.; Vachon, Celine M.; Ziv, Elad; Stram, Daniel O.; Hazelett, Dennis J.; Haiman, Christopher A.; Cozen, Wendy

2017-01-01

Background Genome-wide association studies (GWAS) in European populations have identified genetic risk variants associated with multiple myeloma (MM). Methods We performed association testing of common variation in eight regions in 1,264 MM patients and 1,479 controls of European ancestry (EA) and 1,305 MM patients and 7,078 controls of African ancestry (AA) and conducted a meta-analysis to localize the signals, with epigenetic annotation used to predict functionality. Results We found that variants in 7p15.3, 17p11.2, 22q13.1 were statistically significantly (p<0.05) associated with MM risk in AAs and EAs and the variant in 3p22.1 was associated in EAs only. In a combined AA-EA meta-analysis, variation in five regions (2p23.3, 3p22.1, 7p15.3, 17p11.2, 22q13.1) was statistically signficantly associated with MM risk. In 3p22.1, the correlated variants clustered within the gene body of ULK4. Correlated variants in 7p15.3 clustered around an enhancer at the 3′ end of the CDCA7L transcription termination site. A missense variant at 17p11.2 (rs34562254, Pro251Leu, OR=1.32, p=2.93×10−7) in TNFRSF13B, encodes a lymphocyte-specific protein in the tumor necrosis factor receptor family that interacts with the NF-κB pathway. SNPs correlated with the index signal in 22q13.1 cluster around the promoter and enhancer regions of CBX7. Conclusions We found that reported MM susceptibility regions contain risk variants important across populations supporting the use of multiple racial/ethnic groups with different underlying genetic architecture to enhance the localization and identification of putatively functional alleles. Impact A subset of reported risk loci for multiple myeloma have consistent affects across populations and are likely to be functional. PMID:27587788

Structural modelling and comparative analysis of homologous, analogous and specific proteins from Trypanosoma cruzi versus Homo sapiens: putative drug targets for chagas' disease treatment

PubMed Central

2010-01-01

Background Trypanosoma cruzi is the etiological agent of Chagas' disease, an endemic infection that causes thousands of deaths every year in Latin America. Therapeutic options remain inefficient, demanding the search for new drugs and/or new molecular targets. Such efforts can focus on proteins that are specific to the parasite, but analogous enzymes and enzymes with a three-dimensional (3D) structure sufficiently different from the corresponding host proteins may represent equally interesting targets. In order to find these targets we used the workflows MHOLline and AnEnΠ obtaining 3D models from homologous, analogous and specific proteins of Trypanosoma cruzi versus Homo sapiens. Results We applied genome wide comparative modelling techniques to obtain 3D models for 3,286 predicted proteins of T. cruzi. In combination with comparative genome analysis to Homo sapiens, we were able to identify a subset of 397 enzyme sequences, of which 356 are homologous, 3 analogous and 38 specific to the parasite. Conclusions In this work, we present a set of 397 enzyme models of T. cruzi that can constitute potential structure-based drug targets to be investigated for the development of new strategies to fight Chagas' disease. The strategies presented here support the concept of structural analysis in conjunction with protein functional analysis as an interesting computational methodology to detect potential targets for structure-based rational drug design. For example, 2,4-dienoyl-CoA reductase (EC 1.3.1.34) and triacylglycerol lipase (EC 3.1.1.3), classified as analogous proteins in relation to H. sapiens enzymes, were identified as new potential molecular targets. PMID:21034488

Determination of the full-genome sequence of hepatitis E virus (HEV) SAAS-FX17 and use as a reference to identify putative HEV genotype 4 virulence determinants.

PubMed

Zhu, Yumin; Yu, Xiaoming; Huang, Fenfen; Yu, Ruisong; Dong, Shijuan; Si, Fusheng; Zhang, Yuanshu; Li, Zhen

2012-11-08

Four major genotypes of hepatitis E virus (HEV), the causative agent of hepatitis E, have so far been recognized. While genotypes 3 and 4 are both zoonotic, the disease symptoms caused by the latter tend to be more severe. To examine if specific nucleotide/amino acid variations between genotypes 3 and 4 play a role in determining the severity of hepatitis E disease, the complete genome of one swine HEV genotype 4 isolate, SAAS-FX17, was determined and compared with other genotype 4 and genotype 3 genomes to identify putative HEV genotype 4 virulence determinants. A total of 42 conformable nt/aa variations between genotype 3 and 4 HEVs were detected, of which 19 were proposed to be potential disease severity determinants for genotype 4 strains. One potential determinant was located in each of the 5'-UTR and 3'-UTR, 3 and 12 within ORF1 and ORF2 respectively, and 2 in the junction region.

MiR-181b targets Six2 and inhibits the proliferation of metanephric mesenchymal cells in vitro

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

Lyu, Zhongshi; Mao, Zhaomin; Wang, Honglian

2013-11-01

Highlights: •We do bio-informatics websites to analysis of Six2 3′UTR. •MiR181b is a putative miRNA which can targets Six2 3′UTR. •MiR-181b binding site in the 3′UTR of Six2 is functional. •MiR-181b suppresses MK3 cells cell proliferation by targeting Six2. -- Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that down-regulate gene expression by binding to target mRNA for cleavage or translational repression, and play important regulatory roles in renal development. Despite increasing genes have been predicted to be miRNA targets by bioinformatic analysis during kidney development, few of them have been verified by experiment. The objective of our study is tomore » identify the miRNAs targeting Six2, a critical transcription factor that maintains the mesenchymal progenitor pool via self-renewal (proliferation) during renal development. We initially analyzed the 3′UTR of Six2 and found 37 binding sites targeted by 50 putative miRNAs in the 3′UTR of Six2. Among the 50 miRNAs, miR-181b is the miRNAs predicted by the three used websites. In our study, the results of luciferase reporter assay, realtime-PCR and Western blot demonstrated that miR-181b directly targeted on the 3′UTR of Six2 and down-regulate the expression of Six2 at mRNA and protein levels. Furthermore, EdU proliferation assay along with the Six2 rescue strategy showed that miR-181b suppresses the proliferation of metanephric mesenchymal by targeting Six2 in part. In our research, we concluded that by targeting the transcription factor gene Six2, miR-181b inhibits the proliferation of metanephric mesenchymal cells in vitro and might play an important role in the formation of nephrons.« less

Identifying On-Orbit Test Targets for Space Fence Operational Testing

NASA Astrophysics Data System (ADS)

Pechkis, D.; Pacheco, N.; Botting, T.

2014-09-01

Space Fence will be an integrated system of two ground-based, S-band (2 to 4 GHz) phased-array radars located in Kwajalein and perhaps Western Australia [1]. Space Fence will cooperate with other Space Surveillance Network sensors to provide space object tracking and radar characterization data to support U.S. Strategic Command space object catalog maintenance and other space situational awareness needs. We present a rigorous statistical test design intended to test Space Fence to the letter of the program requirements as well as to characterize the system performance across the entire operational envelope. The design uses altitude, size, and inclination as independent factors in statistical tests of dependent variables (e.g., observation accuracy) linked to requirements. The analysis derives the type and number of necessary test targets. Comparing the resulting sample sizes with the number of currently known targets, we identify those areas where modelling and simulation methods are needed. Assuming hypothetical Kwajalein radar coverage and a conservative number of radar passes per object per day, we conclude that tests involving real-world space objects should take no more than 25 days to evaluate all operational requirements; almost 60 percent of the requirements can be tested in a single day and nearly 90 percent can be tested in one week or less. Reference: [1] L. Haines and P. Phu, Space Fence PDR Concept Development Phase, 2011 AMOS Conference Technical Papers.

High-throughput screening identifies microRNAs that target Nox2 and improve function after acute myocardial infarction.

PubMed

Yang, Junyu; Brown, Milton E; Zhang, Hanshuo; Martinez, Mario; Zhao, Zhihua; Bhutani, Srishti; Yin, Shenyi; Trac, David; Xi, Jianzhong Jeff; Davis, Michael E

2017-05-01

Myocardial infarction (MI) is the most common cause of heart failure. Excessive production of ROS plays a key role in the pathogenesis of cardiac remodeling after MI. NADPH with NADPH oxidase (Nox)2 as the catalytic subunit is a major source of superoxide production, and expression is significantly increased in the infarcted myocardium, especially by infiltrating macrophages. While microRNAs (miRNAs) are potent regulators of gene expression and play an important role in heart disease, there still lacks efficient ways to identify miRNAs that target important pathological genes for treating MI. Thus, the overall objective was to establish a miRNA screening and delivery system for improving heart function after MI using Nox2 as a critical target. With the use of the miRNA-target screening system composed of a self-assembled cell microarray (SAMcell), three miRNAs, miR-106b, miR-148b, and miR-204, were identified that could regulate Nox2 expression and its downstream products in both human and mouse macrophages. Each of these miRNAs were encapsulated into polyketal (PK3) nanoparticles that could effectively deliver miRNAs into macrophages. Both in vitro and in vivo studies in mice confirmed that PK3-miRNAs particles could inhibit Nox2 expression and activity and significantly improve infarct size and acute cardiac function after MI. In conclusion, our results show that miR-106b, miR-148b, and miR-204 were able to improve heart function after myocardial infarction in mice by targeting Nox2 and possibly altering inflammatory cytokine production. This screening system and delivery method could have broader implications for miRNA-mediated therapeutics for cardiovascular and other diseases. NEW & NOTEWORTHY NADPH oxidase (Nox)2 is a promising target for treating cardiovascular disease, but there are no specific inhibitors. Finding endogenous signals that can target Nox2 and other inflammatory molecules is of great interest. In this study, we used high-throughput screening

Distribution and innervation of putative peripheral arterial chemoreceptors in the red-eared slider (Trachemys scripta elegans).

PubMed

Reyes, Catalina; Fong, Angelina Y; Milsom, William K

2015-06-15

Peripheral arterial chemoreceptors have been isolated to the common carotid artery, aorta, and pulmonary artery of turtles. However, the putative neurotransmitters associated with these chemoreceptors have not yet been described. The goal of the present study was to determine the neurochemical content, innervations, and distribution of putative oxygen-sensing cells in the central vasculature of turtles and to derive homologies with peripheral arterial chemoreceptors of other vertebrates. We used tract tracing together with immunohistochemical markers for cholinergic cells (vesicular acetylcholine transporter [VAChT]), tyrosine hydroxylase (TH; the rate-limiting enzyme in catecholamine synthesis), and serotonin (5HT) to identify putative oxygen-sensing cells and to determine their anatomical relation to branches of the vagus nerve (Xth cranial nerve). We found potential oxygen-sensing cells in all three chemosensory areas innervated by branches of the Xth cranial nerve. Cells containing either 5HT or VAChT were found in all three sites. The morphology and size of these cells resemble glomus cells found in amphibians, mammals, tortoises, and lizards. Furthermore, we found populations of cholinergic cells located at the base of the aorta and pulmonary artery that are likely involved in efferent regulation of vessel resistance. Catecholamine-containing cells were not found in any of the putative chemosensitive areas. The presence of 5HT- and VAChT-immunoreactive cells in segments of the common carotid artery, aorta, and pulmonary artery appears to reflect a transition between cells containing the major neurotransmitters seen in fish (5HT) and mammals (ACh and adenosine). © 2015 Wiley Periodicals, Inc.

Identification and use of the putative Bacteroides ovatus xylanase promoter for the inducible production of recombinant human proteins.

PubMed

Hamady, Zaed Z R; Farrar, Mark D; Whitehead, Terence R; Holland, Keith T; Lodge, J Peter A; Carding, Simon R

2008-10-01

The use of genetically modified bacteria to deliver biologically active molecules directly to the gut has become an increasingly attractive area of investigation. The challenge of regulation of production of the therapeutic molecule and colonization of the bowel led us to investigate Bacteroides ovatus for the production of these molecules, due to its ability to colonize the colon and xylan utilization properties. Here we have identified the putative xylanase promoter. The 5' region of the corresponding mRNA was determined by 5'RACE analysis and the transcription initiation site was identified 216 bp upstream of the ATG start codon. The putative xylanase promoter was regulated by xylan in a dose- and time-dependent manner, and repressed by glucose. This promoter was subsequently used to direct the controlled expression of a gene encoding the human intestinal trefoil factor (TFF-3) after integration as a single copy into the chromosome of B. ovatus. The resulting strain produced biologically active TFF-3 in the presence of xylan. These findings identify the B. ovatus xylanase operon promoter and show that it can be utilized to direct xylan-inducible expression of heterologous eukaryotic genes in B. ovatus.

The Druggable Pocketome of Corynebacterium diphtheriae: A New Approach for in silico Putative Druggable Targets

PubMed Central

Hassan, Syed S.; Jamal, Syed B.; Radusky, Leandro G.; Tiwari, Sandeep; Ullah, Asad; Ali, Javed; Behramand; de Carvalho, Paulo V. S. D.; Shams, Rida; Khan, Sabir; Figueiredo, Henrique C. P.; Barh, Debmalya; Ghosh, Preetam; Silva, Artur; Baumbach, Jan; Röttger, Richard; Turjanski, Adrián G.; Azevedo, Vasco A. C.

2018-01-01

Diphtheria is an acute and highly infectious disease, previously regarded as endemic in nature but vaccine-preventable, is caused by Corynebacterium diphtheriae (Cd). In this work, we used an in silico approach along the 13 complete genome sequences of C. diphtheriae followed by a computational assessment of structural information of the binding sites to characterize the “pocketome druggability.” To this end, we first computed the “modelome” (3D structures of a complete genome) of a randomly selected reference strain Cd NCTC13129; that had 13,763 open reading frames (ORFs) and resulted in 1,253 (∼9%) structure models. The amino acid sequences of these modeled structures were compared with the remaining 12 genomes and consequently, 438 conserved protein sequences were obtained. The RCSB-PDB database was consulted to check the template structures for these conserved proteins and as a result, 401 adequate 3D models were obtained. We subsequently predicted the protein pockets for the obtained set of models and kept only the conserved pockets that had highly druggable (HD) values (137 across all strains). Later, an off-target host homology analyses was performed considering the human proteome using NCBI database. Furthermore, the gene essentiality analysis was carried out that gave a final set of 10-conserved targets possessing highly druggable protein pockets. To check the target identification robustness of the pipeline used in this work, we crosschecked the final target list with another in-house target identification approach for C. diphtheriae thereby obtaining three common targets, these were; hisE-phosphoribosyl-ATP pyrophosphatase, glpX-fructose 1,6-bisphosphatase II, and rpsH-30S ribosomal protein S8. Our predicted results suggest that the in silico approach used could potentially aid in experimental polypharmacological target determination in C. diphtheriae and other pathogens, thereby, might complement the existing and new drug-discovery pipelines

A chemical screen for medulloblastoma identifies quercetin as a putative radiosensitizer.

PubMed

Lagerweij, Tonny; Hiddingh, Lotte; Biesmans, Dennis; Crommentuijn, Matheus H W; Cloos, Jacqueline; Li, Xiao-Nan; Kogiso, Mari; Tannous, Bakhos A; Vandertop, W Peter; Noske, David P; Kaspers, Gertjan J L; Würdinger, Tom; Hulleman, Esther

2016-06-14

Treatment of medulloblastoma in children fails in approximately 30% of patients, and is often accompanied by severe late sequelae. Therefore, more effective drugs are needed that spare normal tissue and diminish long-term side effects. Since radiotherapy plays a pivotal role in the treatment of medulloblastoma, we set out to identify novel drugs that could potentiate the effect of ionizing radiation.Thereto, a small molecule library, consisting of 960 chemical compounds, was screened for its ability to sensitize towards irradiation. This small molecule screen identified the flavonoid quercetin as a novel radiosensitizer for the medulloblastoma cell lines DAOY, D283-med, and, to a lesser extent, D458-med at low micromolar concentrations and irradiation doses used in fractionated radiation schemes. Quercetin did not affect the proliferation of neural precursor cells or normal human fibroblasts. Importantly, in vivo experiments confirmed the radiosensitizing properties of quercetin. Administration of this flavonoid at the time of irradiation significantly prolonged survival in orthotopically xenografted mice. Together, these findings indicate that quercetin is a potent radiosensitizer for medulloblastoma cells that may be a promising lead for the treatment of medulloblastoma in patients.

Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci.

PubMed

Canver, Matthew C; Lessard, Samuel; Pinello, Luca; Wu, Yuxuan; Ilboudo, Yann; Stern, Emily N; Needleman, Austen J; Galactéros, Frédéric; Brugnara, Carlo; Kutlar, Abdullah; McKenzie, Colin; Reid, Marvin; Chen, Diane D; Das, Partha Pratim; A Cole, Mitchel; Zeng, Jing; Kurita, Ryo; Nakamura, Yukio; Yuan, Guo-Cheng; Lettre, Guillaume; Bauer, Daniel E; Orkin, Stuart H

2017-04-01

Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, which is associated with red-blood-cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false-positive regions, thus emphasizing the importance of off-target analysis in the design of saturating-mutagenesis experiments. Together, these data establish a widely applicable high-throughput and high-resolution methodology to identify minimal functional sequences within large disease- and trait-associated regions.

Targetable vulnerabilities in T- and NK-cell lymphomas identified through preclinical models.

PubMed

Ng, Samuel Y; Yoshida, Noriaki; Christie, Amanda L; Ghandi, Mahmoud; Dharia, Neekesh V; Dempster, Joshua; Murakami, Mark; Shigemori, Kay; Morrow, Sara N; Van Scoyk, Alexandria; Cordero, Nicolas A; Stevenson, Kristen E; Puligandla, Maneka; Haas, Brian; Lo, Christopher; Meyers, Robin; Gao, Galen; Cherniack, Andrew; Louissaint, Abner; Nardi, Valentina; Thorner, Aaron R; Long, Henry; Qiu, Xintao; Morgan, Elizabeth A; Dorfman, David M; Fiore, Danilo; Jang, Julie; Epstein, Alan L; Dogan, Ahmet; Zhang, Yanming; Horwitz, Steven M; Jacobsen, Eric D; Santiago, Solimar; Ren, Jian-Guo; Guerlavais, Vincent; Annis, D Allen; Aivado, Manuel; Saleh, Mansoor N; Mehta, Amitkumar; Tsherniak, Aviad; Root, David; Vazquez, Francisca; Hahn, William C; Inghirami, Giorgio; Aster, Jon C; Weinstock, David M; Koch, Raphael

2018-05-22

T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53-wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models.

Gene-centric Meta-analysis in 87,736 Individuals of European Ancestry Identifies Multiple Blood-Pressure-Related Loci

PubMed Central

Tragante, Vinicius; Barnes, Michael R.; Ganesh, Santhi K.; Lanktree, Matthew B.; Guo, Wei; Franceschini, Nora; Smith, Erin N.; Johnson, Toby; Holmes, Michael V.; Padmanabhan, Sandosh; Karczewski, Konrad J.; Almoguera, Berta; Barnard, John; Baumert, Jens; Chang, Yen-Pei Christy; Elbers, Clara C.; Farrall, Martin; Fischer, Mary E.; Gaunt, Tom R.; Gho, Johannes M.I.H.; Gieger, Christian; Goel, Anuj; Gong, Yan; Isaacs, Aaron; Kleber, Marcus E.; Leach, Irene Mateo; McDonough, Caitrin W.; Meijs, Matthijs F.L.; Melander, Olle; Nelson, Christopher P.; Nolte, Ilja M.; Pankratz, Nathan; Price, Tom S.; Shaffer, Jonathan; Shah, Sonia; Tomaszewski, Maciej; van der Most, Peter J.; Van Iperen, Erik P.A.; Vonk, Judith M.; Witkowska, Kate; Wong, Caroline O.L.; Zhang, Li; Beitelshees, Amber L.; Berenson, Gerald S.; Bhatt, Deepak L.; Brown, Morris; Burt, Amber; Cooper-DeHoff, Rhonda M.; Connell, John M.; Cruickshanks, Karen J.; Curtis, Sean P.; Davey-Smith, George; Delles, Christian; Gansevoort, Ron T.; Guo, Xiuqing; Haiqing, Shen; Hastie, Claire E.; Hofker, Marten H.; Hovingh, G. Kees; Kim, Daniel S.; Kirkland, Susan A.; Klein, Barbara E.; Klein, Ronald; Li, Yun R.; Maiwald, Steffi; Newton-Cheh, Christopher; O’Brien, Eoin T.; Onland-Moret, N. Charlotte; Palmas, Walter; Parsa, Afshin; Penninx, Brenda W.; Pettinger, Mary; Vasan, Ramachandran S.; Ranchalis, Jane E.; M Ridker, Paul; Rose, Lynda M.; Sever, Peter; Shimbo, Daichi; Steele, Laura; Stolk, Ronald P.; Thorand, Barbara; Trip, Mieke D.; van Duijn, Cornelia M.; Verschuren, W. Monique; Wijmenga, Cisca; Wyatt, Sharon; Young, J. Hunter; Zwinderman, Aeilko H.; Bezzina, Connie R.; Boerwinkle, Eric; Casas, Juan P.; Caulfield, Mark J.; Chakravarti, Aravinda; Chasman, Daniel I.; Davidson, Karina W.; Doevendans, Pieter A.; Dominiczak, Anna F.; FitzGerald, Garret A.; Gums, John G.; Fornage, Myriam; Hakonarson, Hakon; Halder, Indrani; Hillege, Hans L.; Illig, Thomas; Jarvik, Gail P.; Johnson, Julie A.; Kastelein, John J.P.; Koenig, Wolfgang; Kumari, Meena; März, Winfried; Murray, Sarah S.; O’Connell, Jeffery R.; Oldehinkel, Albertine J.; Pankow, James S.; Rader, Daniel J.; Redline, Susan; Reilly, Muredach P.; Schadt, Eric E.; Kottke-Marchant, Kandice; Snieder, Harold; Snyder, Michael; Stanton, Alice V.; Tobin, Martin D.; Uitterlinden, André G.; van der Harst, Pim; van der Schouw, Yvonne T.; Samani, Nilesh J.; Watkins, Hugh; Johnson, Andrew D.; Reiner, Alex P.; Zhu, Xiaofeng; de Bakker, Paul I.W.; Levy, Daniel; Asselbergs, Folkert W.; Munroe, Patricia B.; Keating, Brendan J.

2014-01-01

Blood pressure (BP) is a heritable risk factor for cardiovascular disease. To investigate genetic associations with systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP), and pulse pressure (PP), we genotyped ∼50,000 SNPs in up to 87,736 individuals of European ancestry and combined these in a meta-analysis. We replicated findings in an independent set of 68,368 individuals of European ancestry. Our analyses identified 11 previously undescribed associations in independent loci containing 31 genes including PDE1A, HLA-DQB1, CDK6, PRKAG2, VCL, H19, NUCB2, RELA, HOXC@ complex, FBN1, and NFAT5 at the Bonferroni-corrected array-wide significance threshold (p < 6 × 10−7) and confirmed 27 previously reported associations. Bioinformatic analysis of the 11 loci provided support for a putative role in hypertension of several genes, such as CDK6 and NUCB2. Analysis of potential pharmacological targets in databases of small molecules showed that ten of the genes are predicted to be a target for small molecules. In summary, we identified previously unknown loci associated with BP. Our findings extend our understanding of genes involved in BP regulation, which may provide new targets for therapeutic intervention or drug response stratification. PMID:24560520

Acoustic Parametric Array for Identifying Standoff Targets

NASA Astrophysics Data System (ADS)

Hinders, M. K.; Rudd, K. E.

2010-02-01

An integrated simulation method for investigating nonlinear sound beams and 3D acoustic scattering from any combination of complicated objects is presented. A standard finite-difference simulation method is used to model pulsed nonlinear sound propagation from a source to a scattering target via the KZK equation. Then, a parallel 3D acoustic simulation method based on the finite integration technique is used to model the acoustic wave interaction with the target. Any combination of objects and material layers can be placed into the 3D simulation space to study the resulting interaction. Several example simulations are presented to demonstrate the simulation method and 3D visualization techniques. The combined simulation method is validated by comparing experimental and simulation data and a demonstration of how this combined simulation method assisted in the development of a nonlinear acoustic concealed weapons detector is also presented.

Bioinformatic identification and expression analysis of banana microRNAs and their targets.

PubMed

Chai, Juan; Feng, Renjun; Shi, Hourui; Ren, Mengyun; Zhang, Yindong; Wang, Jingyi

2015-01-01

MicroRNAs (miRNAs) represent a class of endogenous non-coding small RNAs that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Thousands of miRNAs have been identified in many plant species, whereas only a limited number of miRNAs have been predicted in M. acuminata (A genome) and M. balbisiana (B genome). Here, previously known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS), a database of banana genes. A total of 32 potential miRNAs belonging to 13 miRNAs families were detected using a range of filtering criteria. 244 miRNA:target pairs were subsequently predicted, most of which encode transcription factors or enzymes that participate in the regulation of development, growth, metabolism, and other physiological processes. In order to validate the predicted miRNAs and the mutual relationship between miRNAs and their target genes, qRT-PCR was applied to detect the tissue-specific expression levels of 12 putative miRNAs and 6 target genes in roots, leaves, flowers, and fruits. This study provides some important information about banana pre-miRNAs, mature miRNAs, and miRNA target genes and these findings can be applied to future research of miRNA functions.

Localization and expression of putative circadian clock transcripts in the brain of the nudibranch Melibe leonina.

PubMed

Duback, Victoria E; Sabrina Pankey, M; Thomas, Rachel I; Huyck, Taylor L; Mbarani, Izhar M; Bernier, Kyle R; Cook, Geoffrey M; O'Dowd, Colleen A; Newcomb, James M; Watson, Winsor H

2018-09-01

The nudibranch, Melibe leonina, expresses a circadian rhythm of locomotion, and we recently determined the sequences of multiple circadian clock transcripts that may play a role in controlling these daily patterns of behavior. In this study, we used these genomic data to help us: 1) identify putative clock neurons using fluorescent in situ hybridization (FISH); and 2) determine if there is a daily rhythm of expression of clock transcripts in the M. leonina brain, using quantitative PCR. FISH indicated the presence of the clock-related transcripts clock, period, and photoreceptive and non-photoreceptive cryptochrome (pcry and npcry, respectively) in two bilateral neurons in each cerebropleural ganglion and a group of <10 neurons in the anterolateral region of each pedal ganglion. Double-label experiments confirmed colocalization of all four clock transcripts with each other. Quantitative PCR demonstrated that the genes clock, period, pcry and npcry exhibited significant differences in expression levels over 24 h. These data suggest that the putative circadian clock network in M. leonina consists of a small number of identifiable neurons that express circadian genes with a daily rhythm. Copyright © 2018 Elsevier Inc. All rights reserved.

Use of eQTL Analysis for the Discovery of Target Genes Identified by GWAS

DTIC Science & Technology

2013-04-01

the biologic pathways affected by these inherited factors, and ultimately to identify targets for disease prediction, risk stratification and...quality using an Agilent chip technology. Cases having a RIN number of 7.0 or greater were considered good quality. Once completed, the optimum set of...AD_________________ Award Number: W81XWH-11-1-0261 TITLE: Use of eQTL Analysis for the Discovery of

Update on apelin peptides as putative targets for cardiovascular drug discovery.

PubMed

Charles, Christopher J

2011-06-01

The physiological importance of GPCR/ligand pathways is highlighted by the fact that numerous pathologies are attributed to their signaling dysfunction. Over 50% of the pharmaceutical drugs currently used to treat human disease are based on compounds that interact with GPCRs. Apelin/APJ constitutes a novel endogenous peptide/GPCR system proposed to be involved in a wide range of physiological functions. Early evidence suggests that apelin/APJ may hold promise as a target for development of novel therapeutic agents which may counteract a number of pathologies including cardiovascular disease. Despite advances in treatment of cardiovascular disease, incidence, prevalence, morbidity and economic costs remain high necessitating the development of new treatment paradigms. This review summarizes apelin/APJ structure, distribution and regulation; presents evidence for a role of apelin in pressure/volume homeostasis and in the pathophysiology of cardiovascular disease; summarizes data on beneficial effects of apelin in preclinical, animal models of cardiovascular disease and measurement of plasma levels of apelin across the full spectrum of cardiovascular disease in humans; and notes the first studies describing bioactivity of apelin peptides in human healthy volunteers and patients with heart failure. More clarity is needed on the precise physiological/pathophysiological role of the apelin/APJ system in human health and disease. Nonetheless, preclinical studies and initial studies in humans show that APJ antagonism may represent a novel therapeutic target for patients with cardiovascular disease. Development of appropriately validated assays for apelin will clarify circulating levels of the peptide in health and disease. Development of suitable agonists/antagonists will pave the way for much needed future studies essential for advancing this promising field of drug discovery.

Immunogenicity and protective efficacy of recombinant Haemophilus parasuis SH0165 putative outer membrane proteins.

PubMed

Fu, Shulin; Zhang, Minmin; Xu, Juan; Ou, Jiwen; Wang, Yan; Liu, Huazhen; Liu, Jinlin; Chen, Huanchun; Bei, Weicheng

2013-01-02

Haemophilus parasuis (H. parasuis), the causative agent of swine polyserositis, polyarthritis, and meningitis, is one of the most important bacterial diseases of pigs worldwide. Little vaccines currently exist that have a significant effect on infections with all pathogenic serovars of H. parasuis. H. parasuis putative outer membrane proteins (OMPs) are potentially essential components of more effective vaccines. Recently, the genomic sequence of H. parasuis serovar 5 strain SH0165 was completed in our laboratory, which allow us to target OMPs for the development of recombinant vaccines. In this study, we focused on 10 putative OMPs and all the putative OMPs were cloned, expressed and purified as HIS fusion proteins. Primary screening for immunoprotective potential was performed in mice challenged with an LD50 challenge. Out of these 10 OMPs three fusion proteins rGAPDH, rOapA, and rHPS-0675 were found to be protective in a mouse model of H. parasuis infection. We further evaluated the immune responses and protective efficacy of rGAPDH, rOapA, and rHPS-0675 in pig models. All three proteins elicited humoral antibody responses and conferred different levels of protection against challenge with a lethal dose of H. parasuis SH0165 in pig models. In addition, the antisera against the three individual proteins and the synergistic protein efficiently inhibited bacterial growth in a whole blood assay. The data demonstrated that the three proteins showed high value individually and the combination of rGAPDH, rOapA, and rHPS-0675 offered the best protection. Our results indicate that rGAPDH, rOapA, and rHPS-0675 induced protection against H. parasuis SH0165 infection, which may facilitate the development of a multi-component vaccine. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of Putative Coffee Rust Mycoparasites via Single-Molecule DNA Sequencing of Infected Pustules

PubMed Central

Marino, John A.; Perfecto, Ivette; Vandermeer, John

2015-01-01

The interaction of crop pests with their natural enemies is a fundament to their control. Natural enemies of fungal pathogens of crops are poorly known relative to those of insect pests, despite the diversity of fungal pathogens and their economic importance. Currently, many regions across Latin America are experiencing unprecedented epidemics of coffee rust (Hemileia vastatrix). Identification of natural enemies of coffee rust could aid in developing management strategies or in pinpointing species that could be used for biocontrol. In the present study, we characterized fungal communities associated with coffee rust lesions by single-molecule DNA sequencing of fungal rRNA gene bar codes from leaf discs (≈28 mm2) containing rust lesions and control discs with no rust lesions. The leaf disc communities were hyperdiverse in terms of fungi, with up to 69 operational taxonomic units (putative species) per control disc, and the diversity was only slightly reduced in rust-infected discs, with up to 63 putative species. However, geography had a greater influence on the fungal community than whether the disc was infected by coffee rust. Through comparisons between control and rust-infected leaf discs, as well as taxonomic criteria, we identified 15 putative mycoparasitic fungi. These fungi are concentrated in the fungal family Cordycipitaceae and the order Tremellales. These data emphasize the complexity of diverse fungi of unknown ecological function within a leaf that might influence plant disease epidemics or lead to the development of species for biocontrol of fungal disease. PMID:26567299

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.

A Behavioral Intervention to Reduce Child Exposure to Indoor Air Pollution: Identifying Possible Target Behaviors

ERIC Educational Resources Information Center

Barnes, Brendon R.; Mathee, Angela; Shafritz, Lonna B.; Krieger, Laurie; Zimicki, Susan

2004-01-01

Indoor air pollution has been causally linked to acute lower respiratory infections in children younger than 5. The aim of this study was to identify target behaviors for a behavioral intervention to reduce child exposure to indoor air pollution by attempting to answer two research questions: Which behaviors are protective of child respiratory…

Kidney disease models: tools to identify mechanisms and potential therapeutic targets

PubMed Central

Bao, Yin-Wu; Yuan, Yuan; Chen, Jiang-Hua; Lin, Wei-Qiang

2018-01-01

Acute kidney injury (AKI) and chronic kidney disease (CKD) are worldwide public health problems affecting millions of people and have rapidly increased in prevalence in recent years. Due to the multiple causes of renal failure, many animal models have been developed to advance our understanding of human nephropathy. Among these experimental models, rodents have been extensively used to enable mechanistic understanding of kidney disease induction and progression, as well as to identify potential targets for therapy. In this review, we discuss AKI models induced by surgical operation and drugs or toxins, as well as a variety of CKD models (mainly genetically modified mouse models). Results from recent and ongoing clinical trials and conceptual advances derived from animal models are also explored. PMID:29515089

Everyday stress response targets in the science of behavior change.

PubMed

Smyth, Joshua M; Sliwinski, Martin J; Zawadzki, Matthew J; Scott, Stacey B; Conroy, David E; Lanza, Stephanie T; Marcusson-Clavertz, David; Kim, Jinhyuk; Stawski, Robert S; Stoney, Catherine M; Buxton, Orfeu M; Sciamanna, Christopher N; Green, Paige M; Almeida, David M

2018-02-01

Stress is an established risk factor for negative health outcomes, and responses to everyday stress can interfere with health behaviors such as exercise and sleep. In accordance with the Science of Behavior Change (SOBC) program, we apply an experimental medicine approach to identifying stress response targets, developing stress response assays, intervening upon these targets, and testing intervention effectiveness. We evaluate an ecologically valid, within-person approach to measuring the deleterious effects of everyday stress on physical activity and sleep patterns, examining multiple stress response components (i.e., stress reactivity, stress recovery, and stress pile-up) as indexed by two key response indicators (negative affect and perseverative cognition). Our everyday stress response assay thus measures multiple malleable stress response targets that putatively shape daily health behaviors (physical activity and sleep). We hypothesize that larger reactivity, incomplete recovery, and more frequent stress responses (pile-up) will negatively impact health behavior enactment in daily life. We will identify stress-related reactivity, recovery, and response in the indicators using coordinated analyses across multiple naturalistic studies. These results are the basis for developing a new stress assay and replicating the initial findings in a new sample. This approach will advance our understanding of how specific aspects of everyday stress responses influence health behaviors, and can be used to develop and test an innovative ambulatory intervention for stress reduction in daily life to enhance health behaviors. Copyright © 2017 Elsevier Ltd. All rights reserved.

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 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

Analysis of SOX10 mutations identified in Waardenburg-Hirschsprung patients: Differential effects on target gene regulation.

PubMed

Chan, Kwok Keung; Wong, Corinne Kung Yen; Lui, Vincent Chi Hang; Tam, Paul Kwong Hang; Sham, Mai Har

2003-10-15

SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis

Visual Tuning Properties of Genetically Identified Layer 2/3 Neuronal Types in the Primary Visual Cortex of Cre-Transgenic Mice

PubMed Central

Zariwala, Hatim A.; Madisen, Linda; Ahrens, Kurt F.; Bernard, Amy; Lein, Edward S.; Jones, Allan R.; Zeng, Hongkui

2011-01-01

The putative excitatory and inhibitory cell classes within the mouse primary visual cortex V1 have different functional properties as studied using recording microelectrode. Excitatory neurons show high selectivity for the orientation angle of moving gratings while the putative inhibitory neurons show poor selectivity. However, the study of selectivity of the genetically identified interneurons and their subtypes remain controversial. Here we use novel Cre-driver and reporter mice to identify genetic subpopulations in vivo for two-photon calcium dye imaging: Wfs1(+)/Gad1(−) mice that labels layer 2/3 excitatory cell population and Pvalb(+)/Gad1(+) mice that labels a genetic subpopulation of inhibitory neurons. The cells in both mice were identically labeled with a tdTomato protein, visible in vivo, using a Cre-reporter line. We found that the Wfs1(+) cells exhibited visual tuning properties comparable to the excitatory population, i.e., high selectivity and tuning to the angle, direction, and spatial frequency of oriented moving gratings. The functional tuning of Pvalb(+) neurons was consistent with previously reported narrow-spiking interneurons in microelectrode studies, exhibiting poorer selectivity than the excitatory neurons. This study demonstrates the utility of Cre-transgenic mouse technology in selective targeting of subpopulations of neurons and makes them amenable to structural, functional, and connectivity studies. PMID:21283555

Widespread genetic heterogeneity in multiple myeloma: implications for targeted therapy

PubMed Central

Lohr, Jens G.; Stojanov, Petar; Carter, Scott L.; Cruz-Gordillo, Peter; Lawrence, Michael S.; Auclair, Daniel; Sougnez, Carrie; Knoechel, Birgit; Gould, Joshua; Saksena, Gordon; Cibulskis, Kristian; McKenna, Aaron; Chapman, Michael A.; Straussman, Ravid; Levy, Joan; Perkins, Louise M.; Keats, Jonathan J.; Schumacher, Steven E.; Rosenberg, Mara; Getz, Gad

2014-01-01

SUMMARY We performed massively parallel sequencing of paired tumor/normal samples from 203 multiple myeloma (MM) patients and identified significantly mutated genes and copy number alterations, and discovered putative tumor suppressor genes by determining homozygous deletions and loss-of-heterozygosity. We observed frequent mutations in KRAS (particularly in previously treated patients), NRAS, BRAF, FAM46C, TP53 and DIS3 (particularly in non-hyperdiploid MM). Mutations were often present in subclonal populations, and multiple mutations within the same pathway (e.g. KRAS, NRAS and BRAF) were observed in the same patient. In vitro modeling predicts only partial treatment efficacy of targeting subclonal mutations, and even growth promotion of non-mutated subclones in some cases. These results emphasize the importance of heterogeneity analysis for treatment decisions. PMID:24434212

Widespread genetic heterogeneity in multiple myeloma: implications for targeted therapy.

PubMed

Lohr, Jens G; Stojanov, Petar; Carter, Scott L; Cruz-Gordillo, Peter; Lawrence, Michael S; Auclair, Daniel; Sougnez, Carrie; Knoechel, Birgit; Gould, Joshua; Saksena, Gordon; Cibulskis, Kristian; McKenna, Aaron; Chapman, Michael A; Straussman, Ravid; Levy, Joan; Perkins, Louise M; Keats, Jonathan J; Schumacher, Steven E; Rosenberg, Mara; Getz, Gad; Golub, Todd R

2014-01-13

We performed massively parallel sequencing of paired tumor/normal samples from 203 multiple myeloma (MM) patients and identified significantly mutated genes and copy number alterations and discovered putative tumor suppressor genes by determining homozygous deletions and loss of heterozygosity. We observed frequent mutations in KRAS (particularly in previously treated patients), NRAS, BRAF, FAM46C, TP53, and DIS3 (particularly in nonhyperdiploid MM). Mutations were often present in subclonal populations, and multiple mutations within the same pathway (e.g., KRAS, NRAS, and BRAF) were observed in the same patient. In vitro modeling predicts only partial treatment efficacy of targeting subclonal mutations, and even growth promotion of nonmutated subclones in some cases. These results emphasize the importance of heterogeneity analysis for treatment decisions. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification and Phylogenetic Analysis of Tityus pachyurus and Tityus obscurus Novel Putative Na+-Channel Scorpion Toxins

PubMed Central

Guerrero-Vargas, Jimmy A.; Mourão, Caroline B. F.; Quintero-Hernández, Verónica; Possani, Lourival D.; Schwartz, Elisabeth F.

2012-01-01

Background Colombia and Brazil are affected by severe cases of scorpionism. In Colombia the most dangerous accidents are caused by Tityus pachyurus that is widely distributed around this country. In the Brazilian Amazonian region scorpion stings are a common event caused by Tityus obscurus. The main objective of this work was to perform the molecular cloning of the putative Na+-channel scorpion toxins (NaScTxs) from T. pachyurus and T. obscurus venom glands and to analyze their phylogenetic relationship with other known NaScTxs from Tityus species. Methodology/Principal Findings cDNA libraries from venom glands of these two species were constructed and five nucleotide sequences from T. pachyurus were identified as putative modulators of Na+-channels, and were named Tpa4, Tpa5, Tpa6, Tpa7 and Tpa8; the latter being the first anti-insect excitatory β-class NaScTx in Tityus scorpion venom to be described. Fifteen sequences from T. obscurus were identified as putative NaScTxs, among which three had been previously described, and the others were named To4 to To15. The peptides Tpa4, Tpa5, Tpa6, To6, To7, To9, To10 and To14 are closely related to the α-class NaScTxs, whereas Tpa7, Tpa8, To4, To8, To12 and To15 sequences are more related to the β-class NaScTxs. To5 is possibly an arthropod specific toxin. To11 and To13 share sequence similarities with both α and β NaScTxs. By means of phylogenetic analysis using the Maximum Parsimony method and the known NaScTxs from Tityus species, these toxins were clustered into 14 distinct groups. Conclusions/Significance This communication describes new putative NaScTxs from T. pachyurus and T. obscurus and their phylogenetic analysis. The results indicate clear geographic separation between scorpions of Tityus genus inhabiting the Amazonian and Mountain Andes regions and those distributed over the Southern of the Amazonian rainforest. Based on the consensus sequences for the different clusters, a new nomenclature for the Na

Ranking targets in structure-based virtual screening of three-dimensional protein libraries: methods and problems.

PubMed

Kellenberger, Esther; Foata, Nicolas; Rognan, Didier

2008-05-01

Structure-based virtual screening is a promising tool to identify putative targets for a specific ligand. Instead of docking multiple ligands into a single protein cavity, a single ligand is docked in a collection of binding sites. In inverse screening, hits are in fact targets which have been prioritized within the pool of best ranked proteins. The target rate depends on specificity and promiscuity in protein-ligand interactions and, to a considerable extent, on the effectiveness of the scoring function, which still is the Achilles' heel of molecular docking. In the present retrospective study, virtual screening of the sc-PDB target library by GOLD docking was carried out for four compounds (biotin, 4-hydroxy-tamoxifen, 6-hydroxy-1,6-dihydropurine ribonucleoside, and methotrexate) of known sc-PDB targets and, several ranking protocols based on GOLD fitness score and topological molecular interaction fingerprint (IFP) comparison were evaluated. For the four investigated ligands, the fusion of GOLD fitness and two IFP scores allowed the recovery of most targets, including the rare proteins which are not readily suitable for statistical analysis, while significantly filtering out most false positive entries. The current survey suggests that selecting a small number of targets (<20) for experimental evaluation is achievable with a pure structure-based approach.

FOXP2 targets show evidence of positive selection in European populations.

PubMed

Ayub, Qasim; Yngvadottir, Bryndis; Chen, Yuan; Xue, Yali; Hu, Min; Vernes, Sonja C; Fisher, Simon E; Tyler-Smith, Chris

2013-05-02

Forkhead box P2 (FOXP2) is a highly conserved transcription factor that has been implicated in human speech and language disorders and plays important roles in the plasticity of the developing brain. The pattern of nucleotide polymorphisms in FOXP2 in modern populations suggests that it has been the target of positive (Darwinian) selection during recent human evolution. In our study, we searched for evidence of selection that might have followed FOXP2 adaptations in modern humans. We examined whether or not putative FOXP2 targets identified by chromatin-immunoprecipitation genomic screening show evidence of positive selection. We developed an algorithm that, for any given gene list, systematically generates matched lists of control genes from the Ensembl database, collates summary statistics for three frequency-spectrum-based neutrality tests from the low-coverage resequencing data of the 1000 Genomes Project, and determines whether these statistics are significantly different between the given gene targets and the set of controls. Overall, there was strong evidence of selection of FOXP2 targets in Europeans, but not in the Han Chinese, Japanese, or Yoruba populations. Significant outliers included several genes linked to cellular movement, reproduction, development, and immune cell trafficking, and 13 of these constituted a significant network associated with cardiac arteriopathy. Strong signals of selection were observed for CNTNAP2 and RBFOX1, key neurally expressed genes that have been consistently identified as direct FOXP2 targets in multiple studies and that have themselves been associated with neurodevelopmental disorders involving language dysfunction. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Turmeric (Curcuma longa): miRNAs and their regulating targets are involved in development and secondary metabolite pathways.

PubMed

Singh, Noopur; Sharma, Ashok

Turmeric has been used as a therapeutic herb over centuries in traditional medicinal systems due to the presence of several secondary metabolite compounds. microRNAs are known to regulate gene expression at the post-transcriptional level by transcriptional cleavage or translation repression. miRNAs have been demonstrated to play an active role in secondary metabolism regulation. The present work was focused on the identification of the miRNAs involved in the regulation of secondary metabolite and development process of turmeric. Eighteen miRNA families were identified for turmeric. Sixteen miRNA families were observed to regulate 238 target transcripts. LncRNAs targets of the putative miRNA candidates were also predicted. Our results indicated their role in binding, reproduction, stress, and other developmental processes. Gene annotation and pathway analysis illustrated the biological function of the targets regulated by the putative miRNAs. The miRNA-mediated gene regulatory network also revealed co-regulated targets that were regulated by two or more miRNA families. miR156 and miR5015 were observed to be involved in rhizome development. miR5021 showed regulation for terpenoid backbone biosynthesis and isoquinoline alkaloid biosynthesis pathways. The flavonoid biosynthesis pathway was observed to be regulated by miR2919. The analysis revealed the probable involvement of three miRNAs (miR1168.2, miR156b and miR1858) in curcumin biosynthesis. Other miRNAs were found to be involved in the growth and developmental process of turmeric. Phylogenetic analysis of selective miRNAs was also performed. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

A new method of identifying target groups for pronatalist policy applied to Australia.

PubMed

Chen, Mengni; Lloyd, Chris J; Yip, Paul S F

2018-01-01

A country's total fertility rate (TFR) depends on many factors. Attributing changes in TFR to changes of policy is difficult, as they could easily be correlated with changes in the unmeasured drivers of TFR. A case in point is Australia where both pronatalist effort and TFR increased in lock step from 2001 to 2008 and then decreased. The global financial crisis or other unobserved confounders might explain both the reducing TFR and pronatalist incentives after 2008. Therefore, it is difficult to estimate causal effects of policy using econometric techniques. The aim of this study is to instead look at the structure of the population to identify which subgroups most influence TFR. Specifically, we build a stochastic model relating TFR to the fertility rates of various subgroups and calculate elasticity of TFR with respect to each rate. For each subgroup, the ratio of its elasticity to its group size is used to evaluate the subgroup's potential cost effectiveness as a pronatalist target. In addition, we measure the historical stability of group fertility rates, which measures propensity to change. Groups with a high effectiveness ratio and also high propensity to change are natural policy targets. We applied this new method to Australian data on fertility rates broken down by parity, age and marital status. The results show that targeting parity 3+ is more cost-effective than lower parities. This study contributes to the literature on pronatalist policies by investigating the targeting of policies, and generates important implications for formulating cost-effective policies.

A new method of identifying target groups for pronatalist policy applied to Australia

PubMed Central

Chen, Mengni; Lloyd, Chris J.

2018-01-01

A country’s total fertility rate (TFR) depends on many factors. Attributing changes in TFR to changes of policy is difficult, as they could easily be correlated with changes in the unmeasured drivers of TFR. A case in point is Australia where both pronatalist effort and TFR increased in lock step from 2001 to 2008 and then decreased. The global financial crisis or other unobserved confounders might explain both the reducing TFR and pronatalist incentives after 2008. Therefore, it is difficult to estimate causal effects of policy using econometric techniques. The aim of this study is to instead look at the structure of the population to identify which subgroups most influence TFR. Specifically, we build a stochastic model relating TFR to the fertility rates of various subgroups and calculate elasticity of TFR with respect to each rate. For each subgroup, the ratio of its elasticity to its group size is used to evaluate the subgroup’s potential cost effectiveness as a pronatalist target. In addition, we measure the historical stability of group fertility rates, which measures propensity to change. Groups with a high effectiveness ratio and also high propensity to change are natural policy targets. We applied this new method to Australian data on fertility rates broken down by parity, age and marital status. The results show that targeting parity 3+ is more cost-effective than lower parities. This study contributes to the literature on pronatalist policies by investigating the targeting of policies, and generates important implications for formulating cost-effective policies. PMID:29425220

Tiered High-Throughput Screening Approach to Identify ...

EPA Pesticide Factsheets

High-throughput screening (HTS) for potential thyroid–disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the US EPA ToxCast screening assay portfolio. To fill one critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast Phase I and II chemical libraries, comprised of 1,074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single concentration screen were retested in concentration-response. Due to high false positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed two additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using

Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

NASA Astrophysics Data System (ADS)

Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

2015-11-01

Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

A Putative Multiple-Demand System in the Macaque Brain.

PubMed

Mitchell, Daniel J; Bell, Andrew H; Buckley, Mark J; Mitchell, Anna S; Sallet, Jerome; Duncan, John

2016-08-17

In humans, cognitively demanding tasks of many types recruit common frontoparietal brain areas. Pervasive activation of this "multiple-demand" (MD) network suggests a core function in supporting goal-oriented behavior. A similar network might therefore be predicted in nonhuman primates that readily perform similar tasks after training. However, an MD network in nonhuman primates has not been described. Single-cell recordings from macaque frontal and parietal cortex show some similar properties to human MD fMRI responses (e.g., adaptive coding of task-relevant information). Invasive recordings, however, come from limited prespecified locations, so they do not delineate a macaque homolog of the MD system and their positioning could benefit from knowledge of where MD foci lie. Challenges of scanning behaving animals mean that few macaque fMRI studies specifically contrast levels of cognitive demand, so we sought to identify a macaque counterpart to the human MD system using fMRI connectivity in 35 rhesus macaques. Putative macaque MD regions, mapped from frontoparietal MD regions defined in humans, were found to be functionally connected under anesthesia. To further refine these regions, an iterative process was used to maximize their connectivity cross-validated across animals. Finally, whole-brain connectivity analyses identified voxels that were robustly connected to MD regions, revealing seven clusters across frontoparietal and insular cortex comparable to human MD regions and one unexpected cluster in the lateral fissure. The proposed macaque MD regions can be used to guide future electrophysiological investigation of MD neural coding and in task-based fMRI to test predictions of similar functional properties to human MD cortex. In humans, a frontoparietal "multiple-demand" (MD) brain network is recruited during a wide range of cognitively demanding tasks. Because this suggests a fundamental function, one might expect a similar network to exist in nonhuman

The Near-Earth Object Human Space Flight Accessible Targets Study (NHATS) List of Near-Earth Asteroids: Identifying Potential Targets for Future Exploration

NASA Astrophysics Data System (ADS)

Abell, Paul; Barbee, B. W.; Mink, R. G.; Adamo, D. R.; Alberding, C. M.; Mazanek, D. D.; Johnson, L. N.; Yeomans, D. K.; Chodas, P. W.; Chamberlin, A. B.; Benner, L. A. M.; Drake, B. G.; Friedensen, V. P.

2012-10-01

Introduction: Much attention has recently been focused on human exploration of near-Earth asteroids (NEAs). Detailed planning for deep space exploration and identification of potential NEA targets for human space flight requires selecting objects from the growing list of known NEAs. NASA therefore initiated the Near-Earth Object Human Space Flight Accessible Target Study (NHATS), which uses dynamical trajectory performance constraints to identify potentially accessible NEAs. Accessibility Criteria: Future NASA human space flight capability is being defined while the Orion Multi-Purpose Crew Vehicle and Space Launch System are under development. Velocity change and mission duration are two of the most critical factors in any human spaceflight endeavor, so the most accessible NEAs tend to be those with orbits similar to Earth’s. To be classified as NHATS-compliant, a NEA must offer at least one round-trip trajectory solution satisfying purposely inclusive constraints, including total mission change in velocity ≤ 12 km/s, mission duration ≤ 450 days (with at least 8 days at the NEA), Earth departure between Jan 1, 2015 and Dec 31, 2040, Earth departure C3 ≤ 60 km2/s2, and Earth return atmospheric entry speed ≤ 12 km/s. Monitoring and Updates: The NHATS list of potentially accessible targets is continuously updated as NEAs are discovered and orbit solutions for known NEAs are improved. The current list of accessible NEAs identified as potentially viable for future human exploration under the NHATS criteria is available to the international community via a website maintained by NASA’s NEO Program Office (http://neo.jpl.nasa.gov/nhats/). This website also lists predicted optical and radar observing opportunities for each NHATS-compliant NEA to facilitate acquisition of follow-up observations. Conclusions: This list of NEAs will be useful for analyzing robotic mission opportunities, identifying optimal round trip human space flight trajectories, and

Putative Bronchopulmonary Flagellated Protozoa in Immunosuppressed Patients

PubMed Central

Kilimcioglu, Ali Ahmet; Havlucu, Yavuz; Çelik, Pınar; Özbilgin, Ahmet

2014-01-01

Flagellated protozoa that cause bronchopulmonary symptoms in humans are commonly neglected. These protozoal forms which were presumed to be “flagellated protozoa” have been previously identified in immunosuppressed patients in a number of studies, but have not been certainly classified so far. Since no human cases of bronchopulmonary flagellated protozoa were reported from Turkey, we aimed to investigate these putative protozoa in immunosuppressed patients who are particularly at risk of infectious diseases. Bronchoalveolar lavage fluid samples of 110 immunosuppressed adult patients who were admitted to the Department of Chest Diseases, Hafsa Sultan Hospital of Celal Bayar University, Manisa, Turkey, were examined in terms of parasites by light microscopy. Flagellated protozoal forms were detected in nine (8.2%) of 110 cases. Metronidazole (500 mg b.i.d. for 30 days) was given to all positive cases and a second bronchoscopy was performed at the end of the treatment, which revealed no parasites. In conclusion, immunosuppressed patients with bronchopulmonary symptoms should attentively be examined with regard to flagellated protozoa which can easily be misidentified as epithelial cells. PMID:24804259

Putative bronchopulmonary flagellated protozoa in immunosuppressed patients.

PubMed

Kilimcioglu, Ali Ahmet; Havlucu, Yavuz; Girginkardesler, Nogay; Celik, Pınar; Yereli, Kor; Özbilgin, Ahmet

2014-01-01

Flagellated protozoa that cause bronchopulmonary symptoms in humans are commonly neglected. These protozoal forms which were presumed to be "flagellated protozoa" have been previously identified in immunosuppressed patients in a number of studies, but have not been certainly classified so far. Since no human cases of bronchopulmonary flagellated protozoa were reported from Turkey, we aimed to investigate these putative protozoa in immunosuppressed patients who are particularly at risk of infectious diseases. Bronchoalveolar lavage fluid samples of 110 immunosuppressed adult patients who were admitted to the Department of Chest Diseases, Hafsa Sultan Hospital of Celal Bayar University, Manisa, Turkey, were examined in terms of parasites by light microscopy. Flagellated protozoal forms were detected in nine (8.2%) of 110 cases. Metronidazole (500 mg b.i.d. for 30 days) was given to all positive cases and a second bronchoscopy was performed at the end of the treatment, which revealed no parasites. In conclusion, immunosuppressed patients with bronchopulmonary symptoms should attentively be examined with regard to flagellated protozoa which can easily be misidentified as epithelial cells.

Identification and Characterization of Putative Integron-Like Elements of the Heavy-Metal-Hypertolerant Strains of Pseudomonas spp.

PubMed

Ciok, Anna; Adamczuk, Marcin; Bartosik, Dariusz; Dziewit, Lukasz

2016-11-28

Pseudomonas strains isolated from the heavily contaminated Lubin copper mine and Zelazny Most post-flotation waste reservoir in Poland were screened for the presence of integrons. This analysis revealed that two strains carried homologous DNA regions composed of a gene encoding a DNA_BRE_C domain-containing tyrosine recombinase (with no significant sequence similarity to other integrases of integrons) plus a three-component array of putative integron gene cassettes. The predicted gene cassettes encode three putative polypeptides with homology to (i) transmembrane proteins, (ii) GCN5 family acetyltransferases, and (iii) hypothetical proteins of unknown function (homologous proteins are encoded by the gene cassettes of several class 1 integrons). Comparative sequence analyses identified three structural variants of these novel integron-like elements within the sequenced bacterial genomes. Analysis of their distribution revealed that they are found exclusively in strains of the genus Pseudomonas .

Uncovering the Putative B-Star Binary Companion of the SN 1993J Progenitor

NASA Technical Reports Server (NTRS)

Fox, Ori D.; Bostroem, K. Azalee; Van Dyk, Schuyler D.; Filippenko, Alexei V.; Fransson, Claes; Matheson, Thomas; Cenko, S. Bradley; Chandra, Poonam; Dwarkadas, Vikram; Li, Weidong;

In-situ Click Reaction Coupled with Quantitative Proteomics for Identifying Protein Targets of Catechol Estrogens.

PubMed

Liang, Huei-Chen; Liu, Yi-Chen; Chen, Hsin; Ku, Ming Chun; Do, Quynh-Trang; Wang, Chih-Yen; Tzeng, Shun-Fen; Chen, Shu-Hui

2018-06-13

Catechol estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in-situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography-mass spectrometry (LC-MS). A total of 334 liver proteins were repeatedly identified (n  2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 non-specific binders (keratins) had D/H < 1. These results were confirmed using spiked covalent control (strong binder) and noncovalent control (weak binder), as well as in vitro testing of cytochrome c (D/H = 5.9) which showed covalent conjugation with CEs. Many identified strong binders, such as glutathione transferase, catechol-O-methyl transferase, superoxide dismutase, catalase, glutathione peroxidase, and cytochrome c, are involved in cellular redox processes or detoxification activities. CE conjugation was shown to suppress the superoxide oxidase activity of cytochrome c, suggesting that CEs modification may alter the redox action of cellular proteins. Due to structural similarity and inert alkyne group, EE2 probe is very likely to capture protein targets of CEs in general. Thus, this strategy can be adopted to explore the biological impact of CEs modification in living systems.

Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

DOE PAGES

Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica; ...

2016-04-28

Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and thatmore » they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all

Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

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

Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica

Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and thatmore » they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all

Integrated genomic analysis identifies the mitotic checkpoint kinase WEE1 as a novel therapeutic target in medulloblastoma

PubMed Central

2014-01-01

Background Medulloblastoma is the most common type of malignant brain tumor that afflicts children. Although recent advances in chemotherapy and radiation have improved outcomes, high-risk patients do poorly with significant morbidity. Methods To identify new molecular targets, we performed an integrated genomic analysis using structural and functional methods. Gene expression profiling in 16 medulloblastoma patient samples and subsequent gene set enrichment analysis indicated that cell cycle-related kinases were associated with disease development. In addition a kinome-wide small interfering RNA (siRNA) screen was performed to identify kinases that, when inhibited, could prevent cell proliferation. The two genome-scale analyses were combined to identify key vulnerabilities in medulloblastoma. The inhibition of one of the identified targets was further investigated using RNAi and a small molecule inhibitor. Results Combining the two analyses revealed that mitosis-related kinases were critical determinants of medulloblastoma cell proliferation. RNA interference (RNAi)-mediated knockdown of WEE1 kinase and other mitotic kinases was sufficient to reduce medulloblastoma cell proliferation. These data prompted us to examine the effects of inhibiting WEE1 by RNAi and by a small molecule inhibitor of WEE1, MK-1775, in medulloblastoma cell lines. MK-1775 inhibited the growth of medulloblastoma cell lines, induced apoptosis and increased DNA damage at nanomolar concentrations. Further, MK-1775 was synergistic with cisplatin in reducing medulloblastoma cell proliferation and resulted in an associated increase in cell death. In vivo MK-1775 suppressed medulloblastoma tumor growth as a single agent. Conclusions Taken together, these findings highlight mitotic kinases and, in particular, WEE1 as a rational therapeutic target for medulloblastoma. PMID:24661910

A Meta-analysis of Multiple Myeloma Risk Regions in African and European Ancestry Populations Identifies Putatively Functional Loci.

PubMed

Rand, Kristin A; Song, Chi; Dean, Eric; Serie, Daniel J; Curtin, Karen; Sheng, Xin; Hu, Donglei; Huff, Carol Ann; Bernal-Mizrachi, Leon; Tomasson, Michael H; Ailawadhi, Sikander; Singhal, Seema; Pawlish, Karen; Peters, Edward S; Bock, Cathryn H; Stram, Alex; Van Den Berg, David J; Edlund, Christopher K; Conti, David V; Zimmerman, Todd; Hwang, Amie E; Huntsman, Scott; Graff, John; Nooka, Ajay; Kong, Yinfei; Pregja, Silvana L; Berndt, Sonja I; Blot, William J; Carpten, John; Casey, Graham; Chu, Lisa; Diver, W Ryan; Stevens, Victoria L; Lieber, Michael R; Goodman, Phyllis J; Hennis, Anselm J M; Hsing, Ann W; Mehta, Jayesh; Kittles, Rick A; Kolb, Suzanne; Klein, Eric A; Leske, Cristina; Murphy, Adam B; Nemesure, Barbara; Neslund-Dudas, Christine; Strom, Sara S; Vij, Ravi; Rybicki, Benjamin A; Stanford, Janet L; Signorello, Lisa B; Witte, John S; Ambrosone, Christine B; Bhatti, Parveen; John, Esther M; Bernstein, Leslie; Zheng, Wei; Olshan, Andrew F; Hu, Jennifer J; Ziegler, Regina G; Nyante, Sarah J; Bandera, Elisa V; Birmann, Brenda M; Ingles, Sue A; Press, Michael F; Atanackovic, Djordje; Glenn, Martha J; Cannon-Albright, Lisa A; Jones, Brandt; Tricot, Guido; Martin, Thomas G; Kumar, Shaji K; Wolf, Jeffrey L; Deming Halverson, Sandra L; Rothman, Nathaniel; Brooks-Wilson, Angela R; Rajkumar, S Vincent; Kolonel, Laurence N; Chanock, Stephen J; Slager, Susan L; Severson, Richard K; Janakiraman, Nalini; Terebelo, Howard R; Brown, Elizabeth E; De Roos, Anneclaire J; Mohrbacher, Ann F; Colditz, Graham A; Giles, Graham G; Spinelli, John J; Chiu, Brian C; Munshi, Nikhil C; Anderson, Kenneth C; Levy, Joan; Zonder, Jeffrey A; Orlowski, Robert Z; Lonial, Sagar; Camp, Nicola J; Vachon, Celine M; Ziv, Elad; Stram, Daniel O; Hazelett, Dennis J; Haiman, Christopher A; Cozen, Wendy

2016-12-01

Genome-wide association studies (GWAS) in European populations have identified genetic risk variants associated with multiple myeloma. We performed association testing of common variation in eight regions in 1,318 patients with multiple myeloma and 1,480 controls of European ancestry and 1,305 patients with multiple myeloma and 7,078 controls of African ancestry and conducted a meta-analysis to localize the signals, with epigenetic annotation used to predict functionality. We found that variants in 7p15.3, 17p11.2, 22q13.1 were statistically significantly (P < 0.05) associated with multiple myeloma risk in persons of African ancestry and persons of European ancestry, and the variant in 3p22.1 was associated in European ancestry only. In a combined African ancestry-European ancestry meta-analysis, variation in five regions (2p23.3, 3p22.1, 7p15.3, 17p11.2, 22q13.1) was statistically significantly associated with multiple myeloma risk. In 3p22.1, the correlated variants clustered within the gene body of ULK4 Correlated variants in 7p15.3 clustered around an enhancer at the 3' end of the CDCA7L transcription termination site. A missense variant at 17p11.2 (rs34562254, Pro251Leu, OR, 1.32; P = 2.93 × 10 -7 ) in TNFRSF13B encodes a lymphocyte-specific protein in the TNF receptor family that interacts with the NF-κB pathway. SNPs correlated with the index signal in 22q13.1 cluster around the promoter and enhancer regions of CBX7 CONCLUSIONS: We found that reported multiple myeloma susceptibility regions contain risk variants important across populations, supporting the use of multiple racial/ethnic groups with different underlying genetic architecture to enhance the localization and identification of putatively functional alleles. A subset of reported risk loci for multiple myeloma has consistent effects across populations and is likely to be functional. Cancer Epidemiol Biomarkers Prev; 25(12); 1609-18. ©2016 AACR. ©2016 American Association for Cancer Research.

Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species.

PubMed

Zhao, Xin; Kuipers, Oscar P

2016-11-07

Gram-positive bacteria of the Bacillales are important producers of antimicrobial compounds that might be utilized for medical, food or agricultural applications. Thanks to the wide availability of whole genome sequence data and the development of specific genome mining tools, novel antimicrobial compounds, either ribosomally- or non-ribosomally produced, of various Bacillales species can be predicted and classified. Here, we provide a classification scheme of known and putative antimicrobial compounds in the specific context of Bacillales species. We identify and describe known and putative bacteriocins, non-ribosomally synthesized peptides (NRPs), polyketides (PKs) and other antimicrobials from 328 whole-genome sequenced strains of 57 species of Bacillales by using web based genome-mining prediction tools. We provide a classification scheme for these bacteriocins, update the findings of NRPs and PKs and investigate their characteristics and suitability for biocontrol by describing per class their genetic organization and structure. Moreover, we highlight the potential of several known and novel antimicrobials from various species of Bacillales. Our extended classification of antimicrobial compounds demonstrates that Bacillales provide a rich source of novel antimicrobials that can now readily be tapped experimentally, since many new gene clusters are identified.

Phenome-genome association studies of pancreatic cancer: new targets for therapy and diagnosis.

PubMed

Narayanan, Ramaswamy

2015-01-01

Pancreatic cancer, has a very high mortality rate and requires novel molecular targets for diagnosis and therapy. Genetic association studies over databases offer an attractive starting point for gene discovery. The National Center for Biotechnology Information (NCBI) Phenome Genome Integrator (PheGenI) tool was enriched for pancreatic cancer-associated traits. The genes associated with the trait were characterized using diverse bioinformatics tools for Genome-Wide Association (GWA), transcriptome and proteome profile and protein classes for motif and domain. Two hundred twenty-six genes were identified that had a genetic association with pancreatic cancer in the human genome. This included 25 uncharacterized open reading frames (ORFs). Bioinformatics analysis of these ORFs identified putative druggable proteins and biomarkers including enzymes, transporters and G-protein-coupled receptor signaling proteins. Secreted proteins including a neuroendocrine factor and a chemokine were identified. Five out of these ORFs encompassed non coding RNAs. The ORF protein expression was detected in numerous body fluids, such as ascites, bile, pancreatic juice, milk, plasma, serum and saliva. Transcriptome and proteome analyses showed a correlation of mRNA and protein expression for nine ORFs. Analysis of the Catalogue of Somatic Mutations in Cancer (COSMIC) database revealed a strong correlation across copy number variations and mRNA over-expression for four ORFs. Mining of the International Cancer Gene Consortium (ICGC) database identified somatic mutations in a significant number of pancreatic patients' tumors for most of these ORFs. The pancreatic cancer-associated ORFs were also found to be genetically associated with other neoplasms, including leukemia, malignant melanoma, neuroblastoma and prostate carcinomas, as well as other unrelated diseases and disorders, such as Alzheimer's disease, Crohn's disease, coronary diseases, attention deficit disorder and addiction. Based

Identification of Putative Coffee Rust Mycoparasites via Single-Molecule DNA Sequencing of Infected Pustules.

PubMed

James, Timothy Y; Marino, John A; Perfecto, Ivette; Vandermeer, John

2016-01-15

The interaction of crop pests with their natural enemies is a fundament to their control. Natural enemies of fungal pathogens of crops are poorly known relative to those of insect pests, despite the diversity of fungal pathogens and their economic importance. Currently, many regions across Latin America are experiencing unprecedented epidemics of coffee rust (Hemileia vastatrix). Identification of natural enemies of coffee rust could aid in developing management strategies or in pinpointing species that could be used for biocontrol. In the present study, we characterized fungal communities associated with coffee rust lesions by single-molecule DNA sequencing of fungal rRNA gene bar codes from leaf discs (≈28 mm(2)) containing rust lesions and control discs with no rust lesions. The leaf disc communities were hyperdiverse in terms of fungi, with up to 69 operational taxonomic units (putative species) per control disc, and the diversity was only slightly reduced in rust-infected discs, with up to 63 putative species. However, geography had a greater influence on the fungal community than whether the disc was infected by coffee rust. Through comparisons between control and rust-infected leaf discs, as well as taxonomic criteria, we identified 15 putative mycoparasitic fungi. These fungi are concentrated in the fungal family Cordycipitaceae and the order Tremellales. These data emphasize the complexity of diverse fungi of unknown ecological function within a leaf that might influence plant disease epidemics or lead to the development of species for biocontrol of fungal disease. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A screen of cell-surface molecules identifies leucine-rich repeat proteins as key mediators of synaptic target selection in the Drosophila neuromuscular system

PubMed Central

Kurusu, Mitsuhiko; Cording, Amy; Taniguchi, Misako; Menon, Kaushiki; Suzuki, Emiko; Zinn, Kai

2008-01-01

Summary In Drosophila embryos and larvae, a small number of identified motor neurons innervate body wall muscles in a highly stereotyped pattern. Although genetic screens have identified many proteins that are required for axon guidance and synaptogenesis in this system, little is known about the mechanisms by which muscle fibers are defined as targets for specific motor axons. To identify potential target labels, we screened 410 genes encoding cell-surface and secreted proteins, searching for those whose overexpression on all muscle fibers causes motor axons to make targeting errors. Thirty such genes were identified, and a number of these were members of a large gene family encoding proteins whose extracellular domains contain leucine-rich repeat (LRR) sequences, which are protein interaction modules. By manipulating gene expression in muscle 12, we showed that four LRR proteins participate in the selection of this muscle as the appropriate synaptic target for the RP5 motor neuron. PMID:18817735

Characterization of two new putative adhesins of Leptospira interrogans.

PubMed

Figueredo, Jupciana M; Siqueira, Gabriela H; de Souza, Gisele O; Heinemann, Marcos B; Vasconcellos, Silvio A; Chapola, Erica G B; Nascimento, Ana L T O

2017-01-01

We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host -pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.

Identifying mechanisms for facilitating knowledge to action strategies targeting the built environment.

PubMed

Fazli, Ghazal S; Creatore, Maria I; Matheson, Flora I; Guilcher, Sara; Kaufman-Shriqui, Vered; Manson, Heather; Johns, Ashley; Booth, Gillian L

2017-01-03

In recent years, obesity-related diseases have been on the rise globally resulting in major challenges for health systems and society as a whole. Emerging research in population health suggests that interventions targeting the built environment may help reduce the burden of obesity and type 2 diabetes. However, translation of the evidence on the built environment into effective policy and planning changes requires engagement and collaboration between multiple sectors and government agencies for designing neighborhoods that are more conducive to healthy and active living. In this study, we identified knowledge gaps and other barriers to evidence-based decision-making and policy development related to the built environment; as well as the infrastructure, processes, and mechanisms needed to drive policy changes in this area. We conducted a qualitative thematic analysis of data collected through consultations with a broad group of stakeholders (N = 42) from Southern Ontario, Canada, within various sectors (public health, urban planning, and transportation) and levels of government (federal, provincial, and municipalities). Relevant themes were classified based on the specific phase of the knowledge-to-action cycle (research, translation, and implementation) in which they were most closely aligned. We identified 5 themes including: 1) the need for policy-informed and actionable research (e.g. health economic analyses and policy evaluations); 2) impactful messaging that targets all relevant sectors to create the political will necessary to drive policy change; 3) common measures and tools to increase capacity for monitoring and surveillance of built environment changes; (4) intersectoral collaboration and alignment within and between levels of government to enable collective actions and provide mechanisms for sharing of resources and expertise, (5) aligning public and private sector priorities to generate public demand and support for community action; and, (6

Functional precision medicine identifies novel druggable targets and therapeutic options in head and neck cancer. | Office of Cancer Genomics

Cancer.gov

Purpose: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with high mortality and a lack of targeted therapies. To identify and prioritize druggable targets, we performed genome analysis together with genome-scale siRNA and oncology drug profiling using low passage tumor cells derived from a patient with a treatmentresistant HPV-negative HNSCC.

Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma

PubMed Central

Bartel, Sabine; Schulz, Nikola; Alessandrini, Francesca; Schamberger, Andrea C.; Pagel, Philipp; Theis, Fabian J.; Milger, Katrin; Noessner, Elfriede; Stick, Stephen M.; Kicic, Anthony; Eickelberg, Oliver; Freishtat, Robert J.; Krauss-Etschmann, Susanne

2017-01-01

Asthma is highly prevalent, but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study, we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore, deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17, miR-144, and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment, while it’s expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally, we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies. PMID:28383034

Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma.

PubMed

Bartel, Sabine; Schulz, Nikola; Alessandrini, Francesca; Schamberger, Andrea C; Pagel, Philipp; Theis, Fabian J; Milger, Katrin; Noessner, Elfriede; Stick, Stephen M; Kicic, Anthony; Eickelberg, Oliver; Freishtat, Robert J; Krauss-Etschmann, Susanne

2017-04-06

Asthma is highly prevalent, but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study, we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore, deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17, miR-144, and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment, while it's expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally, we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies.

Novel Modeling of Combinatorial miRNA Targeting Identifies SNP with Potential Role in Bone Density

PubMed Central

Coronnello, Claudia; Hartmaier, Ryan; Arora, Arshi; Huleihel, Luai; Pandit, Kusum V.; Bais, Abha S.; Butterworth, Michael; Kaminski, Naftali; Stormo, Gary D.; Oesterreich, Steffi; Benos, Panayiotis V.

2012-01-01

MicroRNAs (miRNAs) are post-transcriptional regulators that bind to their target mRNAs through base complementarity. Predicting miRNA targets is a challenging task and various studies showed that existing algorithms suffer from high number of false predictions and low to moderate overlap in their predictions. Until recently, very few algorithms considered the dynamic nature of the interactions, including the effect of less specific interactions, the miRNA expression level, and the effect of combinatorial miRNA binding. Addressing these issues can result in a more accurate miRNA:mRNA modeling with many applications, including efficient miRNA-related SNP evaluation. We present a novel thermodynamic model based on the Fermi-Dirac equation that incorporates miRNA expression in the prediction of target occupancy and we show that it improves the performance of two popular single miRNA target finders. Modeling combinatorial miRNA targeting is a natural extension of this model. Two other algorithms show improved prediction efficiency when combinatorial binding models were considered. ComiR (Combinatorial miRNA targeting), a novel algorithm we developed, incorporates the improved predictions of the four target finders into a single probabilistic score using ensemble learning. Combining target scores of multiple miRNAs using ComiR improves predictions over the naïve method for target combination. ComiR scoring scheme can be used for identification of SNPs affecting miRNA binding. As proof of principle, ComiR identified rs17737058 as disruptive to the miR-488-5p:NCOA1 interaction, which we confirmed in vitro. We also found rs17737058 to be significantly associated with decreased bone mineral density (BMD) in two independent cohorts indicating that the miR-488-5p/NCOA1 regulatory axis is likely critical in maintaining BMD in women. With increasing availability of comprehensive high-throughput datasets from patients ComiR is expected to become an essential tool for mi

Pharmacophore based design of some multi-targeted compounds targeted against pathways of diabetic complications.

PubMed

Chadha, Navriti; Silakari, Om

2017-09-01

Diabetic complications is a complex metabolic disorder developed primarily due to prolonged hyperglycemia in the body. The complexity of the disease state as well as the unifying pathophysiology discussed in the literature reports exhibited that the use of multi-targeted agents with multiple complementary biological activities may offer promising therapy for the intervention of the disease over the single-target drugs. In the present study, novel thiazolidine-2,4-dione analogues were designed as multi-targeted agents implicated against the molecular pathways involved in diabetic complications using knowledge based as well as in-silico approaches such as pharmacophore mapping, molecular docking etc. The hit molecules were duly synthesized and biochemical estimation of these molecules against aldose reductase (ALR2), protein kinase Cβ (PKCβ) and poly (ADP-ribose) polymerase 1 (PARP-1) led to identification of compound 2 that showed good potency against PARP-1 and ALR2 enzymes. These positive results support the progress of a low cost multi-targeted agent with putative roles in diabetic complications. Copyright © 2017 Elsevier Inc. All rights reserved.

Deep sequencing and in silico analysis of small RNA library reveals novel miRNA from leaf Persicaria minor transcriptome.

PubMed

Samad, Abdul Fatah A; Nazaruddin, Nazaruddin; Murad, Abdul Munir Abdul; Jani, Jaeyres; Zainal, Zamri; Ismail, Ismanizan

2018-03-01

In current era, majority of microRNA (miRNA) are being discovered through computational approaches which are more confined towards model plants. Here, for the first time, we have described the identification and characterization of novel miRNA in a non-model plant, Persicaria minor ( P . minor ) using computational approach. Unannotated sequences from deep sequencing were analyzed based on previous well-established parameters. Around 24 putative novel miRNAs were identified from 6,417,780 reads of the unannotated sequence which represented 11 unique putative miRNA sequences. PsRobot target prediction tool was deployed to identify the target transcripts of putative novel miRNAs. Most of the predicted target transcripts (mRNAs) were known to be involved in plant development and stress responses. Gene ontology showed that majority of the putative novel miRNA targets involved in cellular component (69.07%), followed by molecular function (30.08%) and biological process (0.85%). Out of 11 unique putative miRNAs, 7 miRNAs were validated through semi-quantitative PCR. These novel miRNAs discoveries in P . minor may develop and update the current public miRNA database.

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.

COMPARATIVE DIVERSITY OF FECAL BACTERIA IN AGRICULTURALLY SIGNIFICANT ANIMALS TO IDENTIFY ALTERNATIVE TARGETS FOR MICROBIAL SOURCE TRACKING

EPA Science Inventory

Animals of agricultural significance contribute a large percentage of fecal pollution to waterways via runoff contamination. The premise of microbial source tracking is to utilize fecal bacteria to identify target populations which are directly correlated to specific animal feces...

SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea.

PubMed

Kirm, Benjamin; Magdevska, Vasilka; Tome, Miha; Horvat, Marinka; Karničar, Katarina; Petek, Marko; Vidmar, Robert; Baebler, Spela; Jamnik, Polona; Fujs, Štefan; Horvat, Jaka; Fonovič, Marko; Turk, Boris; Gruden, Kristina; Petković, Hrvoje; Kosec, Gregor

2013-12-17

Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bld

SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea

PubMed Central

2013-01-01

Background Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. Results We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. Conclusions SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence

iTAR: a web server for identifying target genes of transcription factors using ChIP-seq or ChIP-chip data.

PubMed

Yang, Chia-Chun; Andrews, Erik H; Chen, Min-Hsuan; Wang, Wan-Yu; Chen, Jeremy J W; Gerstein, Mark; Liu, Chun-Chi; Cheng, Chao

2016-08-12

Chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) or microarray hybridization (ChIP-chip) has been widely used to determine the genomic occupation of transcription factors (TFs). We have previously developed a probabilistic method, called TIP (Target Identification from Profiles), to identify TF target genes using ChIP-seq/ChIP-chip data. To achieve high specificity, TIP applies a conservative method to estimate significance of target genes, with the trade-off being a relatively low sensitivity of target gene identification compared to other methods. Additionally, TIP's output does not render binding-peak locations or intensity, information highly useful for visualization and general experimental biological use, while the variability of ChIP-seq/ChIP-chip file formats has made input into TIP more difficult than desired. To improve upon these facets, here we present are fined TIP with key extensions. First, it implements a Gaussian mixture model for p-value estimation, increasing target gene identification sensitivity and more accurately capturing the shape of TF binding profile distributions. Second, it enables the incorporation of TF binding-peak data by identifying their locations in significant target gene promoter regions and quantifies their strengths. Finally, for full ease of implementation we have incorporated it into a web server ( http://syslab3.nchu.edu.tw/iTAR/ ) that enables flexibility of input file format, can be used across multiple species and genome assembly versions, and is freely available for public use. The web server additionally performs GO enrichment analysis for the identified target genes to reveal the potential function of the corresponding TF. The iTAR web server provides a user-friendly interface and supports target gene identification in seven species, ranging from yeast to human. To facilitate investigating the quality of ChIP-seq/ChIP-chip data, the web server generates the chart of the

Strategic incorporation of fluorine in the drug discovery of new-generation antitubercular agents targeting bacterial cell division protein FtsZ⋆

PubMed Central

Ojima, Iwao; Awasthi, Divya; Wei, Longfei; Haranahalli, Krupanandan

2016-01-01

This article presents an account of our research on the discovery and development of new-generation fluorine-containing antibacterial agents against drug-resistant tuberculosis, targeting FtsZ. FtsZ is an essential protein for bacterial cell division and a highly promising therapeutic target for antibacterial drug discovery. Through design, synthesis and semi-HTP screening of libraries of novel benzimidazoles, followed by SAR studies, we identified highly potent lead compounds. However, these lead compounds were found to lack sufficient metabolic and plasma stabilities. Accordingly, we have performed extensive study on the strategic incorporation of fluorine into lead compounds to improve pharmacological properties. This study has led to the development of highly efficacious fluorine-containing benzimidazoles as potential drug candidates. We have also performed computational docking analysis of these novel FtsZ inhibitors to identify their putative binding site. Based on the structural data and docking analysis, a plausible mode-of-action for this novel class of FtsZ inhibitors is proposed. PMID:28555087

Meta-Analysis Identifies NF-κB as a Therapeutic Target in Renal Cancer

PubMed Central

Peri, Suraj; Devarajan, Karthik; Yang, Dong-Hua; Knudson, Alfred G.; Balachandran, Siddharth

2013-01-01

Objective To determine the expression patterns of NF-κB regulators and target genes in clear cell renal cell carcinoma (ccRCC), their correlation with von Hippel Lindau (VHL) mutational status, and their association with survival outcomes. Methods Meta-analyses were carried out on published ccRCC gene expression datasets by RankProd, a non-parametric statistical method. DEGs with a False Discovery Rate of < 0.05 by this method were considered significant, and intersected with a curated list of NF-κB regulators and targets to determine the nature and extent of NF-κB deregulation in ccRCC. Results A highly-disproportionate fraction (~40%; p < 0.001) of NF-κB regulators and target genes were found to be up-regulated in ccRCC, indicative of elevated NF-κB activity in this cancer. A subset of these genes, comprising a key NF-κB regulator (IKBKB) and established mediators of the NF-κB cell-survival and pro-inflammatory responses (MMP9, PSMB9, and SOD2), correlated with higher relative risk, poorer prognosis, and reduced overall patient survival. Surprisingly, levels of several interferon regulatory factors (IRFs) and interferon target genes were also elevated in ccRCC, indicating that an ‘interferon signature’ may represent a novel feature of this disease. Loss of VHL gene expression correlated strongly with the appearance of NF-κB- and interferon gene signatures in both familial and sporadic cases of ccRCC. As NF-κB controls expression of key interferon signaling nodes, our results suggest a causal link between VHL loss, elevated NF-κB activity, and the appearance of an interferon signature during ccRCC tumorigenesis. Conclusions These findings identify NF-κB and interferon signatures as clinical features of ccRCC, provide strong rationale for the incorporation of NF-κB inhibitors and/or and the exploitation of interferon signaling in the treatment of ccRCC, and supply new NF-κB targets for potential therapeutic intervention in this currently

CD24 can be used to isolate Lgr5+ putative colonic epithelial stem cells in mice

PubMed Central

King, Jeffrey B.; von Furstenberg, Richard J.; Smith, Brian J.; McNaughton, Kirk K.; Galanko, Joseph A.

2012-01-01

A growing body of evidence has implicated CD24, a cell-surface protein, as a marker of colorectal cancer stem cells and target for antitumor therapy, although its presence in normal colonic epithelium has not been fully characterized. Previously, our group showed that CD24-based cell sorting can be used to isolate a fraction of murine small intestinal epithelial cells enriched in actively cycling stem cells. Similarly, we hypothesized that CD24-based isolation of colonic epithelial cells would generate a fraction enriched in actively cycling colonic epithelial stem cells (CESCs). Immunohistochemistry performed on mouse colonic tissue showed CD24 expression in the bottom half of proximal colon crypts and the crypt base in the distal colon. This pattern of distribution was similar to enhanced green fluorescent protein (EGFP) expression in Lgr5-EGFP mice. Areas expressing CD24 contained actively proliferating cells as determined by ethynyl deoxyuridine (EdU) incorporation, with a distinct difference between the proximal colon, where EdU-labeled cells were most frequent in the midcrypt, and the distal colon, where they were primarily at the crypt base. Flow cytometric analyses of single epithelial cells, identified by epithelial cell adhesion molecule (EpCAM) positivity, from mouse colon revealed an actively cycling CD24+ fraction that contained the majority of Lgr5-EGFP+ putative CESCs. Transcript analysis by quantitative RT-PCR confirmed enrichment of active CESC markers [leucine-rich-repeat-containing G protein-coupled receptor 5 (Lgr5), ephrin type B receptor 2 (EphB2), and CD166] in the CD24+EpCAM+ fraction but also showed enrichment of quiescent CESC markers [leucine-rich repeats and immunoglobin domains (Lrig), doublecortin and calmodulin kinase-like 1 (DCAMKL-1), and murine telomerase reverse transcriptase (mTert)]. We conclude that CD24-based sorting in wild-type mice isolates a colonic epithelial fraction highly enriched in actively cycling and quiescent

Bioinformatic Identification and Expression Analysis of Banana MicroRNAs and Their Targets

PubMed Central

Shi, Hourui; Ren, Mengyun; Zhang, Yindong; Wang, Jingyi

2015-01-01

MicroRNAs (miRNAs) represent a class of endogenous non-coding small RNAs that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Thousands of miRNAs have been identified in many plant species, whereas only a limited number of miRNAs have been predicted in M. acuminata (A genome) and M. balbisiana (B genome). Here, previously known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS), a database of banana genes. A total of 32 potential miRNAs belonging to 13 miRNAs families were detected using a range of filtering criteria. 244 miRNA:target pairs were subsequently predicted, most of which encode transcription factors or enzymes that participate in the regulation of development, growth, metabolism, and other physiological processes. In order to validate the predicted miRNAs and the mutual relationship between miRNAs and their target genes, qRT-PCR was applied to detect the tissue-specific expression levels of 12 putative miRNAs and 6 target genes in roots, leaves, flowers, and fruits. This study provides some important information about banana pre-miRNAs, mature miRNAs, and miRNA target genes and these findings can be applied to future research of miRNA functions. PMID:25856313

Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies

PubMed Central

Santiago-Rodriguez, Tasha M.; Luciani, Stefania; Toranzos, Gary A.; Marota, Isolina; Giuffra, Valentina; Cano, Raul J.

2017-01-01

Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA) gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes) may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era. PMID:29112136

Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies.

PubMed

Santiago-Rodriguez, Tasha M; Fornaciari, Gino; Luciani, Stefania; Toranzos, Gary A; Marota, Isolina; Giuffra, Valentina; Cano, Raul J

2017-11-07

Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA) gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes) may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era.

Arabidopsis thaliana DNA gyrase is targeted to chloroplasts and mitochondria.

PubMed

Wall, Melisa K; Mitchenall, Lesley A; Maxwell, Anthony

2004-05-18

DNA gyrase is the bacterial DNA topoisomerase (topo) that supercoils DNA by using the free energy of ATP hydrolysis. The enzyme, an A(2)B(2) tetramer encoded by the gyrA and gyrB genes, catalyses topological changes in DNA during replication and transcription, and is the only topo that is able to introduce negative supercoils. Gyrase is essential in bacteria and apparently absent from eukaryotes and is, consequently, an important target for antibacterial agents (e.g., quinolones and coumarins). We have identified four putative gyrase genes in the model plant Arabidopsis thaliana; one gyrA and three gyrB homologues. DNA gyrase protein A (GyrA) has a dual translational initiation site targeting the mature protein to both chloroplasts and mitochondria, and there are individual targeting sequences for two of the DNA gyrase protein B (GyrB) homologues. N-terminal fusions of the organellar targeting sequences to GFPs support the hypothesis that one enzyme is targeted to the chloroplast and another to the mitochondrion, which correlates with supercoiling activity in isolated organelles. Treatment of seedlings and cultured cells with gyrase-specific drugs leads to growth inhibition. Knockout of A. thaliana gyrA is embryo-lethal whereas knockouts in the gyrB genes lead to seedling-lethal phenotypes or severely stunted growth and development. The A. thaliana genes have been cloned in Escherichia coli and found to complement gyrase temperature-sensitive strains. This report confirms the existence of DNA gyrase in eukaryotes and has important implications for drug targeting, organelle replication, and the evolution of topos in plants.

Gene-centric meta-analysis in 87,736 individuals of European ancestry identifies multiple blood-pressure-related loci.

PubMed

Tragante, Vinicius; Barnes, Michael R; Ganesh, Santhi K; Lanktree, Matthew B; Guo, Wei; Franceschini, Nora; Smith, Erin N; Johnson, Toby; Holmes, Michael V; Padmanabhan, Sandosh; Karczewski, Konrad J; Almoguera, Berta; Barnard, John; Baumert, Jens; Chang, Yen-Pei Christy; Elbers, Clara C; Farrall, Martin; Fischer, Mary E; Gaunt, Tom R; Gho, Johannes M I H; Gieger, Christian; Goel, Anuj; Gong, Yan; Isaacs, Aaron; Kleber, Marcus E; Mateo Leach, Irene; McDonough, Caitrin W; Meijs, Matthijs F L; Melander, Olle; Nelson, Christopher P; Nolte, Ilja M; Pankratz, Nathan; Price, Tom S; Shaffer, Jonathan; Shah, Sonia; Tomaszewski, Maciej; van der Most, Peter J; Van Iperen, Erik P A; Vonk, Judith M; Witkowska, Kate; Wong, Caroline O L; Zhang, Li; Beitelshees, Amber L; Berenson, Gerald S; Bhatt, Deepak L; Brown, Morris; Burt, Amber; Cooper-DeHoff, Rhonda M; Connell, John M; Cruickshanks, Karen J; Curtis, Sean P; Davey-Smith, George; Delles, Christian; Gansevoort, Ron T; Guo, Xiuqing; Haiqing, Shen; Hastie, Claire E; Hofker, Marten H; Hovingh, G Kees; Kim, Daniel S; Kirkland, Susan A; Klein, Barbara E; Klein, Ronald; Li, Yun R; Maiwald, Steffi; Newton-Cheh, Christopher; O'Brien, Eoin T; Onland-Moret, N Charlotte; Palmas, Walter; Parsa, Afshin; Penninx, Brenda W; Pettinger, Mary; Vasan, Ramachandran S; Ranchalis, Jane E; M Ridker, Paul; Rose, Lynda M; Sever, Peter; Shimbo, Daichi; Steele, Laura; Stolk, Ronald P; Thorand, Barbara; Trip, Mieke D; van Duijn, Cornelia M; Verschuren, W Monique; Wijmenga, Cisca; Wyatt, Sharon; Young, J Hunter; Zwinderman, Aeilko H; Bezzina, Connie R; Boerwinkle, Eric; Casas, Juan P; Caulfield, Mark J; Chakravarti, Aravinda; Chasman, Daniel I; Davidson, Karina W; Doevendans, Pieter A; Dominiczak, Anna F; FitzGerald, Garret A; Gums, John G; Fornage, Myriam; Hakonarson, Hakon; Halder, Indrani; Hillege, Hans L; Illig, Thomas; Jarvik, Gail P; Johnson, Julie A; Kastelein, John J P; Koenig, Wolfgang; Kumari, Meena; März, Winfried; Murray, Sarah S; O'Connell, Jeffery R; Oldehinkel, Albertine J; Pankow, James S; Rader, Daniel J; Redline, Susan; Reilly, Muredach P; Schadt, Eric E; Kottke-Marchant, Kandice; Snieder, Harold; Snyder, Michael; Stanton, Alice V; Tobin, Martin D; Uitterlinden, André G; van der Harst, Pim; van der Schouw, Yvonne T; Samani, Nilesh J; Watkins, Hugh; Johnson, Andrew D; Reiner, Alex P; Zhu, Xiaofeng; de Bakker, Paul I W; Levy, Daniel; Asselbergs, Folkert W; Munroe, Patricia B; Keating, Brendan J

2014-03-06

Blood pressure (BP) is a heritable risk factor for cardiovascular disease. To investigate genetic associations with systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP), and pulse pressure (PP), we genotyped ~50,000 SNPs in up to 87,736 individuals of European ancestry and combined these in a meta-analysis. We replicated findings in an independent set of 68,368 individuals of European ancestry. Our analyses identified 11 previously undescribed associations in independent loci containing 31 genes including PDE1A, HLA-DQB1, CDK6, PRKAG2, VCL, H19, NUCB2, RELA, HOXC@ complex, FBN1, and NFAT5 at the Bonferroni-corrected array-wide significance threshold (p < 6 × 10(-7)) and confirmed 27 previously reported associations. Bioinformatic analysis of the 11 loci provided support for a putative role in hypertension of several genes, such as CDK6 and NUCB2. Analysis of potential pharmacological targets in databases of small molecules showed that ten of the genes are predicted to be a target for small molecules. In summary, we identified previously unknown loci associated with BP. Our findings extend our understanding of genes involved in BP regulation, which may provide new targets for therapeutic intervention or drug response stratification. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

FBW7 targets KLF10 for ubiquitin-dependent degradation.

PubMed

Yu, Su; Wang, Feng; Tan, Xiao; Gao, Guo-Li; Pan, Wei-Juan; Luan, Yi; Ge, Xin

2018-01-08

FBW7, a key component of SCF FBW7 E3 ubiquitin ligase, targets various proteins for degradation via the conserved Cdc4 phosphodegron (CPD) in substrates. In this study, we report that KLF10 is degraded by FBW7 via a conserved CPD. Through systematic analysis of the degradation of KLF transcription factors by FBW7, we identified KLF10 as a novel degradation target of FBW7. Ectopic expression of FBW7 markedly promoted the degradation of KLF10 while knockdown of endogenous FBW7 increased the protein levels of KLF10. In addition, simultaneous mutations of both threonine 82 (T82) and serine 86 (S86) significantly reduced the FBW7-mediated KLF10 degradation. Moreover, KLF10 containing a conserved putative CPD (TPPXSP) from amino acids 82 to 87, directly interacted with WD40 domain of FBW7 in a phosphorylation-dependent manner. Importantly, FBW7 could reverse the KLF10-mediated inhibition of Smad7 activity. Thus, our study uncovers a novel regulatory mechanism underlying which KLF10 stability and its biological function are mediated by FBW7. Copyright © 2017 Elsevier Inc. All rights reserved.

Systemic analysis of genome-wide expression profiles identified potential therapeutic targets of demethylation drugs for glioblastoma.

PubMed

Ning, Tongbo; Cui, Hao; Sun, Feng; Zou, Jidian

2017-09-05

Glioblastoma represents one of the most aggressive malignant brain tumors with high morbidity and motility. Demethylation drugs have been developed for its treatment with little efficacy has been observed. The purpose of this study was to screen therapeutic targets of demethylation drugs or bioactive molecules for glioblastoma through systemic bioinformatics analysis. We firstly downloaded genome-wide expression profiles from the Gene Expression Omnibus (GEO) and conducted the primary analysis through R software, mainly including preprocessing of raw microarray data, transformation between probe ID and gene symbol and identification of differential expression genes (DEGs). Secondly, functional enrichment analysis was conducted via the Database for Annotation, Visualization and Integrated Discovery (DAVID) to explore biological processes involved in the development of glioblastoma. Thirdly, we constructed protein-protein interaction (PPI) network of interested genes and conducted cross analysis for multi datasets to obtain potential therapeutic targets for glioblastoma. Finally, we further confirmed the therapeutic targets through real-time RT-PCR. As a result, biological processes that related to cancer development, amino metabolism, immune response and etc. were found to be significantly enriched in genes that differential expression in glioblastoma and regulated by 5'aza-dC. Besides, network and cross analysis identified ACAT2, UFC1 and CYB5R1 as novel therapeutic targets of demethylation drugs which also confirmed by real time RT-PCR. In conclusions, our study identified several biological processes and genes that involved in the development of glioblastoma and regulated by 5'aza-dC, which would be helpful for the treatment of glioblastoma. Copyright © 2017 Elsevier B.V. All rights reserved.

Phosphotyrosine profiling identifies ephrin receptor A2 as a potential therapeutic target in esophageal squamous‐cell carcinoma

PubMed Central

Syed, Nazia; Barbhuiya, Mustafa A.; Pinto, Sneha M.; Nirujogi, Raja Sekhar; Renuse, Santosh; Datta, Keshava K.; Khan, Aafaque Ahmad; Srikumar, Kotteazeth; Prasad, T. S. Keshava; Kumar, M. Vijaya; Kumar, Rekha Vijay; Chatterjee, Aditi; Pandey, Akhilesh

2015-01-01

Esophageal squamous‐cell carcinoma (ESCC) is one of the most common malignancies in Asia. Currently, surgical resection of early‐stage tumor is the best available treatment. However, most patients present late when surgery is not an option. Data suggest that chemotherapy regimens are inadequate for clinical management of advanced cancer. Targeted therapy has emerged as one of the most promising approaches to treat several malignancies. A prerequisite for developing targeted therapy is prior knowledge of proteins and pathways that drive proliferation in malignancies. We carried out phosphotyrosine profiling across four different ESCC cell lines and compared it to non‐neoplastic Het‐1A cell line to identify activated tyrosine kinase signaling pathways in ESCC. A total of 278 unique phosphopeptides were identified across these cell lines. This included several tyrosine kinases and their substrates that were hyperphosphorylated in ESCC. Ephrin receptor A2 (EPHA2), a receptor tyrosine kinase, was hyperphosphorylated in all the ESCC cell lines used in the study. EPHA2 is reported to be oncogenic in several cancers and is also known to promote metastasis. Immunohistochemistry‐based studies have revealed EPHA2 is overexpressed in nearly 50% of ESCC. We demonstrated EPHA2 as a potential therapeutic target in ESCC by carrying out siRNA‐based knockdown studies. Knockdown of EPHA2 in ESCC cell line TE8 resulted in significant decrease in cell proliferation and invasion, suggesting it is a promising therapeutic target in ESCC that warrants further evaluation. PMID:25366905

Classification accuracy of claims-based methods for identifying providers failing to meet performance targets.

PubMed

Hubbard, Rebecca A; Benjamin-Johnson, Rhondee; Onega, Tracy; Smith-Bindman, Rebecca; Zhu, Weiwei; Fenton, Joshua J

2015-01-15

Quality assessment is critical for healthcare reform, but data sources are lacking for measurement of many important healthcare outcomes. With over 49 million people covered by Medicare as of 2010, Medicare claims data offer a potentially valuable source that could be used in targeted health care quality improvement efforts. However, little is known about the operating characteristics of provider profiling methods using claims-based outcome measures that may estimate provider performance with error. Motivated by the example of screening mammography performance, we compared approaches to identifying providers failing to meet guideline targets using Medicare claims data. We used data from the Breast Cancer Surveillance Consortium and linked Medicare claims to compare claims-based and clinical estimates of cancer detection rate. We then demonstrated the performance of claim-based estimates across a broad range of operating characteristics using simulation studies. We found that identification of poor performing providers was extremely sensitive to algorithm specificity, with no approach identifying more than 65% of poor performing providers when claims-based measures had specificity of 0.995 or less. We conclude that claims have the potential to contribute important information on healthcare outcomes to quality improvement efforts. However, to achieve this potential, development of highly accurate claims-based outcome measures should remain a priority. Copyright © 2014 John Wiley & Sons, Ltd.

Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets.

PubMed

Brooks, Lauren E; Ul-Hasan, Sabah; Chan, Benjamin K; Sistrom, Mark J

2018-01-01

Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection-representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments-specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been developed to date

Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets

PubMed Central

Ul-Hasan, Sabah; Chan, Benjamin K.; Sistrom, Mark J.

2018-01-01

ABSTRACT Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection—representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments—specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been

Berberine acts as a putative epigenetic modulator by affecting the histone code.

PubMed

Wang, Zhixiang; Liu, Yuan; Xue, Yong; Hu, Haiyan; Ye, Jieyu; Li, Xiaodong; Lu, Zhigang; Meng, Fanyi; Liang, Shuang

2016-10-01

Berberine, an isoquinoline plant alkaloid, exhibits a wide range of biochemical and pharmacological effects. However, the precise mechanism of these bioactivities remains poorly understood. In this study, we found significant similarity between berberine and two epigenetic modulators (CG-1521 and TSA). Reverse-docking using berberine as a ligand identified lysine-N-methyltransferase as a putative target of berberine. These findings suggested the potential role of berberine in epigenetic modulation. The results of PCR array analysis of epigenetic chromatin modification enzymes supported our hypothesis. Furthermore, the analysis showed that enzymes involved in histone acetylation and methylation were predominantly affected by treatment with berberine. Up-regulation of histone acetyltransferase CREBBP and EP300, histone deacetylase SIRT3, histone demethylase KDM6A as well as histone methyltransferase SETD7, and down-regulation of histone acetyltransferase HDAC8, histone methyltransferase WHSC1I, WHSC1II and SMYD3, in addition to 38 genes from histone clusters 1-3 were observed in berberine-treated cells using real-time PCR. In parallel, western blotting analyses revealed that the expression of H3K4me3, H3K27me3 and H3K36me3 proteins decreased with berberine treatment. These results were further confirmed in acute myelocytic leukemia (AML) cell lines HL-60/ADR and KG1-α. Taken together, this study suggests that berberine might modulate the expression of epigenetic regulators important for many downstream pathways, resulting in the variation of its bioactivities. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Toddlers' Duration of Attention toward Putative Threat

ERIC Educational Resources Information Center

Kiel, Elizabeth J.; Buss, Kristin A.

2011-01-01

Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk of developing anxious behavior, toddlers' attention toward a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined…

Ontology-oriented retrieval of putative microRNAs in Vitis vinifera via GrapeMiRNA: a web database of de novo predicted grape microRNAs.

PubMed

Lazzari, Barbara; Caprera, Andrea; Cestaro, Alessandro; Merelli, Ivan; Del Corvo, Marcello; Fontana, Paolo; Milanesi, Luciano; Velasco, Riccardo; Stella, Alessandra

2009-06-29

Two complete genome sequences are available for Vitis vinifera Pinot noir. Based on the sequence and gene predictions produced by the IASMA, we performed an in silico detection of putative microRNA genes and of their targets, and collected the most reliable microRNA predictions in a web database. The application is available at http://www.itb.cnr.it/ptp/grapemirna/. The program FindMiRNA was used to detect putative microRNA genes in the grape genome. A very high number of predictions was retrieved, calling for validation. Nine parameters were calculated and, based on the grape microRNAs dataset available at miRBase, thresholds were defined and applied to FindMiRNA predictions having targets in gene exons. In the resulting subset, predictions were ranked according to precursor positions and sequence similarity, and to target identity. To further validate FindMiRNA predictions, comparisons to the Arabidopsis genome, to the grape Genoscope genome, and to the grape EST collection were performed. Results were stored in a MySQL database and a web interface was prepared to query the database and retrieve predictions of interest. The GrapeMiRNA database encompasses 5,778 microRNA predictions spanning the whole grape genome. Predictions are integrated with information that can be of use in selection procedures. Tools added in the web interface also allow to inspect predictions according to gene ontology classes and metabolic pathways of targets. The GrapeMiRNA database can be of help in selecting candidate microRNA genes to be validated.

Stimulus selectivity and response latency in putative inhibitory and excitatory neurons of the primate inferior temporal cortex

PubMed Central

Mruczek, Ryan E. B.

2012-01-01

The cerebral cortex is composed of many distinct classes of neurons. Numerous studies have demonstrated corresponding differences in neuronal properties across cell types, but these comparisons have largely been limited to conditions outside of awake, behaving animals. Thus the functional role of the various cell types is not well understood. Here, we investigate differences in the functional properties of two widespread and broad classes of cells in inferior temporal cortex of macaque monkeys: inhibitory interneurons and excitatory projection cells. Cells were classified as putative inhibitory or putative excitatory neurons on the basis of their extracellular waveform characteristics (e.g., spike duration). Consistent with previous intracellular recordings in cortical slices, putative inhibitory neurons had higher spontaneous firing rates and higher stimulus-evoked firing rates than putative excitatory neurons. Additionally, putative excitatory neurons were more susceptible to spike waveform adaptation following very short interspike intervals. Finally, we compared two functional properties of each neuron's stimulus-evoked response: stimulus selectivity and response latency. First, putative excitatory neurons showed stronger stimulus selectivity compared with putative inhibitory neurons. Second, putative inhibitory neurons had shorter response latencies compared with putative excitatory neurons. Selectivity differences were maintained and latency differences were enhanced during a visual search task emulating more natural viewing conditions. Our results suggest that short-latency inhibitory responses are likely to sculpt visual processing in excitatory neurons, yielding a sparser visual representation. PMID:22933717

Researchers Use a Kinome Screen to Identify New Therapeutic Targets | Office of Cancer Genomics

Cancer.gov

The tumor suppressor p53 is mutated in over 50% of head and neck squamous cell carcinomas (HNSCC), yet there are currently no available therapies to target it. CTD2 researchers at the Fred Hutchison Cancer Research Center hypothesized that HNSCC cancer cells with p53 mutations are dependent on particular kinases for survival. In a study published in Clinical Cancer Research, they sought to identify these kinases using RNAi against known kinase genes in mouse and human cell lines.

DLEU2 encodes an antisense RNA for the putative bicistronic RFP2/LEU5 gene in humans and mouse.

PubMed

Corcoran, Martin M; Hammarsund, Marianne; Zhu, Chaoyong; Lerner, Mikael; Kapanadze, Bagrat; Wilson, Bill; Larsson, Catharina; Forsberg, Lars; Ibbotson, Rachel E; Einhorn, Stefan; Oscier, David G; Grandér, Dan; Sangfelt, Olle

2004-08-01

Our group previously identified two novel genes, RFP2/LEU5 and DLEU2, within a 13q14.3 genomic region of loss seen in various malignancies. However, no specific inactivating mutations were found in these or other genes in the vicinity of the deletion, suggesting that a nonclassical tumor-suppressor mechanism may be involved. Here, we present data showing that the DLEU2 gene encodes a putative noncoding antisense RNA, with one exon directly overlapping the first exon of the RFP2/LEU5 gene in the opposite orientation. In addition, the RFP2/LEU5 transcript can be alternatively spliced to produce either several monocistronic transcripts or a putative bicistronic transcript encoding two separate open-reading frames, adding to the complexity of the locus. The finding that these gene structures are conserved in the mouse, including the putative bicistronic RFP2/LEU5 transcript as well as the antisense relationship with DLEU2, further underlines the significance of this unusual organization and suggests a biological function for DLEU2 in the regulation of RFP2/LEU5. Copyright 2004 Wiley-Liss, Inc.

Pharmacological Validation of Candidate Causal Sleep Genes Identified in an N2 Cross

PubMed Central

Brunner, Joseph I.; Gotter, Anthony L.; Millstein, Joshua; Garson, Susan; Binns, Jacquelyn; Fox, Steven V.; Savitz, Alan T.; Yang, He S.; Fitzpatrick, Karrie; Zhou, Lili; Owens, Joseph R.; Webber, Andrea L.; Vitaterna, Martha H.; Kasarskis, Andrew; Uebele, Victor N.; Turek, Fred; Renger, John J.; Winrow, Christopher J.

2013-01-01

Despite the substantial impact of sleep disturbances on human health and the many years of study dedicated to understanding sleep pathologies, the underlying genetic mechanisms that govern sleep and wake largely remain unknown. Recently, we completed large scale genetic and gene expression analyses in a segregating inbred mouse cross and identified candidate causal genes that regulate the mammalian sleep-wake cycle, across multiple traits including total sleep time, amounts of REM, non-REM, sleep bout duration and sleep fragmentation. Here we describe a novel approach toward validating candidate causal genes, while also identifying potential targets for sleep-related indications. Select small molecule antagonists and agonists were used to interrogate candidate causal gene function in rodent sleep polysomnography assays to determine impact on overall sleep architecture and to evaluate alignment with associated sleep-wake traits. Significant effects on sleep architecture were observed in validation studies using compounds targeting the muscarinic acetylcholine receptor M3 subunit (Chrm3)(wake promotion), nicotinic acetylcholine receptor alpha4 subunit (Chrna4)(wake promotion), dopamine receptor D5 subunit (Drd5)(sleep induction), serotonin 1D receptor (Htr1d)(altered REM fragmentation), glucagon-like peptide-1 receptor (Glp1r)(light sleep promotion and reduction of deep sleep), and Calcium channel, voltage-dependent, T type, alpha 1I subunit (Cacna1i)(increased bout duration slow wave sleep). Taken together, these results show the complexity of genetic components that regulate sleep-wake traits and highlight the importance of evaluating this complex behavior at a systems level. Pharmacological validation of genetically identified putative targets provides a rapid alternative to generating knock out or transgenic animal models, and may ultimately lead towards new therapeutic opportunities. PMID:22091728

Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: population genetics, human serologic response, and gene transcription.

PubMed

Reid, S D; Green, N M; Buss, J K; Lei, B; Musser, J M

2001-06-19

Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase-PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research.

VISLISI trial, a prospective clinical study allowing identification of a new metalloprotease and putative virulence factor from Staphylococcus lugdunensis.

PubMed

Argemi, X; Prévost, G; Riegel, P; Keller, D; Meyer, N; Baldeyrou, M; Douiri, N; Lefebvre, N; Meghit, K; Ronde Oustau, C; Christmann, D; Cianférani, S; Strub, J M; Hansmann, Y

2017-05-01

Staphylococcus lugdunensis is a coagulase-negative staphylococcus that displays an unusually high virulence rate close to that of Staphylococcus aureus. It also shares phenotypic properties with S. aureus and several studies found putative virulence factors. The objective of the study was to describe the clinical manifestations of S. lugdunensis infections and investigate putative virulence factors. We conducted a prospective study from November 2013 to March 2016 at the University Hospital of Strasbourg. Putative virulence factors were investigated by clumping factor detection, screening for proteolytic activity, and sequence analysis using tandem nano-liquid chromatography-mass spectrometry. In total, 347 positive samples for S. lugdunensis were collected, of which 129 (37.2%) were from confirmed cases of S. lugdunensis infection. Eighty-one of these 129 patients were included in the study. Bone and prosthetic joints (PJI) were the most frequent sites of infection (n=28; 34.6%) followed by skin and soft tissues (n=23; 28.4%). We identified and purified a novel protease secreted by 50 samples (61.7%), most frequently associated with samples from deep infections and PJI (pr 0.97 and pr 0.91, respectively). Protease peptide sequencing by nano-liquid chromatography-mass spectrometry revealed a novel protease bearing 62.42% identity with ShpI, a metalloprotease secreted by Staphylococcus hyicus. This study confirms the pathogenicity of S. lugdunensis, particularly in bone and PJI. We also identified a novel metalloprotease called lugdulysin that may contribute to virulence. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Identification and Analysis of Putative Homologues of Mechanosensitive Channels in Pathogenic Protozoa

PubMed Central

Prole, David L.; Taylor, Colin W.

2013-01-01

Mechanosensitive channels play important roles in the physiology of many organisms, and their dysfunction can affect cell survival. This suggests that they might be therapeutic targets in pathogenic organisms. Pathogenic protozoa lead to diseases such as malaria, dysentery, leishmaniasis and trypanosomiasis that are responsible for millions of deaths each year worldwide. We analyzed the genomes of pathogenic protozoa and show the existence within them of genes encoding putative homologues of mechanosensitive channels. Entamoeba histolytica, Leishmania spp., Trypanosoma cruzi and Trichomonas vaginalis have genes encoding homologues of Piezo channels, while most pathogenic protozoa have genes encoding homologues of mechanosensitive small-conductance (MscS) and K+-dependent (MscK) channels. In contrast, all parasites examined lack genes encoding mechanosensitive large-conductance (MscL), mini-conductance (MscM) and degenerin/epithelial Na+ (DEG/ENaC) channels. Multiple sequence alignments of evolutionarily distant protozoan, amoeban, plant, insect and vertebrate Piezo channel subunits define an absolutely conserved motif that may be involved in channel conductance or gating. MscS channels are not present in humans, and the sequences of protozoan and human homologues of Piezo channels differ substantially. This suggests the possibility for specific targeting of mechanosensitive channels of pathogens by therapeutic drugs. PMID:23785469

Systematic approach identifies RHOA as a potential biomarker therapeutic target for Asian gastric cancer.

PubMed

Chang, Hae Ryung; Nam, Seungyoon; Lee, Jinhyuk; Kim, Jin-Hee; Jung, Hae Rim; Park, Hee Seo; Park, Sungjin; Ahn, Young Zoo; Huh, Iksoo; Balch, Curt; Ku, Ja-Lok; Powis, Garth; Park, Taesung; Jeong, Jin-Hyun; Kim, Yon Hui

2016-12-06

Gastric cancer (GC) is a highly heterogeneous disease, in dire need of specific, biomarker-driven cancer therapies. While the accumulation of cancer "Big Data" has propelled the search for novel molecular targets for GC, its specific subpathway and cellular functions vary from patient to patient. In particular, mutations in the small GTPase gene RHOA have been identified in recent genome-wide sequencing of GC tumors. Moreover, protein overexpression of RHOA was reported in Chinese populations, while RHOA mutations were found in Caucasian GC tumors. To develop evidence-based precision medicine for heterogeneous cancers, we established a systematic approach to integrate transcriptomic and genomic data. Predicted signaling subpathways were then laboratory-validated both in vitro and in vivo, resulting in the identification of new candidate therapeutic targets. Here, we show: i) differences in RHOA expression patterns, and its pathway activity, between Asian and Caucasian GC tumors; ii) in vitro and in vivo perturbed RHOA expression inhibits GC cell growth in high RHOA-expressing cell lines; iii) inverse correlation between RHOA and RHOB expression; and iv) an innovative small molecule design strategy for RHOA inhibitors. In summary, RHOA, and its oncogenic signaling pathway, represent a strong biomarker-driven therapeutic target for Asian GC. This comprehensive strategy represents a promising approach for the development of "hit" compounds.

Deciphering the pharmacological mechanism of the Chinese formula Huanglian-Jie-Du decoction in the treatment of ischemic stroke using a systems biology-based strategy

PubMed Central

Zhang, Yan-qiong; Wang, Song-song; Zhu, Wei-liang; Ma, Yan; Zhang, Fang-bo; Liang, Ri-xin; Xu, Hai-yu; Yang, Hong-jun

2015-01-01

Aim: Huanglian-Jie-Du decoction (HLJDD) is an important multiherb remedy in TCM, which is recently demonstrated to be effective to treat ischemic stroke. Here, we aimed to investigate the pharmacological mechanisms of HLJDD in the treatment of ischemic stroke using systems biology approaches. Methods: Putative targets of HLJDD were predicted using MetaDrug. An interaction network of putative HLJDD targets and known therapeutic targets for the treatment of ischemic stroke was then constructed, and candidate HLJDD targets were identified by calculating topological features, including 'Degree', 'Node-betweenness', 'Closeness', and 'K-coreness'. The binding efficiencies of the candidate HLJDD targets with the corresponding compositive compounds were further validated by a molecular docking simulation. Results: A total of 809 putative targets were obtained for 168 compositive compounds in HLJDD. Additionally, 39 putative targets were common to all four herbs of HLJDD. Next, 49 major nodes were identified as candidate HLJDD targets due to their network topological importance. The enrichment analysis based on the Gene Ontology (GO) annotation system and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway demonstrated that candidate HLJDD targets were more frequently involved in G-protein-coupled receptor signaling pathways, neuroactive ligand-receptor interactions and gap junctions, which all played important roles in the progression of ischemic stroke. Finally, the molecular docking simulation showed that 170 pairs of chemical components and candidate HLJDD targets had strong binding efficiencies. Conclusion: This study has developed for the first time a comprehensive systems approach integrating drug target prediction, network analysis and molecular docking simulation to reveal the relationships between the herbs contained in HLJDD and their putative targets and ischemic stroke-related pathways. PMID:25937634

Emissions of putative isoprene oxidation products from mango branches under abiotic stress

DOE PAGES

Jardine, Kolby J.; Meyers, Kimberly; Abrell, Leif; ...

2013-07-23

Although several per cent of net carbon assimilation can be re-released as isoprene emissions to the atmosphere by many tropical plants, much uncertainty remains regarding its biological significance. In a previous study, we detected emissions of isoprene and its oxidation products methyl vinyl ketone (MVK) and methacrolein (MACR) from tropical plants under high temperature/light stress, suggesting that isoprene is oxidized not only in the atmosphere but also within plants. However, a comprehensive analysis of the suite of isoprene oxidation products in plants has not been performed and production relationships with environmental stress have not been described. In this study, putativemore » isoprene oxidation products from mango (Mangifera indica) branches under abiotic stress were first identified. High temperature/light and freeze–thaw treatments verified direct emissions of the isoprene oxidation products MVK and MACR together with the first observations of 3-methyl furan (3-MF) and 2-methyl-3-buten-2-ol (MBO) as putative novel isoprene oxidation products. Mechanical wounding also stimulated emissions of MVK and MACR. Photosynthesis under 13CO 2 resulted in rapid (<30min) labelling of up to five carbon atoms of isoprene, with a similar labelling pattern observed in the putative oxidation products. These observations highlight the need to investigate further the mechanisms of isoprene oxidation within plants under stress and its biological and atmospheric significance.« less

Emissions of putative isoprene oxidation products from mango branches under abiotic stress

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

Jardine, Kolby J.; Meyers, Kimberly; Abrell, Leif

Although several per cent of net carbon assimilation can be re-released as isoprene emissions to the atmosphere by many tropical plants, much uncertainty remains regarding its biological significance. In a previous study, we detected emissions of isoprene and its oxidation products methyl vinyl ketone (MVK) and methacrolein (MACR) from tropical plants under high temperature/light stress, suggesting that isoprene is oxidized not only in the atmosphere but also within plants. However, a comprehensive analysis of the suite of isoprene oxidation products in plants has not been performed and production relationships with environmental stress have not been described. In this study, putativemore » isoprene oxidation products from mango (Mangifera indica) branches under abiotic stress were first identified. High temperature/light and freeze–thaw treatments verified direct emissions of the isoprene oxidation products MVK and MACR together with the first observations of 3-methyl furan (3-MF) and 2-methyl-3-buten-2-ol (MBO) as putative novel isoprene oxidation products. Mechanical wounding also stimulated emissions of MVK and MACR. Photosynthesis under 13CO 2 resulted in rapid (<30min) labelling of up to five carbon atoms of isoprene, with a similar labelling pattern observed in the putative oxidation products. These observations highlight the need to investigate further the mechanisms of isoprene oxidation within plants under stress and its biological and atmospheric significance.« less

Ten Putative Contributors to the Obesity Epidemic

PubMed Central

McAllister, Emily J.; Dhurandhar, Nikhil V.; Keith, Scott W.; Aronne, Louis J.; Barger, Jamie; Baskin, Monica; Benca, Ruth M.; Biggio, Joseph; Boggiano, Mary M.; Eisenmann, Joe C.; Elobeid, Mai; Fontaine, Kevin R.; Gluckman, Peter; Hanlon, Erin C.; Katzmarzyk, Peter; Pietrobelli, Angelo; Redden, David T.; Ruden, Douglas M.; Wang, Chenxi; Waterland, Robert A.; Wright, Suzanne M.; Allison, David B.

2010-01-01

The obesity epidemic is a global issue and shows no signs of abating, while the cause of this epidemic remains unclear. Marketing practices of energy-dense foods and institutionally-driven declines in physical activity are the alleged perpetrators for the epidemic, despite a lack of solid evidence to demonstrate their causal role. While both may contribute to obesity, we call attention to their unquestioned dominance in program funding and public efforts to reduce obesity, and propose several alternative putative contributors that would benefit from equal consideration and attention. Evidence for microorganisms, epigenetics, increasing maternal age, greater fecundity among people with higher adiposity, assortative mating, sleep debt, endocrine disruptors, pharmaceutical iatrogenesis, reduction in variability of ambient temperatures, and intrauterine and intergenerational effects, as contributing factors to the obesity epidemic are reviewed herein. While the evidence is strong for some contributors such as pharmaceutical-induced weight gain, it is still emerging for other reviewed factors. Considering the role of such putative etiological factors of obesity may lead to comprehensive, cause specific, and effective strategies for prevention and treatment of this global epidemic. PMID:19960394

Using Market Research to Characterize College Students and Identify Potential Targets for Influencing Health Behaviors

PubMed Central

Berg, Carla J.; Ling, Pamela M.; Guo, Hongfei; Windle, Michael; Thomas, Janet L.; Ahluwalia, Jasjit S.; An, Lawrence C.

2013-01-01

Marketing campaigns, such as those developed by the tobacco industry, are based on market research, which defines segments of a population by assessing psychographic characteristics (i.e., attitudes, interests). This study uses a similar approach to define market segments of college smokers, to examine differences in their health behaviors (smoking, drinking, binge drinking, exercise, diet), and to determine the validity of these segments. A total of 2,265 undergraduate students aged 18–25 years completed a 108-item online survey in fall 2008 assessing demographic, psychographic (i.e., attitudes, interests), and health-related variables. Among the 753 students reporting past 30-day smoking, cluster analysis was conducted using 21 psychographic questions and identified three market segments – Stoic Individualists, Responsible Traditionalists, and Thrill-Seeking Socializers. We found that segment membership was related to frequency of alcohol use, binge drinking, and limiting dietary fat. We then developed three messages targeting each segment and conducted message testing to validate the segments on a subset of 73 smokers representing each segment in spring 2009. As hypothesized, each segment indicated greater relevance and salience for their respective message. These findings indicate that identifying qualitatively different subgroups of young adults through market research may inform the development of engaging interventions and health campaigns targeting college students. PMID:25264429

Using Market Research to Characterize College Students and Identify Potential Targets for Influencing Health Behaviors.

PubMed

Berg, Carla J; Ling, Pamela M; Guo, Hongfei; Windle, Michael; Thomas, Janet L; Ahluwalia, Jasjit S; An, Lawrence C

2010-12-01

Marketing campaigns, such as those developed by the tobacco industry, are based on market research, which defines segments of a population by assessing psychographic characteristics (i.e., attitudes, interests). This study uses a similar approach to define market segments of college smokers, to examine differences in their health behaviors (smoking, drinking, binge drinking, exercise, diet), and to determine the validity of these segments. A total of 2,265 undergraduate students aged 18-25 years completed a 108-item online survey in fall 2008 assessing demographic, psychographic (i.e., attitudes, interests), and health-related variables. Among the 753 students reporting past 30-day smoking, cluster analysis was conducted using 21 psychographic questions and identified three market segments - Stoic Individualists, Responsible Traditionalists, and Thrill-Seeking Socializers. We found that segment membership was related to frequency of alcohol use, binge drinking, and limiting dietary fat. We then developed three messages targeting each segment and conducted message testing to validate the segments on a subset of 73 smokers representing each segment in spring 2009. As hypothesized, each segment indicated greater relevance and salience for their respective message. These findings indicate that identifying qualitatively different subgroups of young adults through market research may inform the development of engaging interventions and health campaigns targeting college students.

Transcriptome analysis of Spodoptera frugiperda Sf9 cells reveals putative apoptosis-related genes and a preliminary apoptosis mechanism induced by azadirachtin.

PubMed

Shu, Benshui; Zhang, Jingjing; Sethuraman, Veeran; Cui, Gaofeng; Yi, Xin; Zhong, Guohua

2017-10-16

As an important botanical pesticide, azadirachtin demonstrates broad insecticidal activity against many agricultural pests. The results of a previous study indicated the toxicity and apoptosis induction of azadirachtin in Spodoptera frugiperda Sf9 cells. However, the lack of genomic data has hindered a deeper investigation of apoptosis in Sf9 cells at a molecular level. In the present study, the complete transcriptome data for Sf9 cell line was accomplished using Illumina sequencing technology, and 97 putative apoptosis-related genes were identified through BLAST and KEGG orthologue annotations. Fragments of potential candidate apoptosis-related genes were cloned, and the mRNA expression patterns of ten identified genes regulated by azadirachtin were examined using qRT-PCR. Furthermore, Western blot analysis showed that six putative apoptosis-related proteins were upregulated after being treated with azadirachtin while the protein Bcl-2 were downregulated. These data suggested that both intrinsic and extrinsic apoptotic signal pathways comprising the identified potential apoptosis-related genes were potentially active in S. frugiperda. In addition, the preliminary results revealed that caspase-dependent or caspase-independent apoptotic pathways could function in azadirachtin-induced apoptosis in Sf9 cells.

Effects of drugs of abuse on putative rostromedial tegmental neurons, inhibitory afferents to midbrain dopamine cells.

PubMed

Lecca, Salvatore; Melis, Miriam; Luchicchi, Antonio; Ennas, Maria Grazia; Castelli, Maria Paola; Muntoni, Anna Lisa; Pistis, Marco

2011-02-01

Recent findings have underlined the rostromedial tegmental nucleus (RMTg), a structure located caudally to the ventral tegmental area, as an important site involved in the mechanisms of aversion. RMTg contains γ-aminobutyric acid neurons responding to noxious stimuli, densely innervated by the lateral habenula and providing a major inhibitory projection to reward-encoding midbrain dopamine (DA) neurons. One of the key features of drug addiction is the perseverance of drug seeking in spite of negative and unpleasant consequences, likely mediated by response suppression within neural pathways mediating aversion. To investigate whether the RMTg has a function in the mechanisms of addicting drugs, we studied acute effects of morphine, cocaine, the cannabinoid agonist WIN55212-2 (WIN), and nicotine on putative RMTg neurons. We utilized single unit extracellular recordings in anesthetized rats and whole-cell patch-clamp recordings in brain slices to identify and characterize putative RMTg neurons and their responses to drugs of abuse. Morphine and WIN inhibited both firing rate in vivo and excitatory postsynaptic currents (EPSCs) evoked by stimulation of rostral afferents in vitro, whereas cocaine inhibited discharge activity without affecting EPSC amplitude. Conversely, nicotine robustly excited putative RMTg neurons and enhanced EPSCs, an effect mediated by α7-containing nicotinic acetylcholine receptors. Our results suggest that activity of RMTg neurons is profoundly influenced by drugs of abuse and, as important inhibitory afferents to midbrain DA neurons, they might take place in the complex interplay between the neural circuits mediating aversion and reward.

Identification of estrogen-responsive genes using a genome-wide analysis of promoter elements for transcription factor binding sites.

PubMed

Kamalakaran, Sitharthan; Radhakrishnan, Senthil K; Beck, William T

2005-06-03

We developed a pipeline to identify novel genes regulated by the steroid hormone-dependent transcription factor, estrogen receptor, through a systematic analysis of upstream regions of all human and mouse genes. We built a data base of putative promoter regions for 23,077 human and 19,984 mouse transcripts from National Center for Biotechnology Information annotation and 8793 human and 6785 mouse promoters from the Data Base of Transcriptional Start Sites. We used this data base of putative promoters to identify potential targets of estrogen receptor by identifying estrogen response elements (EREs) in their promoters. Our program correctly identified EREs in genes known to be regulated by estrogen in addition to several new genes whose putative promoters contained EREs. We validated six genes (KIAA1243, NRIP1, MADH9, NME3, TPD52L, and ABCG2) to be estrogen-responsive in MCF7 cells using reverse transcription PCR. To allow for extensibility of our program in identifying targets of other transcription factors, we have built a Web interface to access our data base and programs. Our Web-based program for Promoter Analysis of Genome, PAGen@UIC, allows a user to identify putative target genes for vertebrate transcription factors through the analysis of their upstream sequences. The interface allows the user to search the human and mouse promoter data bases for potential target genes containing one or more listed transcription factor binding sites (TFBSs) in their upstream elements, using either regular expression-based consensus or position weight matrices. The data base can also be searched for promoters harboring user-defined TFBSs given as a consensus or a position weight matrix. Furthermore, the user can retrieve putative promoter sequences for any given gene together with identified TFBSs located on its promoter. Orthologous promoters are also analyzed to determine conserved elements.

Identification of Putative Precursor Genes for the Biosynthesis of Cannabinoid-Like Compound in Radula marginata

PubMed Central

Hussain, Tajammul; Plunkett, Blue; Ejaz, Mahwish; Espley, Richard V.; Kayser, Oliver

2018-01-01

The liverwort Radula marginata belongs to the bryophyte division of land plants and is a prospective alternate source of cannabinoid-like compounds. However, mechanistic insights into the molecular pathways directing the synthesis of these cannabinoid-like compounds have been hindered due to the lack of genetic information. This prompted us to do deep sequencing, de novo assembly and annotation of R. marginata transcriptome, which resulted in the identification and validation of the genes for cannabinoid biosynthetic pathway. In total, we have identified 11,421 putative genes encoding 1,554 enzymes from 145 biosynthetic pathways. Interestingly, we have identified all the upstream genes of the central precursor of cannabinoid biosynthesis, cannabigerolic acid (CBGA), including its two first intermediates, stilbene acid (SA) and geranyl diphosphate (GPP). Expression of all these genes was validated using quantitative real-time PCR. We have characterized the protein structure of stilbene synthase (STS), which is considered as a homolog of olivetolic acid in R. marginata. Moreover, the metabolomics approach enabled us to identify CBGA-analogous compounds using electrospray ionization mass spectrometry (ESI-MS/MS) and gas chromatography mass spectrometry (GC-MS). Transcriptomic analysis revealed 1085 transcription factors (TF) from 39 families. Comparative analysis showed that six TF families have been uniquely predicted in R. marginata. In addition, the bioinformatics analysis predicted a large number of simple sequence repeats (SSRs) and non-coding RNAs (ncRNAs). Our results collectively provide mechanistic insights into the putative precursor genes for the biosynthesis of cannabinoid-like compounds and a novel transcriptomic resource for R. marginata. The large-scale transcriptomic resource generated in this study would further serve as a reference transcriptome to explore the Radulaceae family.

2D and 3D similarity landscape analysis identifies PARP as a novel off-target for the drug Vatalanib.

PubMed

Gohlke, Bjoern-Oliver; Overkamp, Tim; Richter, Anja; Richter, Antje; Daniel, Peter T; Gillissen, Bernd; Preissner, Robert

2015-09-24

Searching for two-dimensional (2D) structural similarities is a useful tool to identify new active compounds in drug-discovery programs. However, as 2D similarity measures neglect important structural and functional features, similarity by 2D might be underestimated. In the present study, we used combined 2D and three-dimensional (3D) similarity comparisons to reveal possible new functions and/or side-effects of known bioactive compounds. We utilised more than 10,000 compounds from the SuperTarget database with known inhibition values for twelve different anti-cancer targets. We performed all-against-all comparisons resulting in 2D similarity landscapes. Among the regions with low 2D similarity scores are inhibitors of vascular endothelial growth factor receptor (VEGFR) and inhibitors of poly ADP-ribose polymerase (PARP). To demonstrate that 3D landscape comparison can identify similarities, which are untraceable in 2D similarity comparisons, we analysed this region in more detail. This 3D analysis showed the unexpected structural similarity between inhibitors of VEGFR and inhibitors of PARP. Among the VEGFR inhibitors that show similarities to PARP inhibitors was Vatalanib, an oral "multi-targeted" small molecule protein kinase inhibitor being studied in phase-III clinical trials in cancer therapy. An in silico docking simulation and an in vitro HT universal colorimetric PARP assay confirmed that the VEGFR inhibitor Vatalanib exhibits off-target activity as a PARP inhibitor, broadening its mode of action. In contrast to the 2D-similarity search, the 3D-similarity landscape comparison identifies new functions and side effects of the known VEGFR inhibitor Vatalanib.

Putative melatonin receptors in a human biological clock

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

Reppert, S.M.; Weaver, D.R.; Rivkees, S.A.

In vitro autoradiography with /sup 125/I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific /sup 125/I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific /sup 125/I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completelymore » inhibited specific /sup 125/I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.« less

An integrated structure- and system-based framework to identify new targets of metabolites and known drugs

PubMed Central

Naveed, Hammad; Hameed, Umar S.; Harrus, Deborah; Bourguet, William; Arold, Stefan T.; Gao, Xin

2015-01-01

Motivation: The inherent promiscuity of small molecules towards protein targets impedes our understanding of healthy versus diseased metabolism. This promiscuity also poses a challenge for the pharmaceutical industry as identifying all protein targets is important to assess (side) effects and repositioning opportunities for a drug. Results: Here, we present a novel integrated structure- and system-based approach of drug-target prediction (iDTP) to enable the large-scale discovery of new targets for small molecules, such as pharmaceutical drugs, co-factors and metabolites (collectively called ‘drugs’). For a given drug, our method uses sequence order–independent structure alignment, hierarchical clustering and probabilistic sequence similarity to construct a probabilistic pocket ensemble (PPE) that captures promiscuous structural features of different binding sites on known targets. A drug’s PPE is combined with an approximation of its delivery profile to reduce false positives. In our cross-validation study, we use iDTP to predict the known targets of 11 drugs, with 63% sensitivity and 81% specificity. We then predicted novel targets for these drugs—two that are of high pharmacological interest, the peroxisome proliferator-activated receptor gamma and the oncogene B-cell lymphoma 2, were successfully validated through in vitro binding experiments. Our method is broadly applicable for the prediction of protein-small molecule interactions with several novel applications to biological research and drug development. Availability and implementation: The program, datasets and results are freely available to academic users at http://sfb.kaust.edu.sa/Pages/Software.aspx. Contact: xin.gao@kaust.edu.sa and stefan.arold@kaust.edu.sa Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26286808

Identification of putative TAL effector targets of the citrus canker pathogens shows functional convergence underlying disease development and defense response

PubMed Central

2014-01-01

Background Transcriptional activator-like (TAL) effectors, formerly known as the AvrBs3/PthA protein family, are DNA-binding effectors broadly found in Xanthomonas spp. that transactivate host genes upon injection via the bacterial type three-secretion system. Biologically relevant targets of TAL effectors, i.e. host genes whose induction is vital to establish a compatible interaction, have been reported for xanthomonads that colonize rice and pepper; however, citrus genes modulated by the TAL effectors PthA“s” and PthC“s” of the citrus canker bacteria Xanthomonas citri (Xc) and Xanthomonas aurantifolii pathotype C (XaC), respectively, are poorly characterized. Of particular interest, XaC causes canker disease in its host lemon (Citrus aurantifolia), but triggers a defense response in sweet orange. Results Based on, 1) the TAL effector-DNA binding code, 2) gene expression data of Xc and XaC-infiltrated sweet orange leaves, and 3) citrus hypocotyls transformed with PthA2, PthA4 or PthC1, we have identified a collection of Citrus sinensis genes potentially targeted by Xc and XaC TAL effectors. Our results suggest that similar with other strains of Xanthomonas TAL effectors, PthA2 and PthA4, and PthC1 to some extent, functionally converge. In particular, towards induction of genes involved in the auxin and gibberellin synthesis and response, cell division, and defense response. We also present evidence indicating that the TAL effectors act as transcriptional repressors and that the best scoring predicted DNA targets of PthA“s” and PthC“s” in citrus promoters predominantly overlap with or localize near to TATA boxes of core promoters, supporting the idea that TAL effectors interact with the host basal transcriptional machinery to recruit the RNA pol II and start transcription. Conclusions The identification of PthA“s” and PthC“s” targets, such as the LOB (LATERAL ORGAN BOUNDARY) and CCNBS genes that we report here, is key for the understanding

Identification of putative TAL effector targets of the citrus canker pathogens shows functional convergence underlying disease development and defense response.

PubMed

Pereira, Andre L A; Carazzolle, Marcelo F; Abe, Valeria Y; de Oliveira, Maria L P; Domingues, Mariane N; Silva, Jaqueline C; Cernadas, Raul A; Benedetti, Celso E

2014-02-25

Transcriptional activator-like (TAL) effectors, formerly known as the AvrBs3/PthA protein family, are DNA-binding effectors broadly found in Xanthomonas spp. that transactivate host genes upon injection via the bacterial type three-secretion system. Biologically relevant targets of TAL effectors, i.e. host genes whose induction is vital to establish a compatible interaction, have been reported for xanthomonads that colonize rice and pepper; however, citrus genes modulated by the TAL effectors PthA"s" and PthC"s" of the citrus canker bacteria Xanthomonas citri (Xc) and Xanthomonas aurantifolii pathotype C (XaC), respectively, are poorly characterized. Of particular interest, XaC causes canker disease in its host lemon (Citrus aurantifolia), but triggers a defense response in sweet orange. Based on, 1) the TAL effector-DNA binding code, 2) gene expression data of Xc and XaC-infiltrated sweet orange leaves, and 3) citrus hypocotyls transformed with PthA2, PthA4 or PthC1, we have identified a collection of Citrus sinensis genes potentially targeted by Xc and XaC TAL effectors. Our results suggest that similar with other strains of Xanthomonas TAL effectors, PthA2 and PthA4, and PthC1 to some extent, functionally converge. In particular, towards induction of genes involved in the auxin and gibberellin synthesis and response, cell division, and defense response. We also present evidence indicating that the TAL effectors act as transcriptional repressors and that the best scoring predicted DNA targets of PthA"s" and PthC"s" in citrus promoters predominantly overlap with or localize near to TATA boxes of core promoters, supporting the idea that TAL effectors interact with the host basal transcriptional machinery to recruit the RNA pol II and start transcription. The identification of PthA"s" and PthC"s" targets, such as the LOB (lateral organ boundary) and CCNBS genes that we report here, is key for the understanding of the canker symptoms development during host

Cognitive dysfunction in depression - pathophysiology and novel targets.

PubMed

Carvalho, Andre F; Miskowiak, Kamilla K; Hyphantis, Thomas N; Kohler, Cristiano A; Alves, Gilberto S; Bortolato, Beatrice; G Sales, Paulo Marcelo; Machado-Vieira, Rodrigo; Berk, Michael; McIntyre, Roger S

2014-01-01

Major depressive disorder (MDD) is associated with cognitive dysfunction encompassing several domains, including memory, executive function, processing speed and attention. Cognitive deficits persist in a significant proportion of patients even in remission, compromising psychosocial functioning and workforce performance. While monoaminergic antidepressants may improve cognitive performance in MDD, most antidepressants have limited clinical efficacy. The overarching aims of this review were: (1) to synthesize extant literature on putative biological pathways related to cognitive dysfunction in MDD and (2) to review novel neurotherapeutic targets for cognitive enhancement in MDD. We found that reciprocal and overlapping biological pathways may contribute to cognitive dysfunction in MDD, including an hyperactive hypothalamic-pituitary-adrenal axis, an increase in oxidative and nitrosative stress, inflammation (e.g., enhanced production of pro-inflammatory cytokines), mitochondrial dysfunction, increased apoptosis as well as a diminished neurotrophic support. Several promising neurotherapeutic targets were identified such as minocycline, statins, anti-inflammatory compounds, N-acetylcysteine, omega-3 poliunsaturated fatty acids, erythropoietin, thiazolidinediones, glucagon-like peptide-1 analogues, S-adenosyl-l-methionine (SAMe), cocoa flavonols, creatine monohydrate and lithium. Erythropoietin and SAMe had pro-cognitive effects in randomized controlled trials (RCT) involving MDD patients. Despite having preclinical and/or preliminary evidences from trials suggesting possible efficacy as novel cognitive enhancing agents for MDD, no RCT to date was performed for most of the other therapeutic targets reviewed herein. In conclusion, multiple biological pathways are involved in cognitive dysfunction in MDD. RCTs testing genuinely novel pro-cognitive compounds for MDD are warranted.

Non-coding RNAs—Novel targets in neurotoxicity

PubMed Central

Tal, Tamara L.; Tanguay, Robert L.

2012-01-01

Over the past ten years non-coding RNAs (ncRNAs) have emerged as pivotal players in fundamental physiological and cellular processes and have been increasingly implicated in cancer, immune disorders, and cardiovascular, neurodegenerative, and metabolic diseases. MicroRNAs (miRNAs) represent a class of ncRNA molecules that function as negative regulators of post-transcriptional gene expression. miRNAs are predicted to regulate 60% of all human protein-coding genes and as such, play key roles in cellular and developmental processes, human health, and disease. Relative to counterparts that lack bindings sites for miRNAs, genes encoding proteins that are post-transcriptionally regulated by miRNAs are twice as likely to be sensitive to environmental chemical exposure. Not surprisingly, miRNAs have been recognized as targets or effectors of nervous system, developmental, hepatic, and carcinogenic toxicants, and have been identified as putative regulators of phase I xenobiotic-metabolizing enzymes. In this review, we give an overview of the types of ncRNAs and highlight their roles in neurodevelopment, neurological disease, activity-dependent signaling, and drug metabolism. We then delve into specific examples that illustrate their importance as mediators, effectors, or adaptive agents of neurotoxicants or neuroactive pharmaceutical compounds. Finally, we identify a number of outstanding questions regarding ncRNAs and neurotoxicity. PMID:22394481

Astrocytes in the optic nerve head express putative mechanosensitive channels

PubMed Central

Choi, Hee Joo; Sun, Daniel

2015-01-01

Purpose To establish whether optic nerve head astrocytes express candidate molecules to sense tissue stretch. Methods We used conventional PCR, quantitative PCR, and single-cell reverse transcription PCR (RT–PCR) to assess the expression of various members of the transient receptor potential (TRP) channel family and of the recently characterized mechanosensitive channels Piezo1 and 2 in optic nerve head tissue and in single, isolated astrocytes. Results Most TRP subfamilies (TRPC, TRPM, TRPV, TRPA, and TRPP) and Piezo1 and 2 were expressed in the optic nerve head of the mouse. Quantitative real-time PCR analysis showed that TRPC1, TRPM7, TRPV2, TRPP2, and Piezo1 are the dominant isoforms in each subfamily. Single-cell RT–PCR revealed that many TRP isoforms, TRPC1–2, TRPC6, TRPV2, TRPV4, TRPM2, TRPM4, TRPM6–7, TRPP1–2, and Piezo1–2, are expressed in astrocytes of the optic nerve head, and that most astrocytes express TRPC1 and TRPP1–2. Comparisons of the TRPP and Piezo expression levels between different tissue regions showed that Piezo2 expression was higher in the optic nerve head and the optic nerve proper than in the brain and the corpus callosum. TRPP2 also showed higher expression in the optic nerve head. Conclusions Astrocytes in the optic nerve head express multiple putative mechanosensitive channels, in particular the recently identified channels Piezo1 and 2. The expression of putative mechanosensitive channels in these cells may contribute to their responsiveness to traumatic or glaucomatous injury. PMID:26236150

Systematic approach identifies RHOA as a potential biomarker therapeutic target for Asian gastric cancer

PubMed Central

Jung, Hae Rim; Park, Hee Seo; Park, Sungjin; Ahn, Young Zoo; Huh, Iksoo; Balch, Curt; Ku, Ja-Lok; Powis, Garth; Park, Taesung; Jeong, Jin-Hyun; Kim, Yon Hui

2016-01-01

Gastric cancer (GC) is a highly heterogeneous disease, in dire need of specific, biomarker-driven cancer therapies. While the accumulation of cancer “Big Data” has propelled the search for novel molecular targets for GC, its specific subpathway and cellular functions vary from patient to patient. In particular, mutations in the small GTPase gene RHOA have been identified in recent genome-wide sequencing of GC tumors. Moreover, protein overexpression of RHOA was reported in Chinese populations, while RHOA mutations were found in Caucasian GC tumors. To develop evidence-based precision medicine for heterogeneous cancers, we established a systematic approach to integrate transcriptomic and genomic data. Predicted signaling subpathways were then laboratory-validated both in vitro and in vivo, resulting in the identification of new candidate therapeutic targets. Here, we show: i) differences in RHOA expression patterns, and its pathway activity, between Asian and Caucasian GC tumors; ii) in vitro and in vivo perturbed RHOA expression inhibits GC cell growth in high RHOA-expressing cell lines; iii) inverse correlation between RHOA and RHOB expression; and iv) an innovative small molecule design strategy for RHOA inhibitors. In summary, RHOA, and its oncogenic signaling pathway, represent a strong biomarker-driven therapeutic target for Asian GC. This comprehensive strategy represents a promising approach for the development of “hit” compounds. PMID:27806312

Systematic screening of isogenic cancer cells identifies DUSP6 as context-specific synthetic lethal target in melanoma

PubMed Central

Wittig-Blaich, Stephanie; Wittig, Rainer; Schmidt, Steffen; Lyer, Stefan; Bewerunge-Hudler, Melanie; Gronert-Sum, Sabine; Strobel-Freidekind, Olga; Müller, Carolin; List, Markus; Jaskot, Aleksandra; Christiansen, Helle; Hafner, Mathias; Schadendorf, Dirk; Block, Ines; Mollenhauer, Jan

2017-01-01

Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few technologies are available for the systematic downstream evaluation of these results to identify novel starting points of future cancer therapies. We improved and extended a site-specific recombination-based system for systematic analysis of the individual functions of a large number of candidate genes. This was facilitated by a novel system for the construction of isogenic constitutive and inducible gain- and loss-of-function cell lines. Additionally, we demonstrate the construction of isogenic cell lines with combinations of the traits for advanced functional in vitro analyses. In a proof-of-concept experiment, a library of 108 isogenic melanoma cell lines was constructed and 8 genes were identified that significantly reduced viability in a discovery screen and in an independent validation screen. Here, we demonstrate the broad applicability of this recombination-based method and we proved its potential to identify new drug targets via the identification of the tumor suppressor DUSP6 as potential synthetic lethal target in melanoma cell lines with BRAF V600E mutations and high DUSP6 expression. PMID:28423600

Targeting legume loci: A comparison of three methods for target enrichment bait design in Leguminosae phylogenomics.

PubMed

Vatanparast, Mohammad; Powell, Adrian; Doyle, Jeff J; Egan, Ashley N

2018-03-01

The development of pipelines for locus discovery has spurred the use of target enrichment for plant phylogenomics. However, few studies have compared pipelines from locus discovery and bait design, through validation, to tree inference. We compared three methods within Leguminosae (Fabaceae) and present a workflow for future efforts. Using 30 transcriptomes, we compared Hyb-Seq, MarkerMiner, and the Yang and Smith (Y&S) pipelines for locus discovery, validated 7501 baits targeting 507 loci across 25 genera via Illumina sequencing, and inferred gene and species trees via concatenation- and coalescent-based methods. Hyb-Seq discovered loci with the longest mean length. MarkerMiner discovered the most conserved loci with the least flagged as paralogous. Y&S offered the most parsimony-informative sites and putative orthologs. Target recovery averaged 93% across taxa. We optimized our targeted locus set based on a workflow designed to minimize paralog/ortholog conflation and thus present 423 loci for legume phylogenomics. Methods differed across criteria important for phylogenetic marker development. We recommend Hyb-Seq as a method that may be useful for most phylogenomic projects. Our targeted locus set is a resource for future, community-driven efforts to reconstruct the legume tree of life.

Frameshift mutational target gene analysis identifies similarities and differences in constitutional mismatch repair-deficiency and Lynch syndrome.

PubMed

Maletzki, Claudia; Huehns, Maja; Bauer, Ingrid; Ripperger, Tim; Mork, Maureen M; Vilar, Eduardo; Klöcking, Sabine; Zettl, Heike; Prall, Friedrich; Linnebacher, Michael

2017-07-01

Mismatch-repair deficient (MMR-D) malignancies include Lynch Syndrome (LS), which is secondary to germline mutations in one of the MMR genes, and the rare childhood-form of constitutional mismatch repair-deficiency (CMMR-D); caused by bi-allelic MMR gene mutations. A hallmark of LS-associated cancers is microsatellite instability (MSI), characterized by coding frameshift mutations (cFSM) in target genes. By contrast, tumors arising in CMMR-D patients are thought to display a somatic mutation pattern differing from LS. This study has the main goal to identify cFSM in MSI target genes relevant in CMMR-D and to compare the spectrum of common somatic mutations, including alterations in DNA polymerases POLE and D1 between LS and CMMR-D. CMMR-D-associated tumors harbored more somatic mutations compared to LS cases, especially in the TP53 gene and in POLE and POLD1, where novel mutations were additionally identified. Strikingly, MSI in classical mononucleotide markers BAT40 and CAT25 was frequent in CMMR-D cases. MSI-target gene analysis revealed mutations in CMMR-D-associated tumors, some of them known to be frequently hit in LS, such as RNaseT2, HT001, and TGFβR2. Our results imply a general role for these cFSM as potential new drivers of MMR-D tumorigenesis. © 2017 Wiley Periodicals, Inc.

Genome-wide Expression Profiling, In Vivo DNA Binding Analysis, and Probabilistic Motif Prediction Reveal Novel Abf1 Target Genes during Fermentation, Respiration, and Sporulation in Yeast

PubMed Central

Schlecht, Ulrich; Erb, Ionas; Demougin, Philippe; Robine, Nicolas; Borde, Valérie; van Nimwegen, Erik; Nicolas, Alain

2008-01-01

The autonomously replicating sequence binding factor 1 (Abf1) was initially identified as an essential DNA replication factor and later shown to be a component of the regulatory network controlling mitotic and meiotic cell cycle progression in budding yeast. The protein is thought to exert its functions via specific interaction with its target site as part of distinct protein complexes, but its roles during mitotic growth and meiotic development are only partially understood. Here, we report a comprehensive approach aiming at the identification of direct Abf1-target genes expressed during fermentation, respiration, and sporulation. Computational prediction of the protein's target sites was integrated with a genome-wide DNA binding assay in growing and sporulating cells. The resulting data were combined with the output of expression profiling studies using wild-type versus temperature-sensitive alleles. This work identified 434 protein-coding loci as being transcriptionally dependent on Abf1. More than 60% of their putative promoter regions contained a computationally predicted Abf1 binding site and/or were bound by Abf1 in vivo, identifying them as direct targets. The present study revealed numerous loci previously unknown to be under Abf1 control, and it yielded evidence for the protein's variable DNA binding pattern during mitotic growth and meiotic development. PMID:18305101

Treponema pallidum Putative Novel Drug Target Identification and Validation: Rethinking Syphilis Therapeutics with Plant-Derived Terpenoids

PubMed Central

Tiwari, Sameeksha; Singh, Priyanka; Singh, Swati; Awasthi, Manika; Pandey, Veda P.

2015-01-01

Abstract Syphilis, a slow progressive and the third most common sexually transmitted disease found worldwide, is caused by a spirochete gram negative bacteria Treponema pallidum. Emergence of antibiotic resistant T. pallidum has led to a search for novel drugs and their targets. Subtractive genomics analyses of pathogen T. pallidum and host Homo sapiens resulted in identification of 126 proteins essential for survival and viability of the pathogen. Metabolic pathway analyses of these essential proteins led to discovery of nineteen proteins distributed among six metabolic pathways unique to T. pallidum. One hundred plant-derived terpenoids, as potential therapeutic molecules against T. pallidum, were screened for their drug likeness and ADMET (absorption, distribution, metabolism, and toxicity) properties. Subsequently the resulting nine terpenoids were docked with five unique T. pallidum targets through molecular modeling approaches. Out of five targets analyzed, D-alanine:D-alanine ligase was found to be the most promising target, while terpenoid salvicine was the most potent inhibitor. A comparison of the inhibitory potential of the best docked readily available natural compound, namely pomiferin (flavonoid) with that of the best docked terpenoid salvicine, revealed that salvicine was a more potent inhibitor than that of pomiferin. To the best of our knowledge, this is the first report of a terpenoid as a potential therapeutic molecule against T. pallidum with D-alanine:D-alanine ligase as a novel target. Further studies are warranted to evaluate and explore the potential clinical ramifications of these findings in relation to syphilis that has public health importance worldwide. PMID:25683888

An integrated approach to identify normal tissue expression of targets for antibody-drug conjugates: case study of TENB2

PubMed Central

Boswell, C Andrew; Mundo, Eduardo E; Firestein, Ron; Zhang, Crystal; Mao, Weiguang; Gill, Herman; Young, Cynthia; Ljumanovic, Nina; Stainton, Shannon; Ulufatu, Sheila; Fourie, Aimee; Kozak, Katherine R; Fuji, Reina; Polakis, Paul; Khawli, Leslie A; Lin, Kedan

2013-01-01

Background and Purpose The success of antibody-drug conjugates (ADCs) depends on the therapeutic window rendered by the differential expression between normal and pathological tissues. The ability to identify and visualize target expression in normal tissues could reveal causes for target-mediated clearance observed in pharmacokinetic characterization. TENB2 is a prostate cancer target associated with the progression of poorly differentiated and androgen-independent tumour types, and ADCs specific for TENB2 are candidate therapeutics. The objective of this study was to locate antigen expression of TENB2 in normal tissues, thereby elucidating the underlying causes of target-mediated clearance. Experimental Approach A series of pharmacokinetics, tissue distribution and mass balance studies were conducted in mice using a radiolabelled anti-TENB2 ADC. These data were complemented by non-invasive single photon emission computed tomography – X-ray computed tomography imaging and immunohistochemistry. Key Results The intestines were identified as a saturable and specific antigen sink that contributes, at least in part, to the rapid target-mediated clearance of the anti-TENB2 antibody and its drug conjugate in rodents. As a proof of concept, we also demonstrated the selective disposition of the ADC in a tumoural environment in vivo using the LuCaP 77 transplant mouse model. High tumour uptake was observed despite the presence of the antigen sink, and antigen specificity was confirmed by antigen blockade. Conclusions and Implications Our findings provide the anatomical location and biological interpretation of target-mediated clearance of anti-TENB2 antibodies and corresponding drug conjugates. Further investigations may be beneficial in addressing the relative contributions to ADC disposition from antigen expression in both normal and pathological tissues. PMID:22889168

An integrated approach to identify normal tissue expression of targets for antibody-drug conjugates: case study of TENB2.

PubMed

Boswell, C Andrew; Mundo, Eduardo E; Firestein, Ron; Zhang, Crystal; Mao, Weiguang; Gill, Herman; Young, Cynthia; Ljumanovic, Nina; Stainton, Shannon; Ulufatu, Sheila; Fourie, Aimee; Kozak, Katherine R; Fuji, Reina; Polakis, Paul; Khawli, Leslie A; Lin, Kedan

2013-01-01

The success of antibody-drug conjugates (ADCs) depends on the therapeutic window rendered by the differential expression between normal and pathological tissues. The ability to identify and visualize target expression in normal tissues could reveal causes for target-mediated clearance observed in pharmacokinetic characterization. TENB2 is a prostate cancer target associated with the progression of poorly differentiated and androgen-independent tumour types, and ADCs specific for TENB2 are candidate therapeutics. The objective of this study was to locate antigen expression of TENB2 in normal tissues, thereby elucidating the underlying causes of target-mediated clearance. A series of pharmacokinetics, tissue distribution and mass balance studies were conducted in mice using a radiolabelled anti-TENB2 ADC. These data were complemented by non-invasive single photon emission computed tomography - X-ray computed tomography imaging and immunohistochemistry. The intestines were identified as a saturable and specific antigen sink that contributes, at least in part, to the rapid target-mediated clearance of the anti-TENB2 antibody and its drug conjugate in rodents. As a proof of concept, we also demonstrated the selective disposition of the ADC in a tumoural environment in vivo using the LuCaP 77 transplant mouse model. High tumour uptake was observed despite the presence of the antigen sink, and antigen specificity was confirmed by antigen blockade. Our findings provide the anatomical location and biological interpretation of target-mediated clearance of anti-TENB2 antibodies and corresponding drug conjugates. Further investigations may be beneficial in addressing the relative contributions to ADC disposition from antigen expression in both normal and pathological tissues. © 2012 Genentech, Inc.. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Characterization of a putative Xylella fastidiosa diffusible signal factor by HRGC-EI-MS.

PubMed

Colnaghi Simionato, Ana Valéria; da Silva, Denise Santos; Lambais, Marcio Rodrigues; Carrilho, Emanuel

2007-10-01

Xylella fastidiosa (X.f.) is a plant pathogen with high levels of genomic similarity to Xanthomonas campestris pv. campestris (X.c.c.). It has been shown that X. fastidiosa synthesizes a putative diffusible signal factor (X.f.-DSF) that activates regulation of pathogenicity factor (rpf) genes in a X.c.c. reporter system, which might be involved in the regulation of pathogenesis associated genes as in X.c.c., as well as in quorum-sensing. The nature of the X.f.-DSF is not known, whereas the X.c.c.-DSF has been identified as cis-11-methyl-2-dodecenoic acid. In this work, the chemical nature of a putative X.f.-DSF molecule, able to restore endoglucanase activity in a X.c.c. rpfF mutant, was investigated as if it was a fatty acid derivative. Bioassays with X.c.c. reporter bacterium and X.f. culture extracts, based on endoglucanase restoration activity, were also carried out in order to confirm the DSFs molecules similarities. For this reason, a gas chromatography-mass spectrometry method was developed with standard fatty acids methyl esters mixtures. The retention time, as well as the fragmentation patterns, of each standard was used to identify the DSF molecule synthesized by X.f. in the culture medium. Typical ester fragmentation patterns (the derivatized analyte) were observed, such as: McLafferty rearrangement and migration of the Hdelta followed by 1,4-hydrogen shift and cleavage of the bond Cbeta--Cgamma, confirming the nature of this molecule. This confirmation was corroborated by the common peaks in both spectra. Besides, the observed retention time reinforces our conclusion since it corresponds to a methyl ester with 15 carbons. Since the X.f.-DSF molecule was tentatively identified as 12-methyl-tetradecanoic acid (by mass spectra library comparison), this standard compound was also analyzed, strongly suggesting that this is the identification of such a molecule. To our knowledge, this is the first time a DSF produced by X.f. has been characterized.

Characterization of a putative Xylella fastidiosa diffusible signal factor by HRGC-EI-MS.

PubMed

Colnaghi Simionato, Ana Valéria; da Silva, Denise Santos; Lambais, Marcio Rodrigues; Carrilho, Emanuel

2007-04-01

Xylella fastidiosa (X.f.) is a plant pathogen with high levels of genomic similarity to Xanthomonas campestris pv. campestris (X.c.c.). It has been shown that X. fastidiosa synthesizes a putative diffusible signal factor (X.f.-DSF) that activates regulation of pathogenicity factor (rpf) genes in a X.c.c. reporter system, which might be involved in the regulation of pathogenesis associated genes as in X.c.c., as well as in quorum-sensing. The nature of the X.f.-DSF is not known, whereas the X.c.c.-DSF has been identified as cis-11-methyl-2-dodecenoic acid. In this work, the chemical nature of a putative X.f.-DSF molecule, able to restore endoglucanase activity in a X.c.c. rpfF mutant, was investigated as if it was a fatty acid derivative. Bioassays with X.c.c. reporter bacterium and X.f. culture extracts, based on endoglucanase restoration activity, were also carried out in order to confirm the DSFs molecules similarities. For this reason, a gas chromatography-mass spectrometry method was developed with standard fatty acids methyl esters mixtures. The retention time, as well as the fragmentation patterns, of each standard was used to identify the DSF molecule synthesized by X.f. in the culture medium. Typical ester fragmentation patterns (the derivatized analyte) were observed, such as: McLafferty rearrangement and migration of the Hdelta followed by 1,4-hydrogen shift and cleavage of the bond Cbeta-Cgamma, confirming the nature of this molecule. This confirmation was corroborated by the common peaks in both spectra. Besides, the observed retention time reinforces our conclusion since it corresponds to a methyl ester with 15 carbons. Since the X.f.-DSF molecule was tentatively identified as 12-methyl-tetradecanoic acid (by mass spectra library comparison), this standard compound was also analyzed, strongly suggesting that this is the identification of such a molecule. To our knowledge, this is the first time a DSF produced by X.f. has been characterized. Copyright

Can we use genetic and genomic approaches to identify candidate animals for targeted selective treatment.

PubMed

Laurenson, Yan C S M; Kyriazakis, Ilias; Bishop, Stephen C

2013-10-18

Estimated breeding values (EBV) for faecal egg count (FEC) and genetic markers for host resistance to nematodes may be used to identify resistant animals for selective breeding programmes. Similarly, targeted selective treatment (TST) requires the ability to identify the animals that will benefit most from anthelmintic treatment. A mathematical model was used to combine the concepts and evaluate the potential of using genetic-based methods to identify animals for a TST regime. EBVs obtained by genomic prediction were predicted to be the best determinant criterion for TST in terms of the impact on average empty body weight and average FEC, whereas pedigree-based EBVs for FEC were predicted to be marginally worse than using phenotypic FEC as a determinant criterion. Whilst each method has financial implications, if the identification of host resistance is incorporated into a wider genomic selection indices or selective breeding programmes, then genetic or genomic information may be plausibly included in TST regimes. Copyright © 2013 Elsevier B.V. All rights reserved.

Quasi-targeted analysis of hydroxylation-related metabolites of polycyclic aromatic hydrocarbons in human urine by liquid chromatography-mass spectrometry.

PubMed

Tang, Caiming; Tan, Jianhua; Fan, Ruifang; Zhao, Bo; Tang, Caixing; Ou, Weihui; Jin, Jiabin; Peng, Xianzhi

2016-08-26

Metabolite identification is crucial for revealing metabolic pathways and comprehensive potential toxicities of polycyclic aromatic hydrocarbons (PAHs) in human body. In this work, a quasi-targeted analysis strategy was proposed for metabolite identification of monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in human urine using liquid chromatography triple quadruple mass spectrometry (LC-QqQ-MS/MS) combined with liquid chromatography high resolution mass spectrometry (LC-HRMS). Potential metabolites of OH-PAHs were preliminarily screened out by LC-QqQ-MS/MS in association with filtering in a self-constructed information list of possible metabolites, followed by further identification and confirmation with LC-HRMS. The developed method can provide more reliable and systematic results compared with traditional untargeted analysis using LC-HRMS. In addition, data processing for LC-HRMS analysis were greatly simplified. This quasi-targeted analysis method was successfully applied to identifying phase I and phase II metabolites of OH-PAHs in human urine. Five metabolites of hydroxynaphthalene, seven of hydroxyfluorene, four of hydroxyphenanthrene, and three of hydroxypyrene were tentatively identified. Metabolic pathways of PAHs in human body were putatively revealed based on the identified metabolites. The experimental results will be valuable for investigating the metabolic processes of PAHs in human body, and the quasi-targeted analysis strategy can be expanded to the metabolite identification and profiling of other compounds in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Manual method of visually identifying candidate signals for a targeted peptide.

PubMed

Filimonov, Aleksey; Kopylov, Arthur; Lisitsa, Andrey; Archakov, Alexander

2018-04-15

The purpose of this study is to improve peptide signal identification in groups of extracted ion chromatograms (XICs) obtained with the liquid chromatography-selected reaction monitoring (LC-SRM) technique and a triple quadrupole mass spectrometer (QqQ) operating in one of the supported multiple reaction monitoring (MRM) modes. The imperfection of quadrupole mass analyzers causes ion interference, which impedes the identification of peptide signals as chromatographic peak groups in relevant retention time intervals. To investigate this problem in depth, the QqQ conversion of the eluate into XIC groups was considered as the consecutive transformations of the particles' abundances as the corresponding functions of retention time. In this study, the hypothesis that, during this conversion, the same chromatographic profile should be preserved as an implicit sign in each chromatographic peak of the signal was confirmed for peptides. To examine chromatographic profiles, continuous transformations of XIC groups were derived and implemented in srm2prot Express software (s2pe, http://msr.ibmc.msk.ru/s2pe). Because of ion interference, several peptide-like signals may appear in one XIC group. Therefore, these signals must be considered candidates for a targeted peptide's signal and should be resolved after identification. The theoretical investigation of intensity functions as XICs that are not distorted by noise produced three rules for Identifying Candidate Signals for a targeted Peptide (ICSP, http://msr.ibmc.msk.ru/ICSP) that constitute the proposed manual visual method. We theoretically and experimentally compared this method with the conventional semiempirical intuitive technique and found that the former significantly streamlines peptide signal identification and avoids typical errors. Copyright © 2018 Elsevier B.V. All rights reserved.

High-throughput screening to identify selective inhibitors of microbial sulfate reduction (and beyond)

NASA Astrophysics Data System (ADS)

Carlson, H. K.; Coates, J. D.; Deutschbauer, A. M.

2015-12-01

The selective perturbation of complex microbial ecosystems to predictably influence outcomes in engineered and industrial environments remains a grand challenge for geomicrobiology. In some industrial ecosystems, such as oil reservoirs, sulfate reducing microorganisms (SRM) produce hydrogen sulfide which is toxic, explosive and corrosive. Current strategies to selectively inhibit sulfidogenesis are based on non-specific biocide treatments, bio-competitive exclusion by alternative electron acceptors or sulfate-analogs which are competitive inhibitors or futile/alternative substrates of the sulfate reduction pathway. Despite the economic cost of sulfidogenesis, there has been minimal exploration of the chemical space of possible inhibitory compounds, and very little work has quantitatively assessed the selectivity of putative souring treatments. We have developed a high-throughput screening strategy to target SRM, quantitatively ranked the selectivity and potency of hundreds of compounds and identified previously unrecognized SRM selective inhibitors and synergistic interactions between inhibitors. Once inhibitor selectivity is defined, high-throughput characterization of microbial community structure across compound gradients and identification of fitness determinants using isolate bar-coded transposon mutant libraries can give insights into the genetic mechanisms whereby compounds structure microbial communities. The high-throughput (HT) approach we present can be readily applied to target SRM in diverse environments and more broadly, could be used to identify and quantify the potency and selectivity of inhibitors of a variety of microbial metabolisms. Our findings and approach are relevant for engineering environmental ecosystems and also to understand the role of natural gradients in shaping microbial niche space.

microRNA expression profiling in fetal single ventricle malformation identified by deep sequencing.

PubMed

Yu, Zhang-Bin; Han, Shu-Ping; Bai, Yun-Fei; Zhu, Chun; Pan, Ya; Guo, Xi-Rong

2012-01-01

microRNAs (miRNAs) have emerged as key regulators in many biological processes, particularly cardiac growth and development, although the specific miRNA expression profile associated with this process remains to be elucidated. This study aimed to characterize the cellular microRNA profile involved in the development of congenital heart malformation, through the investigation of single ventricle (SV) defects. Comprehensive miRNA profiling in human fetal SV cardiac tissue was performed by deep sequencing. Differential expression of 48 miRNAs was revealed by sequencing by oligonucleotide ligation and detection (SOLiD) analysis. Of these, 38 were down-regulated and 10 were up-regulated in differentiated SV cardiac tissue, compared to control cardiac tissue. This was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Predicted target genes of the 48 differentially expressed miRNAs were analyzed by gene ontology and categorized according to cellular process, regulation of biological process and metabolic process. Pathway-Express analysis identified the WNT and mTOR signaling pathways as the most significant processes putatively affected by the differential expression of these miRNAs. The candidate genes involved in cardiac development were identified as potential targets for these differentially expressed microRNAs and the collaborative network of microRNAs and cardiac development related-mRNAs was constructed. These data provide the basis for future investigation of the mechanism of the occurrence and development of fetal SV malformations.

An Integrated Approach to Change the Outcome Part II: Targeted Neuromuscular Training Techniques to Reduce Identified ACL Injury Risk Factors

PubMed Central

Myer, Gregory D.; Ford, Kevin R.; Brent, Jensen L.; Hewett, Timothy E.

2014-01-01

Prior reports indicate that female athletes who demonstrate high knee abduction moments (KAMs) during landing are more responsive to neuromuscular training designed to reduce KAM. Identification of female athletes who demonstrate high KAM, which accurately identifies those at risk for noncontact anterior cruciate ligament (ACL) injury, may be ideal for targeted neuromuscular training. Specific neuromuscular training targeted to the underlying biomechanical components that increase KAM may provide the most efficient and effective training strategy to reduce noncontact ACL injury risk. The purpose of the current commentary is to provide an integrative approach to identify and target mechanistic underpinnings to increased ACL injury in female athletes. Specific neuromuscular training techniques will be presented that address individual algorithm components related to high knee load landing patterns. If these integrated techniques are employed on a widespread basis, prevention strategies for noncontact ACL injury among young female athletes may prove both more effective and efficient. PMID:22580980

High-Throughput Screening To Identify Potent and Specific Inhibitors of Microbial Sulfate Reduction.

PubMed

Carlson, Hans K; Mullan, Mark R; Mosqueda, Lorraine A; Chen, Steven; Arkin, Michelle R; Coates, John D

2017-06-20

The selective perturbation of complex microbial ecosystems to predictably influence outcomes in engineered and industrial environments remains a grand challenge for geomicrobiology. In some industrial ecosystems, such as oil reservoirs, sulfate reducing microorganisms (SRM) produce hydrogen sulfide which is toxic, explosive, and corrosive. Despite the economic cost of sulfidogenesis, there has been minimal exploration of the chemical space of possible inhibitory compounds, and very little work has quantitatively assessed the selectivity of putative souring treatments. We have developed a high-throughput screening strategy to identify potent and selective inhibitors of SRM, quantitatively ranked the selectivity and potency of hundreds of compounds and identified previously unrecognized SRM selective inhibitors and synergistic interactions between inhibitors. Zinc pyrithione is the most potent inhibitor of sulfidogenesis that we identified, and is several orders of magnitude more potent than commonly used industrial biocides. Both zinc and copper pyrithione are also moderately selective against SRM. The high-throughput (HT) approach we present can be readily adapted to target SRM in diverse environments and similar strategies could be used to quantify the potency and selectivity of inhibitors of a variety of microbial metabolisms. Our findings and approach are relevant to efforts to engineer environmental ecosystems and also to understand the role of natural gradients in shaping microbial niche space.

High-Resolution Genetics Identifies the Lipid Transfer Protein Sec14p as Target for Antifungal Ergolines

PubMed Central

Cotesta, Simona; Perruccio, Francesca; Knapp, Britta; Fu, Yue; Studer, Christian; Pries, Verena; Riedl, Ralph; Helliwell, Stephen B.; Petrovic, Katarina T.; Movva, N. Rao; Sanglard, Dominique; Tao, Jianshi; Hoepfner, Dominic

2016-01-01

Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point. PMID:27855158

Characterization of a Spontaneous Nonmagnetic Mutant of Magnetospirillum gryphiswaldense Reveals a Large Deletion Comprising a Putative Magnetosome Island

PubMed Central

Schübbe, Sabrina; Kube, Michael; Scheffel, André; Wawer, Cathrin; Heyen, Udo; Meyerdierks, Anke; Madkour, Mohamed H.; Mayer, Frank; Reinhardt, Richard; Schüler, Dirk

2003-01-01

Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosome-associated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria. PMID:13129949

Controlling range expansion in habitat networks by adaptively targeting source populations.

PubMed

Hock, Karlo; Wolff, Nicholas H; Beeden, Roger; Hoey, Jessica; Condie, Scott A; Anthony, Kenneth R N; Possingham, Hugh P; Mumby, Peter J

2016-08-01

Controlling the spread of invasive species, pests, and pathogens is often logistically limited to interventions that target specific locations at specific periods. However, in complex, highly connected systems, such as marine environments connected by ocean currents, populations spread dynamically in both space and time via transient connectivity links. This results in nondeterministic future distributions of species in which local populations emerge dynamically and concurrently over a large area. The challenge, therefore, is to choose intervention locations that will maximize the effectiveness of the control efforts. We propose a novel method to manage dynamic species invasions and outbreaks that identifies the intervention locations most likely to curtail population expansion by selectively targeting local populations most likely to expand their future range. Critically, at any point during the development of the invasion or outbreak, the method identifies the local intervention that maximizes the long-term benefit across the ecosystem by restricting species' potential to spread. In so doing, the method adaptively selects the intervention targets under dynamically changing circumstances. To illustrate the effectiveness of the method we applied it to controlling the spread of crown-of-thorns starfish (Acanthaster sp.) outbreaks across Australia's Great Barrier Reef. Application of our method resulted in an 18-fold relative improvement in management outcomes compared with a random targeting of reefs in putative starfish control scenarios. Although we focused on applying the method to reducing the spread of an unwanted species, it can also be used to facilitate the spread of desirable species through connectivity networks. For example, the method could be used to select those fragments of habitat most likely to rebuild a population if they were sufficiently well protected. © 2016 Society for Conservation Biology.

Recurrent Targeted Genes of Hepatitis B Virus in the Liver Cancer Genomes Identified by a Next-Generation Sequencing–Based Approach

PubMed Central

Ding, Dong; Lou, Xiaoyan; Hua, Dasong; Yu, Wei; Li, Lisha; Wang, Jun; Gao, Feng; Zhao, Na; Ren, Guoping; Li, Lanjuan; Lin, Biaoyang

2012-01-01

Integration of the viral DNA into host chromosomes was found in most of the hepatitis B virus (HBV)–related hepatocellular carcinomas (HCCs). Here we devised a massive anchored parallel sequencing (MAPS) method using next-generation sequencing to isolate and sequence HBV integrants. Applying MAPS to 40 pairs of HBV–related HCC tissues (cancer and adjacent tissues), we identified 296 HBV integration events corresponding to 286 unique integration sites (UISs) with precise HBV–Human DNA junctions. HBV integration favored chromosome 17 and preferentially integrated into human transcript units. HBV targeted genes were enriched in GO terms: cAMP metabolic processes, T cell differentiation and activation, TGF beta receptor pathway, ncRNA catabolic process, and dsRNA fragmentation and cellular response to dsRNA. The HBV targeted genes include 7 genes (PTPRJ, CNTN6, IL12B, MYOM1, FNDC3B, LRFN2, FN1) containing IPR003961 (Fibronectin, type III domain), 7 genes (NRG3, MASP2, NELL1, LRP1B, ADAM21, NRXN1, FN1) containing IPR013032 (EGF-like region, conserved site), and three genes (PDE7A, PDE4B, PDE11A) containing IPR002073 (3′, 5′-cyclic-nucleotide phosphodiesterase). Enriched pathways include hsa04512 (ECM-receptor interaction), hsa04510 (Focal adhesion), and hsa04012 (ErbB signaling pathway). Fewer integration events were found in cancers compared to cancer-adjacent tissues, suggesting a clonal expansion model in HCC development. Finally, we identified 8 genes that were recurrent target genes by HBV integration including fibronectin 1 (FN1) and telomerase reverse transcriptase (TERT1), two known recurrent target genes, and additional novel target genes such as SMAD family member 5 (SMAD5), phosphatase and actin regulator 4 (PHACTR4), and RNA binding protein fox-1 homolog (C. elegans) 1 (RBFOX1). Integrating analysis with recently published whole-genome sequencing analysis, we identified 14 additional recurrent HBV target genes, greatly expanding the HBV recurrent

Phylogenetic and comparative gene expression analysis of barley (Hordeum vulgare)WRKY transcription factor family reveals putatively retained functions betweenmonocots and dicots

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

Mangelsen, Elke; Kilian, Joachim; Berendzen, Kenneth W.

2008-02-01

WRKY proteins belong to the WRKY-GCM1 superfamily of zinc finger transcription factors that have been subject to a large plant-specific diversification. For the cereal crop barley (Hordeum vulgare), three different WRKY proteins have been characterized so far, as regulators in sucrose signaling, in pathogen defense, and in response to cold and drought, respectively. However, their phylogenetic relationship remained unresolved. In this study, we used the available sequence information to identify a minimum number of 45 barley WRKY transcription factor (HvWRKY) genes. According to their structural features the HvWRKY factors were classified into the previously defined polyphyletic WRKY subgroups 1 tomore » 3. Furthermore, we could assign putative orthologs of the HvWRKY proteins in Arabidopsis and rice. While in most cases clades of orthologous proteins were formed within each group or subgroup, other clades were composed of paralogous proteins for the grasses and Arabidopsis only, which is indicative of specific gene radiation events. To gain insight into their putative functions, we examined expression profiles of WRKY genes from publicly available microarray data resources and found group specific expression patterns. While putative orthologs of the HvWRKY transcription factors have been inferred from phylogenetic sequence analysis, we performed a comparative expression analysis of WRKY genes in Arabidopsis and barley. Indeed, highly correlative expression profiles were found between some of the putative orthologs. HvWRKY genes have not only undergone radiation in monocot or dicot species, but exhibit evolutionary traits specific to grasses. HvWRKY proteins exhibited not only sequence similarities between orthologs with Arabidopsis, but also relatedness in their expression patterns. This correlative expression is indicative for a putative conserved function of related WRKY proteins in mono- and dicot species.« less

Targeting phenotypically tolerant Mycobacterium tuberculosis

PubMed Central

Gold, Ben; Nathan, Carl

2016-01-01

While the immune system is credited with averting tuberculosis in billions of individuals exposed to Mycobacterium tuberculosis, the immune system is also culpable for tempering the ability of antibiotics to deliver swift and durable cure of disease. In individuals afflicted with tuberculosis, host immunity produces diverse microenvironmental niches that support suboptimal growth, or complete growth arrest, of M. tuberculosis. The physiological state of nonreplication in bacteria is associated with phenotypic drug tolerance. Many of these host microenvironments, when modeled in vitro by carbon starvation, complete nutrient starvation, stationary phase, acidic pH, reactive nitrogen intermediates, hypoxia, biofilms, and withholding streptomycin from the streptomycin-addicted strain SS18b, render M. tuberculosis profoundly tolerant to many of the antibiotics that are given to tuberculosis patients in a clinical setting. Targeting nonreplicating persisters is anticipated to reduce the duration of antibiotic treatment and rate of post-treatment relapse. Some promising drugs to treat tuberculosis, such as rifampicin and bedaquiline, only kill nonreplicating M. tuberculosis in vitro at concentrations far greater than their minimal inhibitory concentrations against replicating bacilli. There is an urgent demand to identify which of the currently used antibiotics, and which of the molecules in academic and corporate screening collections, have potent bactericidal action on nonreplicating M. tuberculosis. With this goal, we review methods of high throughput screening to target nonreplicating M. tuberculosis and methods to progress candidate molecules. A classification based on structures and putative targets of molecules that have been reported to kill nonreplicating M. tuberculosis revealed a rich diversity in pharmacophores. However, few of these compounds were tested under conditions that would exclude the impact of adsorbed compound acting during the recovery phase of

Rodent Models of Experimental Endometriosis: Identifying Mechanisms of Disease and Therapeutic Targets

PubMed Central

Bruner-Tran, Kaylon L.; Mokshagundam, Shilpa; Herington, Jennifer L.; Ding, Tianbing; Osteen, Kevin G.

2018-01-01

Background: Although it has been more than a century since endometriosis was initially described in the literature, understanding the etiology and natural history of the disease has been challenging. However, the broad utility of murine and rat models of experimental endometriosis has enabled the elucidation of a number of potentially targetable processes which may otherwise promote this disease. Objective: To review a variety of studies utilizing rodent models of endometriosis to illustrate their utility in examining mechanisms associated with development and progression of this disease. Results: Use of rodent models of endometriosis has provided a much broader understanding of the risk factors for the initial development of endometriosis, the cellular pathology of the disease and the identification of potential therapeutic targets. Conclusion: Although there are limitations with any animal model, the variety of experimental endometriosis models that have been developed has enabled investigation into numerous aspects of this disease. Thanks to these models, our under-standing of the early processes of disease development, the role of steroid responsiveness, inflammatory processes and the peritoneal environment has been advanced. More recent models have begun to shed light on how epigenetic alterations con-tribute to the molecular basis of this disease as well as the multiple comorbidities which plague many patients. Continued de-velopments of animal models which aid in unraveling the mechanisms of endometriosis development provide the best oppor-tunity to identify therapeutic strategies to prevent or regress this enigmatic disease.

A Fluorescent Protein Scaffold for Presenting Structurally Constrained Peptides Provides an Effective Screening System to Identify High Affinity Target-Binding Peptides

PubMed Central

Kadonosono, Tetsuya; Yabe, Etsuri; Furuta, Tadaomi; Yamano, Akihiro; Tsubaki, Takuya; Sekine, Takuya; Kuchimaru, Takahiro; Sakurai, Minoru; Kizaka-Kondoh, Shinae

2014-01-01

Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131–L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides. PMID:25084350

Co-Expression of Putative Cancer Stem Cell Markers CD44 and CD133 in Prostate Carcinomas.

PubMed

Kalantari, Elham; Asgari, Mojgan; Nikpanah, Seyedehmoozhan; Salarieh, Naghme; Asadi Lari, Mohammad Hossein; Madjd, Zahra

2017-10-01

Cancer stem cells (CSCs) are the main players of prostate tumorigenesis thus; characterization of CSCs can pave the way for understanding the early detection, drug resistance, metastasis and relapse. The current study was conducted to evaluate the expression level and clinical significance of the potential CSC markers CD44 and CD133 in a series of prostate tissues. One hundred and forty eight prostate tissues composed of prostate cancer (PCa), high-grade prostatic intraepithelial neoplasia (HGPIN), and benign prostate hyperplasia (BPH) were immunostained for the putative CSC markers CD44 and CD133. Subsequently, the correlation between the expression of these markers and the clinicopathological variables was examined. A higher level of CD44 expression was observed in 42% of PCa, 57% of HGPIN, and 42% BPH tissues. In the case of CD133 expression PCa, HGPIN, and BPH samples demonstrated high immunoreactivity in 46%, 43%, and 42% of cells, respectively. Statistical analysis showed an inverse significant correlation between CD44 expression with Gleason score of PCa (P = 0.02), while no significant correlation was observed between CD133 expression and clinicopathological parameters. A significant reciprocal correlation was observed between the expression of two putative CSC markers CD44 and CD133 in PCa specimens while not indicating clinical significance. Further clinical investigation is required to consider these markers as targets of new therapeutic strategies for PCa.

Identification of a novel putative pancreatic stem/progenitor cell marker DCAMKL-1 in normal mouse pancreas.

PubMed

May, Randal; Sureban, Sripathi M; Lightfoot, Stan A; Hoskins, Aimee B; Brackett, Daniel J; Postier, Russell G; Ramanujam, Rama; Rao, Chinthalapally V; Wyche, James H; Anant, Shrikant; Houchen, Courtney W

2010-08-01

Stem cells are critical in maintaining adult homeostasis and have been proposed to be the origin of many solid tumors, including pancreatic cancer. Here we demonstrate the expression patterns of the putative intestinal stem cell marker DCAMKL-1 in the pancreas of uninjured C57BL/6 mice compared with other pancreatic stem/progenitor cell markers. We then determined the viability of isolated pancreatic stem/progenitor cells in isotransplantation assays following DCAMKL-1 antibody-based cell sorting. Sorted cells were grown in suspension culture and injected into the flanks of athymic nude mice. Here we report that DCAMKL-1 is expressed in the main pancreatic duct epithelia and islets, but not within acinar cells. Coexpression was observed with somatostatin, NGN3, and nestin, but not glucagon or insulin. Isolated DCAMKL-1+ cells formed spheroids in suspension culture and induced nodule formation in isotransplantation assays. Analysis of nodules demonstrated markers of early pancreatic development (PDX-1), glandular epithelium (cytokeratin-14 and Ep-CAM), and isletlike structures (somatostatin and secretin). These data taken together suggest that DCAMKL-1 is a novel putative stem/progenitor marker, can be used to isolate normal pancreatic stem/progenitors, and potentially regenerates pancreatic tissues. This may represent a novel tool for regenerative medicine and a target for anti-stem cell-based therapeutics in pancreatic cancer.

Identification of a novel putative pancreatic stem/progenitor cell marker DCAMKL-1 in normal mouse pancreas

PubMed Central

May, Randal; Sureban, Sripathi M.; Lightfoot, Stan A.; Hoskins, Aimee B.; Brackett, Daniel J.; Postier, Russell G.; Ramanujam, Rama; Rao, Chinthalapally V.; Wyche, James H.; Anant, Shrikant

2010-01-01

Stem cells are critical in maintaining adult homeostasis and have been proposed to be the origin of many solid tumors, including pancreatic cancer. Here we demonstrate the expression patterns of the putative intestinal stem cell marker DCAMKL-1 in the pancreas of uninjured C57BL/6 mice compared with other pancreatic stem/progenitor cell markers. We then determined the viability of isolated pancreatic stem/progenitor cells in isotransplantation assays following DCAMKL-1 antibody-based cell sorting. Sorted cells were grown in suspension culture and injected into the flanks of athymic nude mice. Here we report that DCAMKL-1 is expressed in the main pancreatic duct epithelia and islets, but not within acinar cells. Coexpression was observed with somatostatin, NGN3, and nestin, but not glucagon or insulin. Isolated DCAMKL-1+ cells formed spheroids in suspension culture and induced nodule formation in isotransplantation assays. Analysis of nodules demonstrated markers of early pancreatic development (PDX-1), glandular epithelium (cytokeratin-14 and Ep-CAM), and isletlike structures (somatostatin and secretin). These data taken together suggest that DCAMKL-1 is a novel putative stem/progenitor marker, can be used to isolate normal pancreatic stem/progenitors, and potentially regenerates pancreatic tissues. This may represent a novel tool for regenerative medicine and a target for anti-stem cell-based therapeutics in pancreatic cancer. PMID:20522640

Targeted and Untargeted Metabolic Profiling of Wild Grassland Plants identifies Antibiotic and Anthelmintic Compounds Targeting Pathogen Physiology, Metabolism and Reproduction.

PubMed

French, Katherine E; Harvey, Joe; McCullagh, James S O

2018-01-26

Plants traditionally used by farmers to manage livestock ailments could reduce reliance on synthetic antibiotics and anthelmintics but in many cases their chemical composition is unknown. As a case study, we analyzed the metabolite profiles of 17 plant species and 45 biomass samples from agricultural grasslands in England using targeted and untargeted metabolite profiling by liquid-chromatography mass spectrometry. We identified a range of plant secondary metabolites, including 32 compounds with known antimicrobial/anthelmintic properties which varied considerably across the different plant samples. These compounds have been shown previously to target multiple aspects of pathogen physiology and metabolism in vitro and in vivo, including inhibition of quorum sensing in bacteria and egg viability in nematodes. The most abundant bioactive compounds were benzoic acid, myricetin, p-coumaric acid, rhamnetin, and rosmarinic acid. Four wild plants (Filipendula ulmaria (L.) Maxim., Prunella vulgaris L., Centuarea nigra L., and Rhinanthus minor L.) and two forage legumes (Medicago sativa L., Trifolium hybridium L.) contained high levels of these compounds. Forage samples from native high-diversity grasslands had a greater abundance of medicinal compounds than samples from agriculturally improved grasslands. Incorporating plants with antibiotic/anthelmintic compounds into livestock feeds may reduce global drug-resistance and preserve the efficacy of last-resort drugs.

smRNAome profiling to identify conserved and novel microRNAs in Stevia rebaudiana Bertoni

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies on miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa etc. and volume of the miRNA database, mirBASE, has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. Several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana. Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs irrespective of availability of genome sequence data. Results To identify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2,509,190 distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly mRNA encoding enzymes regulating essential plant metabolic and signaling pathways. Conclusions This study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist in stevia species. Our results provided information on stevia miRNAs and their targets building a foundation for future studies to

smRNAome profiling to identify conserved and novel microRNAs in Stevia rebaudiana Bertoni.

PubMed

Mandhan, Vibha; Kaur, Jagdeep; Singh, Kashmir

2012-11-01

MicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies on miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa etc. and volume of the miRNA database, mirBASE, has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. Several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana. Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs irrespective of availability of genome sequence data. To identify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2,509,190 distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly mRNA encoding enzymes regulating essential plant metabolic and signaling pathways. This study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist in stevia species. Our results provided information on stevia miRNAs and their targets building a foundation for future studies to understand their roles in key

Structural connectivity patterns associated with the putative visual word form area and children's reading ability.

PubMed

Fan, Qiuyun; Anderson, Adam W; Davis, Nicole; Cutting, Laurie E

2014-10-24

With the advent of neuroimaging techniques, especially functional MRI (fMRI), studies have mapped brain regions that are associated with good and poor reading, most centrally a region within the left occipito-temporal/fusiform region (L-OT/F) often referred to as the visual word form area (VWFA). Despite an abundance of fMRI studies of the putative VWFA, research about its structural connectivity has just started. Provided that the putative VWFA may be connected to distributed regions in the brain, it remains unclear how this network is engaged in constituting a well-tuned reading circuitry in the brain. Here we used diffusion MRI to study the structural connectivity patterns of the putative VWFA and surrounding areas within the L-OT/F in children with typically developing (TD) reading ability and with word recognition deficits (WRD; sometimes referred to as dyslexia). We found that L-OT/F connectivity varied along a posterior-anterior gradient, with specific structural connectivity patterns related to reading ability in the ROIs centered upon the putative VWFA. Findings suggest that the architecture of the putative VWFA connectivity is fundamentally different between TD and WRD, with TD showing greater connectivity to linguistic regions than WRD, and WRD showing greater connectivity to visual and parahippocampal regions than TD. Findings thus reveal clear structural abnormalities underlying the functional abnormalities in the putative VWFA in WRD. Copyright © 2014 Elsevier B.V. All rights reserved.

Integrated DNA/RNA targeted genomic profiling of diffuse large B-cell lymphoma using a clinical assay.

PubMed

Intlekofer, Andrew M; Joffe, Erel; Batlevi, Connie L; Hilden, Patrick; He, Jie; Seshan, Venkatraman E; Zelenetz, Andrew D; Palomba, M Lia; Moskowitz, Craig H; Portlock, Carol; Straus, David J; Noy, Ariela; Horwitz, Steven M; Gerecitano, John F; Moskowitz, Alison; Hamlin, Paul; Matasar, Matthew J; Kumar, Anita; van den Brink, Marcel R; Knapp, Kristina M; Pichardo, Janine D; Nahas, Michelle K; Trabucco, Sally E; Mughal, Tariq; Copeland, Amanda R; Papaemmanuil, Elli; Moarii, Mathai; Levine, Ross L; Dogan, Ahmet; Miller, Vincent A; Younes, Anas

2018-06-12

We sought to define the genomic landscape of diffuse large B-cell lymphoma (DLBCL) by using formalin-fixed paraffin-embedded (FFPE) biopsy specimens. We used targeted sequencing of genes altered in hematologic malignancies, including DNA coding sequence for 405 genes, noncoding sequence for 31 genes, and RNA coding sequence for 265 genes (FoundationOne-Heme). Short variants, rearrangements, and copy number alterations were determined. We studied 198 samples (114 de novo, 58 previously treated, and 26 large-cell transformation from follicular lymphoma). Median number of GAs per case was 6, with 97% of patients harboring at least one alteration. Recurrent GAs were detected in genes with established roles in DLBCL pathogenesis (e.g. MYD88, CREBBP, CD79B, EZH2), as well as notable differences compared to prior studies such as inactivating mutations in TET2 (5%). Less common GAs identified potential targets for approved or investigational therapies, including BRAF, CD274 (PD-L1), IDH2, and JAK1/2. TP53 mutations were more frequently observed in relapsed/refractory DLBCL, and predicted for lack of response to first-line chemotherapy, identifying a subset of patients that could be prioritized for novel therapies. Overall, 90% (n = 169) of the patients harbored a GA which could be explored for therapeutic intervention, with 54% (n = 107) harboring more than one putative target.

BeReTa: a systematic method for identifying target transcriptional regulators to enhance microbial production of chemicals.

PubMed

Kim, Minsuk; Sun, Gwanggyu; Lee, Dong-Yup; Kim, Byung-Gee

2017-01-01

Modulation of regulatory circuits governing the metabolic processes is a crucial step for developing microbial cell factories. Despite the prevalence of in silico strain design algorithms, most of them are not capable of predicting required modifications in regulatory networks. Although a few algorithms may predict relevant targets for transcriptional regulator (TR) manipulations, they have limited reliability and applicability due to their high dependency on the availability of integrated metabolic/regulatory models. We present BeReTa (Beneficial Regulator Targeting), a new algorithm for prioritization of TR manipulation targets, which makes use of unintegrated network models. BeReTa identifies TR manipulation targets by evaluating regulatory strengths of interactions and beneficial effects of reactions, and subsequently assigning beneficial scores for the TRs. We demonstrate that BeReTa can predict both known and novel TR manipulation targets for enhanced production of various chemicals in Escherichia coli Furthermore, through a case study of antibiotics production in Streptomyces coelicolor, we successfully demonstrate its wide applicability to even less-studied organisms. To the best of our knowledge, BeReTa is the first strain design algorithm exclusively designed for predicting TR manipulation targets. MATLAB code is available at https://github.com/kms1041/BeReTa (github). byungkim@snu.ac.krSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Large-Scale Phylogenetic Classification of Fungal Chitin Synthases and Identification of a Putative Cell-Wall Metabolism Gene Cluster in Aspergillus Genomes

PubMed Central

Pacheco-Arjona, Jose Ramon; Ramirez-Prado, Jorge Humberto

2014-01-01

The cell wall is a protective and versatile structure distributed in all fungi. The component responsible for its rigidity is chitin, a product of chitin synthase (Chsp) enzymes. There are seven classes of chitin synthase genes (CHS) and the amount and type encoded in fungal genomes varies considerably from one species to another. Previous Chsp sequence analyses focused on their study as individual units, regardless of genomic context. The identification of blocks of conserved genes between genomes can provide important clues about the interactions and localization of chitin synthases. On the present study, we carried out an in silico search of all putative Chsp encoded in 54 full fungal genomes, encompassing 21 orders from five phyla. Phylogenetic studies of these Chsp were able to confidently classify 347 out of the 369 Chsp identified (94%). Patterns in the distribution of Chsp related to taxonomy were identified, the most prominent being related to the type of fungal growth. More importantly, a synteny analysis for genomic blocks centered on class IV Chsp (the most abundant and widely distributed Chsp class) identified a putative cell wall metabolism gene cluster in members of the genus Aspergillus, the first such association reported for any fungal genome. PMID:25148134

Identification of potential drug targets by subtractive genome analysis of Escherichia coli O157:H7: an in silico approach

PubMed Central

Mondal, Shakhinur Islam; Ferdous, Sabiha; Jewel, Nurnabi Azad; Akter, Arzuba; Mahmud, Zabed; Islam, Md Muzahidul; Afrin, Tanzila; Karim, Nurul

2015-01-01

Bacterial enteric infections resulting in diarrhea, dysentery, or enteric fever constitute a huge public health problem, with more than a billion episodes of disease annually in developing and developed countries. In this study, the deadly agent of hemorrhagic diarrhea and hemolytic uremic syndrome, Escherichia coli O157:H7 was investigated with extensive computational approaches aimed at identifying novel and broad-spectrum antibiotic targets. A systematic in silico workflow consisting of comparative genomics, metabolic pathways analysis, and additional drug prioritizing parameters was used to identify novel drug targets that were essential for the pathogen’s survival but absent in its human host. Comparative genomic analysis of Kyoto Encyclopedia of Genes and Genomes annotated metabolic pathways identified 350 putative target proteins in E. coli O157:H7 which showed no similarity to human proteins. Further bio-informatic approaches including prediction of subcellular localization, calculation of molecular weight, and web-based investigation of 3D structural characteristics greatly aided in filtering the potential drug targets from 350 to 120. Ultimately, 44 non-homologous essential proteins of E. coli O157:H7 were prioritized and proved to have the eligibility to become novel broad-spectrum antibiotic targets and DNA polymerase III alpha (dnaE) was the top-ranked among these targets. Moreover, druggability of each of the identified drug targets was evaluated by the DrugBank database. In addition, 3D structure of the dnaE was modeled and explored further for in silico docking with ligands having potential druggability. Finally, we confirmed that the compounds N-coeleneterazine and N-(1,4-dihydro-5H-tetrazol-5-ylidene)-9-oxo-9H-xanthene-2-sulfon-amide were the most suitable ligands of dnaE and hence proposed as the potential inhibitors of this target protein. The results of this study could facilitate the discovery and release of new and effective drugs against E

A novel mouse model identifies cooperating mutations and therapeutic targets critical for chronic myeloid leukemia progression

PubMed Central

Giotopoulos, George; van der Weyden, Louise; Osaki, Hikari; Rust, Alistair G.; Gallipoli, Paolo; Meduri, Eshwar; Horton, Sarah J.; Chan, Wai-In; Foster, Donna; Prinjha, Rab K.; Pimanda, John E.; Tenen, Daniel G.; Vassiliou, George S.; Koschmieder, Steffen; Adams, David J.

2015-01-01

The introduction of highly selective ABL-tyrosine kinase inhibitors (TKIs) has revolutionized therapy for chronic myeloid leukemia (CML). However, TKIs are only efficacious in the chronic phase of the disease and effective therapies for TKI-refractory CML, or after progression to blast crisis (BC), are lacking. Whereas the chronic phase of CML is dependent on BCR-ABL, additional mutations are required for progression to BC. However, the identity of these mutations and the pathways they affect are poorly understood, hampering our ability to identify therapeutic targets and improve outcomes. Here, we describe a novel mouse model that allows identification of mechanisms of BC progression in an unbiased and tractable manner, using transposon-based insertional mutagenesis on the background of chronic phase CML. Our BC model is the first to faithfully recapitulate the phenotype, cellular and molecular biology of human CML progression. We report a heterogeneous and unique pattern of insertions identifying known and novel candidate genes and demonstrate that these pathways drive disease progression and provide potential targets for novel therapeutic strategies. Our model greatly informs the biology of CML progression and provides a potent resource for the development of candidate therapies to improve the dismal outcomes in this highly aggressive disease. PMID:26304963

Identification and temporal expression of putative circadian clock transcripts in the amphipod crustacean Talitrus saltator

PubMed Central

O’Grady, Joseph F.; Hoelters, Laura S.; Swain, Martin T.

2016-01-01

Background Talitrus saltator is an amphipod crustacean that inhabits the supralittoral zone on sandy beaches in the Northeast Atlantic and Mediterranean. T. saltator exhibits endogenous locomotor activity rhythms and time-compensated sun and moon orientation, both of which necessitate at least one chronometric mechanism. Whilst their behaviour is well studied, currently there are no descriptions of the underlying molecular components of a biological clock in this animal, and very few in other crustacean species. Methods We harvested brain tissue from animals expressing robust circadian activity rhythms and used homology cloning and Illumina RNAseq approaches to sequence and identify the core circadian clock and clock-related genes in these samples. We assessed the temporal expression of these genes in time-course samples from rhythmic animals using RNAseq. Results We identified a comprehensive suite of circadian clock gene homologues in T. saltator including the ‘core’ clock genes period (Talper), cryptochrome 2 (Talcry2), timeless (Taltim), clock (Talclk), and bmal1 (Talbmal1). In addition we describe the sequence and putative structures of 23 clock-associated genes including two unusual, extended isoforms of pigment dispersing hormone (Talpdh). We examined time-course RNAseq expression data, derived from tissues harvested from behaviourally rhythmic animals, to reveal rhythmic expression of these genes with approximately circadian period in Talper and Talbmal1. Of the clock-related genes, casein kinase IIβ (TalckIIβ), ebony (Talebony), jetlag (Taljetlag), pigment dispensing hormone (Talpdh), protein phosphatase 1 (Talpp1), shaggy (Talshaggy), sirt1 (Talsirt1), sirt7 (Talsirt7) and supernumerary limbs (Talslimb) show temporal changes in expression. Discussion We report the sequences of principle genes that comprise the circadian clock of T. saltator and highlight the conserved structural and functional domains of their deduced cognate proteins. Our

The genetic regulatory network centered on Pto-Wuschela and its targets involved in wood formation revealed by association studies.

PubMed

Yang, Xiaohui; Wei, Zunzheng; Du, Qingzhang; Chen, Jinhui; Wang, Qingshi; Quan, Mingyang; Song, Yuepeng; Xie, Jianbo; Zhang, Deqiang

2015-11-09

Transcription factors (TFs) regulate gene expression and can strongly affect phenotypes. However, few studies have examined TF variants and TF interactions with their targets in plants. Here, we used genetic association in 435 unrelated individuals of Populus tomentosa to explore the variants in Pto-Wuschela and its targets to decipher the genetic regulatory network of Pto-Wuschela. Our bioinformatics and co-expression analysis identified 53 genes with the motif TCACGTGA as putative targets of Pto-Wuschela. Single-marker association analysis showed that Pto-Wuschela was associated with wood properties, which is in agreement with the observation that it has higher expression in stem vascular tissues in Populus. Also, SNPs in the 53 targets were associated with growth or wood properties under additive or dominance effects, suggesting these genes and Pto-Wuschela may act in the same genetic pathways that affect variation in these quantitative traits. Epistasis analysis indicated that 75.5% of these genes directly or indirectly interacted Pto-Wuschela, revealing the coordinated genetic regulatory network formed by Pto-Wuschela and its targets. Thus, our study provides an alternative method for dissection of the interactions between a TF and its targets, which will strength our understanding of the regulatory roles of TFs in complex traits in plants.

Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells

PubMed Central

Linehan, Erin K.; Schrader, Carol E.; Stavnezer, Janet

2015-01-01

Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID. PMID:26263206

Sequence of a cDNA and expression of the gene encoding a putative epidermal chitin synthase of Manduca sexta.

PubMed

Zhu, Yu-Cheng; Specht, Charles A; Dittmer, Neal T; Muthukrishnan, Subbaratnam; Kanost, Michael R; Kramer, Karl J

2002-11-01

Glycosyltransferases are enzymes that synthesize oligosaccharides, polysaccharides and glycoconjugates. One type of glycosyltransferase is chitin synthase, a very important enzyme in biology, which is utilized by insects, fungi, and other invertebrates to produce chitin, a polysaccharide of beta-1,4-linked N-acetylglucosamine. Chitin is an important component of the insect's exoskeletal cuticle and gut lining. To identify and characterize a chitin synthase gene of the tobacco hornworm, Manduca sexta, degenerate primers were designed from two highly conserved regions in fungal and nematode chitin synthase protein sequences and then used to amplify a similar region from Manduca cDNA. A full-length cDNA of 5152 nucleotides was assembled for the putative Manduca chitin synthase gene, MsCHS1, and sequencing of genomic DNA verified the contiguity of the sequence. The MsCHS1 cDNA has an ORF of 4692 nucleotides that encodes a transmembrane protein of 1564 amino acid residues with a mass of approximately 179 kDa (GenBank no. AY062175). It is most similar, over its entire length of protein sequence, to putative chitin synthases from other insects and nematodes, with 68% identity to enzymes from both the blow fly, Lucilia cuprina, and the fruit fly, Drosophila melanogaster. The similarity with fungal chitin synthases is restricted to the putative catalytic domain, and the MsCHS1 protein has, at equivalent positions, several amino acids that are essential for activity as revealed by mutagenesis of the fungal enzymes. A 5.3-kb transcript of MsCHS1 was identified by northern blot hybridization of RNA from larval epidermis, suggesting that the enzyme functions to make chitin deposited in the cuticle. Further examination by RT-PCR showed that MsCHS1 expression is regulated in the epidermis, with the amount of transcript increasing during phases of cuticle deposition.

Role of the liver X receptors in skin physiology: Putative pharmacological targets in human diseases.

PubMed

Ouedraogo, Zangbéwendé Guy; Fouache, Allan; Trousson, Amalia; Baron, Silvère; Lobaccaro, Jean-Marc A

2017-10-01

Liver X receptors (LXRs) are members of the nuclear receptor superfamily that have been shown to regulate various physiological functions such as lipid metabolism and cholesterol homeostasis. Concordant reports have elicited the possibility to target them to cure many human diseases including arteriosclerosis, cancer, arthritis, and diabetes. The high relevance of modulating LXR activities to treat numerous skin diseases, mainly those with exacerbated inflammation processes, contrasts with the lack of approved therapeutic use. This review makes an assessment to sum up the findings regarding the physiological roles of LXRs in skin and help progress towards the therapeutic and safe management of their activities. It focuses on the possible pharmacological targeting of LXRs to cure or prevent selected skin diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Putative identification of new p-coumaroyl glycoside flavonoids in grape by ultra-high performance liquid chromatography/high-resolution mass spectrometry.

PubMed

Panighel, Annarita; De Rosso, Mirko; Dalla Vedova, Antonio; Flamini, Riccardo

2015-02-28

Grape polyphenols are antioxidant compounds, markers in vine chemotaxonomy, and involved in color stabilization of red wines. Sugar acylation usually confers higher stability on glycoside derivatives and this effect is enhanced by an aromatic substituent such as p-coumaric acid. Until now, only p-coumaroyl anthocyanins have been found in grape. A method of 'suspect screening analysis' by ultra-high-performance liquid chromatography/high-resolution mass spectrometry (UHPLC/QTOFMS) has recently been developed to study grape metabolomics. In the present study, this approach was used to identify new polyphenols in grape by accurate mass measurement, MS/MS fragmentation, and study of correlations between fragments observed and putative structures. Three putative p-coumaroyl flavonoids were identified in Raboso Piave grape extract: a dihydrokaempferide-3-O-p-coumaroylhexoside-like flavanone, isorhamnetin-3-O-p-coumaroylglucoside, and a chrysoeriol-p-coumaroylhexoside-like flavone. Accurate MS provided structural characterization of functional groups, and literature data indicates their probable position in the molecule. A fragmentation scheme is proposed for each compound. Compounds were identified by overlapping various analytical methods according to recommendations in the MS-based metabolomics literature. Stereochemistry and the definitive position of substituents in the molecule can only be confirmed by isolation and characterization or synthesis of each compound. These findings suggest addressing research of acylated polyphenol glycosides to other grape varieties. Copyright © 2015 John Wiley & Sons, Ltd.

Therapeutic Inhibition of miR-4260 Suppresses Colorectal Cancer via Targeting MCC and SMAD4.

PubMed

Xiao, Junjie; Lv, Dongchao; Zhou, Jinzhe; Bei, Yihua; Chen, Ting; Hu, Muren; Zhou, Qiulian; Fu, Siyi; Huang, Qi

2017-01-01

Dysregulation of microRNAs (miRNAs, miRs) and their putative target genes have been increasingly reported to contribute to colorectal cancer. However, miRNAs that directly target the mutated in colorectal cancer (MCC) gene, a tumor suppressor which is downregulated or inactivated in colorectal cancer, remain largely unknown. By using an array-based miRNA analysis, we identified a group of miRNAs that were dysregulated in human metastatic versus non-metastatic colorectal cancer tissues. One of these miRNAs, miR-4260, was predicted to target MCC in the miRDB database. Results using human HCT116 and HT29 colorectal cancer cell lines showed that miR-4260 mimic enhanced cell proliferation and migration and reduced apoptosis induced by the chemotherapeutic agent 5-fluorouracil while miR-4260 inhibitor had inverse effects. Furthermore, miR-4260 negatively regulated MCC as well as SMAD4 by directly binding to the 3'untranslational region (3'UTR). Using siRNAs targeting MCC or SMAD4, we showed that upregulation of MCC and SMAD4 was essential to mediate the functional roles of miR-4260 inhibitor in colorectal cancer cells. Our in vivo experiments indicated that inhibition of miR-4260 reduced colorectal tumor growth in nude mice subcutaneously implanted with HCT116 cells. Significantly, miR-4260 was increased in human colorectal cancer tissues with simultaneous downregulation of MCC and SMAD4, strongly suggesting the clinical relevance of targeting miR-4260 in the treatment of colorectal cancer. In summary, we identified miR-4260 as a novel oncomiR for colorectal cancer that targets MCC and SMAD4. Inhibition of miR-4260 can, therefore, be a potential therapeutic strategy for colorectal cancer.

Target cell cyclophilins facilitate human papillomavirus type 16 infection.

PubMed

Bienkowska-Haba, Malgorzata; Patel, Hetalkumar D; Sapp, Martin

2009-07-01

Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

The Putative Chemosignal Androstadienone Makes Women More Generous.

PubMed

Perrotta, Valentina; Graffeo, Michele; Bonini, Nicolao; Gottfried, Jay A

2016-06-01

Putative human chemosignals have been shown to influence mood states and emotional processing, but the connection between these effects and higher-order cognitive processing is not well established. This study utilized an economic game (Dictator Game) to test whether androstadienone (AND), an odorous compound derived from testosterone, impacts on altruistic behavior. We predicted that the female participants would act more generously in the AND condition, exhibiting a significant interaction effect between gender and AND on Dictator Game contributions. We also expected that the presence of AND should increase the positive mood of the female participants, compared to a control odor condition and also compared to the mood of the male participants. The results confirm our hypotheses: for women the subliminal perception of AND led to larger monetary donations, compared to a control odor, and also increased positive mood. These effects were absent or significantly weaker in men. Our findings highlight the capacity of human putative chemosignals to influence emotions and higher cognitive processes - in particular the processes used in the context of economic decisions - in a gender-specific way.

De Novo Transcriptome Assembly and Characterization of Lithospermum officinale to Discover Putative Genes Involved in Specialized Metabolites Biosynthesis.

PubMed

Rai, Amit; Nakaya, Taiki; Shimizu, Yohei; Rai, Megha; Nakamura, Michimi; Suzuki, Hideyuki; Saito, Kazuki; Yamazaki, Mami

2018-05-29

Lithospermum officinale is a valuable source of bioactive metabolites with medicinal and industrial values. However, little is known about genes involved in the biosynthesis of these metabolites, primarily due to the lack of genome or transcriptome resources. This study presents the first effort to establish and characterize de novo transcriptome assembly resource for L. officinale and expression analysis for three of its tissues, namely leaf, stem, and root. Using over 4Gbps of RNA-sequencing datasets, we obtained de novo transcriptome assembly of L. officinale , consisting of 77,047 unigenes with assembly N50 value as 1524 bps. Based on transcriptome annotation and functional classification, 52,766 unigenes were assigned with putative genes functions, gene ontology terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. KEGG pathway and gene ontology enrichment analysis using highly expressed unigenes across three tissues and targeted metabolome analysis showed active secondary metabolic processes enriched specifically in the root of L. officinale . Using co-expression analysis, we also identified 20 and 48 unigenes representing different enzymes of lithospermic/chlorogenic acid and shikonin biosynthesis pathways, respectively. We further identified 15 candidate unigenes annotated as cytochrome P450 with the highest expression in the root of L. officinale as novel genes with a role in key biochemical reactions toward shikonin biosynthesis. Thus, through this study, we not only generated a high-quality genomic resource for L. officinale but also propose candidate genes to be involved in shikonin biosynthesis pathways for further functional characterization. Georg Thieme Verlag KG Stuttgart · New York.

DBZ is a putative PPARγ agonist that prevents high fat diet-induced obesity, insulin resistance and gut dysbiosis.

PubMed

Xu, Pengfei; Hong, Fan; Wang, Jialin; Wang, Jing; Zhao, Xia; Wang, Sheng; Xue, Tingting; Xu, Jingwei; Zheng, Xiaohui; Zhai, Yonggong

2017-11-01

The nuclear receptor PPARγ is an effective pharmacological target for some types of metabolic syndrome, including obesity, diabetes, nonalcoholic fatty liver disease, and cardiovascular disease. However, the current PPARγ-targeting thiazolidinedione drugs have undesirable side effects. Danshensu Bingpian Zhi (DBZ), also known as tanshinol borneol ester derived from Salvia miltiorrhiza, is a synthetic derivative of natural compounds used in traditional Chinese medicine for its anti-inflammatory activity. In vitro, investigations of DBZ using a luciferase reporter assay and molecular docking identified this compound as a novel promising PPARγ agonist. Ten-week-old C57BL/6J mice were fed either a normal chow diet (NCD) or a high-fat diet (HFD). The HFD-fed mice were gavaged daily with either vehicle or DBZ (50mg/kg or 100mg/kg) for 10weeks. The gut microbiota composition was assessed by analyzing the 16S rRNA gene V3+V4 regions via pyrosequencing. DBZ is an efficient natural PPARγ agonist that shows lower PPARγ-responsive luciferase reporter activity than thiazolidinediones, has excellent effects on the metabolic phenotype and exhibits no unwanted adverse effects in a HFD-induced obese mouse model. DBZ protects against HFD-induced body weight gain, insulin resistance, hepatic steatosis and inflammation in mice. DBZ not only stimulates brown adipose tissue (BAT) browning and maintains intestinal barrier integrity but also reverses HFD-induced intestinal microbiota dysbiosis. DBZ is a putative PPARγ agonist that prevents HFD-induced obesity-related metabolic syndrome and reverse gut dysbiosis. DBZ may be used as a beneficial probiotic agent to improve HFD-induced obesity-related metabolic syndrome in obese individuals. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of Novel Gene Targets and Putative Regulators of Arsenic-Associated DNA Methylation in Human Urothelial Cells and Bladder Cancer

PubMed Central

Rager, Julia E.; Miller, Sloane; Tulenko, Samantha E.; Smeester, Lisa; Ray, Paul D.; Yosim, Andrew; Currier, Jenna M.; Ishida, María C.; González-Horta, Maria del Carmen; Sánchez-Ramírez, Blanca; Ballinas-Casarrubias, Lourdes; Gutiérrez-Torres, Daniela S.; Drobná, Zuzana; Del Razo, Luz M.; García-Vargas, Gonzalo G.; Kim, William Y.; Zhou, Yi-Hui; Wright, Fred A.; Stýblo, Miroslav; Fry, Rebecca C.

2016-01-01

There is strong epidemiologic evidence linking chronic exposure to inorganic arsenic (iAs) to a myriad of adverse health effects, including cancer of the bladder. The present study set out to identify DNA methylation patterns associated with iAs and its metabolites in exfoliated urothelial cells (EUCs) that originate primarily from the urinary bladder, one of the targets of arsenic (As)-induced carcinogenesis. Genome-wide, gene-specific promoter DNA methylation levels were assessed in EUCs from 46 residents of Chihuahua, Mexico, and the relationship was examined between promoter methylation profiles and the intracellular concentrations of total As (tAs) and As species. A set of 49 differentially methylated genes was identified with increased promoter methylation associated with EUC tAs, iAs, and/or monomethylated As (MMAs) enriched for their roles in metabolic disease and cancer. Notably, no genes had differential methylation associated with EUC dimethylated As (DMAs), suggesting that DMAs may influence DNA methylation-mediated urothelial cell responses to a lesser extent than iAs or MMAs. Further analysis showed that 22 of the 49 As-associated genes (45%) are also differentially methylated in bladder cancer tissue identified using The Cancer Genome Atlas repository. Both the As- and cancer-associated genes are enriched for the binding sites of common transcription factors known to play roles in carcinogenesis, demonstrating a novel potential mechanistic link between iAs exposure and bladder cancer. PMID:26039340

Identifying novel genes and chemicals related to nasopharyngeal cancer in a heterogeneous network.

PubMed

Li, Zhandong; An, Lifeng; Li, Hao; Wang, ShaoPeng; Zhou, You; Yuan, Fei; Li, Lin

2016-05-05

Nasopharyngeal cancer or nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. The factors that induce nasopharyngeal cancer are still not clear. Additional information about the chemicals or genes related to nasopharyngeal cancer will promote a better understanding of the pathogenesis of this cancer and the factors that induce it. Thus, a computational method NPC-RGCP was proposed in this study to identify the possible relevant chemicals and genes based on the presently known chemicals and genes related to nasopharyngeal cancer. To extensively utilize the functional associations between proteins and chemicals, a heterogeneous network was constructed based on interactions of proteins and chemicals. The NPC-RGCP included two stages: the searching stage and the screening stage. The former stage is for finding new possible genes and chemicals in the heterogeneous network, while the latter stage is for screening and removing false discoveries and selecting the core genes and chemicals. As a result, five putative genes, CXCR3, IRF1, CDK1, GSTP1, and CDH2, and seven putative chemicals, iron, propionic acid, dimethyl sulfoxide, isopropanol, erythrose 4-phosphate, β-D-Fructose 6-phosphate, and flavin adenine dinucleotide, were identified by NPC-RGCP. Extensive analyses provided confirmation that the putative genes and chemicals have significant associations with nasopharyngeal cancer.

Identifying novel genes and chemicals related to nasopharyngeal cancer in a heterogeneous network

PubMed Central

Li, Zhandong; An, Lifeng; Li, Hao; Wang, ShaoPeng; Zhou, You; Yuan, Fei; Li, Lin

2016-01-01

Nasopharyngeal cancer or nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. The factors that induce nasopharyngeal cancer are still not clear. Additional information about the chemicals or genes related to nasopharyngeal cancer will promote a better understanding of the pathogenesis of this cancer and the factors that induce it. Thus, a computational method NPC-RGCP was proposed in this study to identify the possible relevant chemicals and genes based on the presently known chemicals and genes related to nasopharyngeal cancer. To extensively utilize the functional associations between proteins and chemicals, a heterogeneous network was constructed based on interactions of proteins and chemicals. The NPC-RGCP included two stages: the searching stage and the screening stage. The former stage is for finding new possible genes and chemicals in the heterogeneous network, while the latter stage is for screening and removing false discoveries and selecting the core genes and chemicals. As a result, five putative genes, CXCR3, IRF1, CDK1, GSTP1, and CDH2, and seven putative chemicals, iron, propionic acid, dimethyl sulfoxide, isopropanol, erythrose 4-phosphate, β-D-Fructose 6-phosphate, and flavin adenine dinucleotide, were identified by NPC-RGCP. Extensive analyses provided confirmation that the putative genes and chemicals have significant associations with nasopharyngeal cancer. PMID:27149165

An Approach for Identification of Novel Drug Targets in Streptococcus pyogenes SF370 Through Pathway Analysis.

PubMed

Singh, Satendra; Singh, Dev Bukhsh; Singh, Anamika; Gautam, Budhayash; Ram, Gurudayal; Dwivedi, Seema; Ramteke, Pramod W

2016-12-01

Streptococcus pyogenes is one of the most important pathogens as it is involved in various infections affecting upper respiratory tract and skin. Due to the emergence of multidrug resistance and cross-resistance, S. Pyogenes is becoming more pathogenic and dangerous. In the present study, an in silico comparative analysis of total 65 metabolic pathways of the host (Homo sapiens) and the pathogen was performed. Initially, 486 paralogous enzymes were identified so that they can be removed from possible drug target list. The 105 enzymes of the biochemical pathways of S. pyogenes from the KEGG metabolic pathway database were compared with the proteins from the Homo sapiens by performing a BLASTP search against the non-redundant database restricted to the Homo sapiens subset. Out of these, 83 enzymes were identified as non-human homologous while 30 enzymes of inadequate amino acid length were removed for further processing. Essential enzymes were finally mined from remaining 53 enzymes. Finally, 28 essential enzymes were identified in S. pyogenes SF370 (serotype M1). In subcellular localization study, 18 enzymes were predicted with cytoplasmic localization and ten enzymes with the membrane localization. These ten enzymes with putative membrane localization should be of particular interest. Acyl-carrier-protein S-malonyltransferase, DNA polymerase III subunit beta and dihydropteroate synthase are novel drug targets and thus can be used to design potential inhibitors against S. pyogenes infection. 3D structure of dihydropteroate synthase was modeled and validated that can be used for virtual screening and interaction study of potential inhibitors with the target enzyme.

Targeted next generation sequencing of mucosal melanomas identifies frequent NF1 and RAS mutations.

PubMed

Cosgarea, Ioana; Ugurel, Selma; Sucker, Antje; Livingstone, Elisabeth; Zimmer, Lisa; Ziemer, Mirjana; Utikal, Jochen; Mohr, Peter; Pfeiffer, Christiane; Pföhler, Claudia; Hillen, Uwe; Horn, Susanne; Schadendorf, Dirk; Griewank, Klaus G; Roesch, Alexander

2017-06-20

Mucosal melanoma represents ~1% of all melanomas, frequently having a poor prognosis due to diagnosis at a late stage of disease. Mucosal melanoma differs from cutaneous melanoma not only in terms of poorer clinical outcome but also on the molecular level having e.g. less BRAF and more frequent KIT mutations than cutaneous melanomas. For the majority of mucosal melanomas oncogenic driver mutations remain unknown. In our study, 75 tumor tissues from patients diagnosed with mucosal melanoma were analyzed, applying a targeted next generation sequencing panel covering 29 known recurrently mutated genes in melanoma. NF1 and RAS mutations were identified as the most frequently mutated genes occurring in 18.3% and 16.9% of samples, respectively. Mutations in BRAF were identified in 8.4% and KIT in 7.0% of tumor samples. Our study identifies NF1 as the most frequently occurring driver mutation in mucosal melanoma. RAS alterations, consisting of NRAS and KRAS mutations, were the second most frequent mutation type. BRAF and KIT mutations were rare with frequencies below 10% each. Our data indicate that in mucosal melanomas RAS/NF1 alterations are frequent, implying a significant pathogenetic role for MAPK and potentially PI3K pathway activation in these tumors.

Targeted next generation sequencing of mucosal melanomas identifies frequent NF1 and RAS mutations

PubMed Central

Cosgarea, Ioana; Ugurel, Selma; Sucker, Antje; Livingstone, Elisabeth; Zimmer, Lisa; Ziemer, Mirjana; Utikal, Jochen; Mohr, Peter; Pfeiffer, Christiane; Pföhler, Claudia; Hillen, Uwe; Horn, Susanne; Schadendorf, Dirk

2017-01-01

Purpose Mucosal melanoma represents ~1% of all melanomas, frequently having a poor prognosis due to diagnosis at a late stage of disease. Mucosal melanoma differs from cutaneous melanoma not only in terms of poorer clinical outcome but also on the molecular level having e.g. less BRAF and more frequent KIT mutations than cutaneous melanomas. For the majority of mucosal melanomas oncogenic driver mutations remain unknown. Experimental Design and Results In our study, 75 tumor tissues from patients diagnosed with mucosal melanoma were analyzed, applying a targeted next generation sequencing panel covering 29 known recurrently mutated genes in melanoma. NF1 and RAS mutations were identified as the most frequently mutated genes occurring in 18.3% and 16.9% of samples, respectively. Mutations in BRAF were identified in 8.4% and KIT in 7.0% of tumor samples. Conclusions Our study identifies NF1 as the most frequently occurring driver mutation in mucosal melanoma. RAS alterations, consisting of NRAS and KRAS mutations, were the second most frequent mutation type. BRAF and KIT mutations were rare with frequencies below 10% each. Our data indicate that in mucosal melanomas RAS/NF1 alterations are frequent, implying a significant pathogenetic role for MAPK and potentially PI3K pathway activation in these tumors. PMID:28380455

Targeted Metabolomics Identifies Pharmacodynamic Biomarkers for BIO 300 Mitigation of Radiation-Induced Lung Injury.

PubMed

Jones, Jace W; Jackson, Isabel L; Vujaskovic, Zeljko; Kaytor, Michael D; Kane, Maureen A

2017-12-01

Biomarkers serve a number of purposes during drug development including defining the natural history of injury/disease, serving as a secondary endpoint or trigger for intervention, and/or aiding in the selection of an effective dose in humans. BIO 300 is a patent-protected pharmaceutical formulation of nanoparticles of synthetic genistein being developed by Humanetics Corporation. The primary goal of this metabolomic discovery experiment was to identify biomarkers that correlate with radiation-induced lung injury and BIO 300 efficacy for mitigating tissue damage based upon the primary endpoint of survival. High-throughput targeted metabolomics of lung tissue from male C57L/J mice exposed to 12.5 Gy whole thorax lung irradiation, treated daily with 400 mg/kg BIO 300 for either 2 weeks or 6 weeks starting 24 h post radiation exposure, were assayed at 180 d post-radiation to identify potential biomarkers. A panel of lung metabolites that are responsive to radiation and able to distinguish an efficacious treatment schedule of BIO 300 from a non-efficacious treatment schedule in terms of 180 d survival were identified. These metabolites represent potential biomarkers that could be further validated for use in drug development of BIO 300 and in the translation of dose from animal to human.

Search strings for the study of putative occupational determinants of disease

PubMed Central

Mattioli, Stefano; Zanardi, Francesca; Baldasseroni, Alberto; Schaafsma, Frederieke; Cooke, Robin MT; Mancini, Gianpiero; Fierro, Mauro; Santangelo, Chiara; Farioli, Andrea; Fucksia, Serenella; Curti, Stefania; Verbeek, Jos

2010-01-01

Objective To identify efficient PubMed search strategies to retrieve articles regarding putative occupational determinants of conditions not generally considered to be work related. Methods Based on MeSH definitions and expert knowledge, we selected as candidate search terms the four MeSH terms describing ‘occupational disease’, ‘occupational exposure’, ‘occupational health’ and ‘occupational medicine’ (DEHM) alongside 22 other promising terms. We first explored overlaps between the candidate terms in PubMed. Using random samples of abstracts retrieved by each term, we estimated the proportions of articles containing potentially pertinent information regarding occupational aetiology in order to formulate two search strategies (one more ‘specific’, one more ‘sensitive’). We applied these strategies to retrieve information on the possible occupational aetiology of meningioma, pancreatitis and atrial fibrillation. Results Only 20.3% of abstracts were retrieved by more than one DEHM term. The more ‘specific’ search string was based on the combination of terms that yielded the highest proportion (40%) of potentially pertinent abstracts. The more ‘sensitive’ string was based on the use of broader search fields and additional coverage provided by other search terms under study. Using the specific string, the numbers of abstracts needed to read to find one potentially pertinent article were 1.2 for meningioma, 1.9 for pancreatitis and 1.8 for atrial fibrillation. Using the sensitive strategy, the numbers needed to read were 4.4 for meningioma, 8.9 for pancreatitis and 10.5 for atrial fibrillation. Conclusions The proposed strings could help health care professionals explore putative occupational aetiology for diseases that are not generally thought to be work related. PMID:19819858

Actin cytoskeleton as a putative target of the neem limonoid Azadirachtin A.

PubMed

Anuradha, Aritakula; Annadurai, Ramaswamy S; Shashidhara, L S

2007-06-01

Limonoids isolated from the Indian neem tree (Azadirachta indica) have been gaining global acceptance in agricultural applications and in contemporary medicine for their myriad but discrete properties. However, their mode of action is still not very well understood. We have studied the mode of action of Azadirachtin A, the major limonoid of neem seed extracts, using Drosophila melanogaster as the model system. Azadirachtin A induces moderate-to-severe phenotypes in different tissues in a dose-dependent manner. At the cellular level, Azadirachtin A induces depolymerization of Actin leading to arrest of cells and subsequently apoptosis in a caspase-independent manner. Azadirachtin A-induced phenotypes were rescued by the over-expression of Cyclin E in a tissue-dependent manner. Cyclin E, which caused global rescue of Azadirachtin A-induced phenotypes, also effected rearrangement of the actin filaments. These results suggest that probably actin is a target of Azadirachtin A activity.

SLC6A19 is a novel putative gene, induced by dioxins via AhR in human hepatoma HepG2 cells.

PubMed

Tian, Wenjing; Fu, Hualing; Xu, Tuan; Xu, Sherry Li; Guo, Zhiling; Tian, Jijing; Tao, Wuqun; Xie, Heidi Qunhui; Zhao, Bin

2018-06-01

The aryl hydrocarbon receptor (AhR) plays an important role in mediating dioxins toxicity. Currently, genes of P450 families are major research interests in studies on AhR-mediated gene alterations caused by dioxins. Genes related to other metabolic pathways or processes may be also responsive to dioxin exposures. Amino acid transporter B0AT1 (encoded by SLC6A19) plays a decisive role in neutral amino acid transport which is present in kidney, intestine and liver. However, effects of dioxins on its expression are still unknown. In the present study, we focused on the effects of dioxin and dioxin-like compounds on SLC6A19 expression in HepG2 cells. We identified SLC6A19 as a novel putative target gene of AhR activation in HepG2 cells. 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) increased the expression of SLC6A19 in time- and concentration-dependent manners. Using AhR antagonist CH223191 and/or siRNA assays, we demonstrated that certain AhR agonists upregulated SLC6A19 expression via AhR, including TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (1,2,3,7,8-PeCDD), 2,3,4,7,8- pentachlorodibenzofuran (2,3,4,7,8-PeCDF) and PCB126. In addition, the expression of B0AT1 was also significantly induced by TCDD in HepG2 cells. Our study suggested that dioxins might affect the transcription and translation of SLC6A19 in HepG2 cells, which might be a novel putative gene to assess dioxins' toxicity in amino acid transport and metabolism in liver. Copyright © 2018 Elsevier Ltd. All rights reserved.

Trypanosome RNA polymerases and transcription factors: sensible trypanocidal drug targets?

PubMed

Vanhamme, Luc

2008-11-01

Trypanosomes and Leishmaniae are the agents of several important parasitic diseases threatening hundreds of million human beings worldwide. As they diverged early in evolution, they display original molecular characteristics. These peculiarities are each defining putative specific targets for anti-parasitic drugs. Transcription displays its lot of unique characteristics in trypanosomes and will be taken as an example to uncover these targets. Unique features of transcription in trypanosomes include constitutive and poly-cistronic transcription by RNA polymerase II as well as transcription of protein-coding genes by RNA polymerase I. It is becoming clear that these unique mechanisms are performed by dedicated molecular players. The first of them have been recently characterized. They are reviewed and their suitability as drug targets is commented.

MIR-27a regulates the TGF-β signaling pathway by targeting SMAD2 and SMAD4 in lung cancer.

PubMed

Chae, Dong-Kyu; Ban, Eunmi; Yoo, Young Sook; Kim, Eunice EunKyeong; Baik, Ja-Hyun; Song, Eun Joo

2017-08-01

The transforming growth factor-β (TGF-β) signaling pathway is associated with carcinogenesis and various biological processes. SMAD2 and SMAD4, which are putative tumor suppressors, have an important role in TGF-β signaling. The aberrant expression of these genes is implicated in some cancers. However, the mechanisms of SMAD2 and SMAD4 dysregulation are poorly understood. In this study, we observed that miR-27a was upregulated in lung cancer cell lines and patients. In addition, SMAD2 and SMAD4 genes were identified as targets of miR-27a by several target prediction databases and experimental validation. Functional studies revealed that miR-27a overexpression decreased SMAD2 and SMAD4 mRNA and protein levels. Furthermore, miR-27a contributed to cell proliferation and invasion by inhibiting TGF-β-induced cell cycle arrest. These results suggest that miR-27a may function as an oncogene by regulating SMAD2 and SMAD4 in lung cancer. Thus, miR-27a may be a potential target for cancer therapy. © 2017 Wiley Periodicals, Inc.

Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread.

PubMed

Kroj, Thomas; Chanclud, Emilie; Michel-Romiti, Corinne; Grand, Xavier; Morel, Jean-Benoit

2016-04-01

Plant immune receptors of the class of nucleotide-binding and leucine-rich repeat domain (NLR) proteins can contain additional domains besides canonical NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)) and leucine-rich repeat (LRR) domains. Recent research suggests that these additional domains act as integrated decoys recognizing effectors from pathogens. Proteins homologous to integrated decoys are suspected to be effector targets and involved in disease or resistance. Here, we scrutinized 31 entire plant genomes to identify putative integrated decoy domains in NLR proteins using the Interpro search. The involvement of the Zinc Finger-BED type (ZBED) protein containing a putative decoy domain, called BED, in rice (Oryza sativa) resistance was investigated by evaluating susceptibility to the blast fungus Magnaporthe oryzae in rice over-expression and knock-out mutants. This analysis showed that all plants tested had integrated various atypical protein domains into their NLR proteins (on average 3.5% of all NLR proteins). We also demonstrated that modifying the expression of the ZBED gene modified disease susceptibility. This study suggests that integration of decoy domains in NLR immune receptors is widespread and frequent in plants. The integrated decoy model is therefore a powerful concept to identify new proteins involved in disease resistance. Further in-depth examination of additional domains in NLR proteins promises to unravel many new proteins of the plant immune system. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Genomic characterization of putative allergen genes in peach/almond and their synteny with apple

PubMed Central

Chen, Lin; Zhang, Shuiming; Illa, Eudald; Song, Lijuan; Wu, Shandong; Howad, Werner; Arús, Pere; Weg, Eric van de; Chen, Kunsong; Gao, Zhongshan

2008-01-01

Background Fruits from several species of the Rosaceae family are reported to cause allergic reactions in certain populations. The allergens identified belong to mainly four protein families: pathogenesis related 10 proteins, thaumatin-like proteins, lipid transfer proteins and profilins. These families of putative allergen genes in apple (Mal d 1 to 4) have been mapped on linkage maps and subsequent genetic study on allelic diversity and hypoallergenic traits has been carried out recently. In peach (Prunus persica), these allergen gene families are denoted as Pru p 1 to 4 and for almond (Prunus dulcis)Pru du 1 to 4. Genetic analysis using current molecular tools may be helpful to establish the cause of allergenicity differences observed among different peach cultivars. This study was to characterize putative peach allergen genes for their genomic sequences and linkage map positions, and to compare them with previously characterized homologous genes in apple (Malus domestica). Results Eight Pru p/du 1 genes were identified, four of which were new. All the Pru p/du 1 genes were mapped in a single bin on the top of linkage group 1 (G1). Five Pru p/du 2 genes were mapped on four different linkage groups, two very similar Pru p/du 2.01 genes (A and B) were on G3, Pru p/du 2.02 on G7,Pru p/du 2.03 on G8 and Pru p/du 2.04 on G1. There were differences in the intron and exon structure in these Pru p/du 2 genes and in their amino acid composition. Three Pru p/du 3 genes (3.01–3.03) containing an intron and a mini exon of 10 nt were mapped in a cluster on G6. Two Pru p/du 4 genes (Pru p/du 4.01 and 4.02) were located on G1 and G7, respectively. The Pru p/du 1 cluster on G1 aligned to the Mal d 1 clusters on LG16; Pru p/du 2.01A and B on G3 to Mal d 2.01A and B on LG9; the Pru p/du 3 cluster on G6 to Mal d 3.01 on LG12; Pru p/du 4.01 on G1 to Mal d 4.03 on LG2; and Pru p/du 4.02 on G7 to Mal d 4.02 on LG2. Conclusion A total of 18 putative peach/almond allergen genes have

Sulfur Isotope Composition of Putative Primary Troilite in Chondrules

NASA Technical Reports Server (NTRS)

Tachibana, Shogo; Huss, Gary R.

2002-01-01

Sulfur isotope compositions of putative primary troilites in chondrules from Bishunpur were measured by ion probe. These primary troilites have the same S isotope compositions as matrix troilites and thus appear to be isotopically unfractionated. Additional information is contained in the original extended abstract.

Identification of Putative Chemosensory Receptor Genes from the Athetis dissimilis Antennal Transcriptome

PubMed Central

Dong, Junfeng; Song, Yueqin; Li, Wenliang; Shi, Jie; Wang, Zhenying

2016-01-01

Olfaction plays a crucial role in insect population survival and reproduction. Identification of the genes associated with the olfactory system, without the doubt will promote studying the insect chemical communication system. In this study, RNA-seq technology was used to sequence the antennae transcriptome of Athetis dissimilis, an emerging crop pest in China with limited genomic information, with the purpose of identifying the gene set involved in olfactory recognition. Analysis of the transcriptome of female and male antennae generated 13.74 Gb clean reads in total from which 98,001 unigenes were assembled, and 25,930 unigenes were annotated. Total of 60 olfactory receptors (ORs), 18 gustatory receptors (GRs), and 12 ionotropic receptors (IRs) were identified by Blast and sequence similarity analyzes. One obligated olfactory receptor co-receptor (Orco) and four conserved sex pheromone receptors (PRs) were annotated in 60 ORs. Among the putative GRs, five genes (AdisGR1, 6, 7, 8 and 94) clustered in the sugar receptor family, and two genes (AdisGR3 and 93) involved in CO2 detection were identified. Finally, AdisIR8a.1 and AdisIR8a.2 co-receptors were identified in the group of candidate IRs. Furthermore, expression levels of these chemosensory receptor genes in female and male antennae were analyzed by mapping the Illumina reads. PMID:26812239

The putative endocannabinoid transport blocker LY2183240 is a potent inhibitor of FAAH and several other brain serine hydrolases.

PubMed

Alexander, Jessica P; Cravatt, Benjamin F

2006-08-02

How lipid transmitters move within and between cells to communicate signals remains an important and largely unanswered question. Integral membrane transporters, soluble lipid-binding proteins, and metabolic enzymes have all been proposed to collaboratively regulate lipid signaling dynamics in vivo. Assignment of the relative contributions made by each of these classes of proteins requires selective pharmacological agents to perturb their individual functions. Recently, LY2183240, a heterocyclic urea inhibitor of the putative endocannabinoid (EC) transporter, was shown to disrupt the cellular uptake of the lipid EC anandamide and promote analgesia in vivo. Here, we show that LY2183240 is a potent, covalent inhibitor of the EC-degrading enzyme fatty acid amide hydrolase (FAAH). LY2183240 inactivates FAAH by carbamylation of the enzyme's serine nucleophile. More global screens using activity-based proteomic probes identified several additional serine hydrolases that are also inhibited by LY2183240. These results indicate that the blockade of anandamide uptake observed with LY2183240 may be due primarily to the inactivation of FAAH, providing further evidence that this enzyme serves as a metabolic driving force that promotes the diffusion of anandamide into cells. More generally, the proteome-wide target promiscuity of LY2183240 designates the heterocyclic urea as a chemotype with potentially excessive protein reactivity for drug design.

Integration analysis of quantitative proteomics and transcriptomics data identifies potential targets of frizzled-8 protein-related antiproliferative factor in vivo.

PubMed

Yang, Wei; Kim, Yongsoo; Kim, Taek-Kyun; Keay, Susan K; Kim, Kwang Pyo; Steen, Hanno; Freeman, Michael R; Hwang, Daehee; Kim, Jayoung

2012-12-01

What's known on the subject? and What does the study add? Interstitial cystitis (IC) is a prevalent and debilitating pelvic disorder generally accompanied by chronic pain combined with chronic urinating problems. Over one million Americans are affected, especially middle-aged women. However, its aetiology or mechanism remains unclear. No efficient drug has been provided to patients. Several urinary biomarker candidates have been identified for IC; among the most promising is antiproliferative factor (APF), whose biological activity is detectable in urine specimens from >94% of patients with both ulcerative and non-ulcerative IC. The present study identified several important mediators of the effect of APF on bladder cell physiology, suggesting several candidate drug targets against IC. In an attempt to identify potential proteins and genes regulated by APF in vivo, and to possibly expand the APF-regulated network identified by stable isotope labelling by amino acids in cell culture (SILAC), we performed an integration analysis of our own SILAC data and the microarray data of Gamper et al. (2009) BMC Genomics 10: 199. Notably, two of the proteins (i.e. MAPKSP1 and GSPT1) that are down-regulated by APF are involved in the activation of mTORC1, suggesting that the mammalian target of rapamycin (mTOR) pathway is potentially a critical pathway regulated by APF in vivo. Several components of the mTOR pathway are currently being studied as potential therapeutic targets in other diseases. Our analysis suggests that this pathway might also be relevant in the design of diagnostic tools and medications targeting IC. • To enhance our understanding of the interstitial cystitis urine biomarker antiproliferative factor (APF), as well as interstitial cystitis biology more generally at the systems level, we reanalyzed recently published large-scale quantitative proteomics and in vivo transcriptomics data sets using an integration analysis tool that we have developed. • To