Identification of putative transcriptomic biomarkers in irritable bowel syndrome (IBS): Differential gene expression and regulation of TPH1 and SERT by vitamin D.

Irritable bowel syndrome (IBS) is one of the most common gastrointestinal disorders and affects approximately 4% of the global population. The diagnosis of IBS can be made based on symptoms using the validated Rome criteria and ruling out commonly occurring organic diseases. Although biomarkers exist for "IBS mimickers" such as celiac disease and inflammatory bowel disease (IBD), no such test exists for IBS. DNA microarrays of colonic tissue have been used to identify disease-associated variants in other gastrointestinal (GI) disorders. In this study, our objective was to identify biomarkers and unique gene expression patterns that may define the pathological state of IBS. Mucosal tissue samples were collected from the sigmoid colon of 29 participants (11 IBS and 18 healthy controls). DNA microarray analysis was used to assess gene expression profiling. Extraction and purification of RNA were then performed and used to synthesize cDNA. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was employed to identify differentially expressed genes in patients diagnosed with IBS compared to healthy, non-IBS patient-derived cDNA. Additional testing probed vitamin D-mediated regulation of select genes associated with serotonergic metabolism. DNA microarray analyses led to the identification of 858 differentially expressed genes that may characterize the IBS pathological state. After screening a series of genes using a combination of gene ontological analysis and RT-qPCR, this spectrum of potential IBS biomarkers was narrowed to 23 genes, some of which are regulated by vitamin D. Seven putative IBS biomarkers, including genes involved in serotonin metabolism, were identified. This work further supports the hypothesis that IBS pathophysiology is evident within the human transcriptome and that vitamin D modulates differential expression of genes in IBS patients. This suggests that IBS pathophysiology may also involve vitamin D deficiency and/or an irregularity in serotonin metabolism.

Senescence and oxidative stress toxicities induced by lamivudine and tenofovir in Drosophila melanogaster.

BACKGROUND: Lamivudine and tenofovir disoproxil fumarate act against the replication of hepatitis B and human immunodeficiency viruses via inhibition of the reverse transcriptase enzyme activity, thereby preventing the synthesis of viral DNA. Chronic administration of these drugs has been associated with toxicities, including senescence, oxidative stress and premature death. A study of these toxicities in Drosophila melanogaster, which share 75% genomic similarity with humans could help to develop a pharmacologic intervention. METHODS: Susceptibility of D. melanogaster for lamivudine and tenofovir-induced toxicities were investigated. First, flies (≤3 days old) were fed with drugs-supplemented diet at varying concentrations (1mg to 300mg/10-gram diet) or distilled water for seven days to determine LD50. Secondly, five groups of 60 flies were fed with four concentrations of test drugs: 2.9mg, 5.82mg, 11.64mg and 23.28mg each per 10-gram diet for 28 days survival and lifespan assays. Then 5-day treatment plan was utilized to determine drugs toxicities on climbing ability and some biomarkers of oxidative stress. Finally, molecular docking was carried out using the Auto-dock vina mode to predict the biological interactions between the test drugs and D. melanogaster acetylcholinesterase (AChE) or glutathione-S-transferase (GST). RESULTS: The LD50 of lamivudine or tenofovir was 47.07 or 43.95mg/10g diet, respectively. Each drug significantly (P<0.05) reduced the survival rate, longevity and climbing performance of the flies dose-dependently. These drugs also altered levels of biochemical parameters: AChE, GST, superoxide dismutase (SOD), catalase (CAT), total thiol (T-SH), and malondialdehyde (MDA) of the flies significantly (P<0.05). In silico molecular analysis showed that the test drugs interacted with significantly (P<0.05) higher binding affinities at the same catalytic sites of D. melanogaster GST and AChE compared with substrates (glutathione or acetylcholine). CONCLUSION: The significant lamivudine and tenofovir-induced toxicities observed as increased mortality, climbing deficits and compromised antioxidant defence in D. melanogaster demands further research for possible pharmacological intervention.

Determinants of adenine-mutagenesis in diversity-generating retroelements.

Diversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial 'dark matter'. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis.

Biochemical characterization of the Lassa virus L protein.

The L protein of arena- and bunyaviruses is structurally and functionally related to the orthomyxovirus polymerase complex. It plays a central role in the viral life cycle, as it replicates the virus genome and generates viral mRNA via a cap-snatching mechanism. Here, we aimed to biochemically characterize the L protein of Lassa virus, a human-pathogenic arenavirus endemic in West Africa. Full-length 250-kDa L protein was expressed using a baculovirus expression system. A low-resolution structure calculated from small-angle X-ray scattering data revealed a conformation similar to that in the crystal structure of the orthomyxovirus polymerase complex. Although the L protein did not exhibit cap-snatching endonuclease activity, it synthesized RNA in vitro RNA polymerization required manganese rather than magnesium ions, was independent of nucleotide primers, and was inhibited by viral Z protein. Maximum activity was mediated by double-stranded promoter sequences with a minimum length of 17 nucleotides, containing a nontemplated 5'-G overhang, as in the natural genome context, as well as the naturally occurring base mismatches between the complementary promoter strands. Experiments with various short primers revealed the presence of two replication initiation sites at the template strand and evidence for primer translocation as proposed by the prime-and-realign hypothesis. Overall, our findings provide the foundation for a detailed understanding of the mechanistic differences and communalities in the polymerase proteins of segmented negative-strand RNA viruses and for the search for antiviral compounds targeting the RNA polymerase of Lassa virus.

Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex.

Diversity-generating retroelements (DGRs) create unparalleled levels of protein sequence variation through mutagenic retrohoming. Sequence information is transferred from an invariant template region (TR), through an RNA intermediate, to a protein-coding variable region. Selective infidelity at adenines during transfer is a hallmark of DGRs from disparate bacteria, archaea, and microbial viruses. We recapitulated selective infidelity in vitro for the prototypical Bordetella bacteriophage DGR. A complex of the DGR reverse transcriptase bRT and pentameric accessory variability determinant (Avd) protein along with DGR RNA were necessary and sufficient for synthesis of template-primed, covalently linked RNA-cDNA molecules, as observed in vivo. We identified RNA-cDNA molecules to be branched and most plausibly linked through 2'-5' phosphodiester bonds. Adenine-mutagenesis was intrinsic to the bRT-Avd complex, which displayed unprecedented promiscuity while reverse transcribing adenines of either DGR or non-DGR RNA templates. In contrast, bRT-Avd processivity was strictly dependent on the template, occurring only for the DGR RNA. This restriction was mainly due to a noncoding segment downstream of TR, which specifically bound Avd and created a privileged site for processive polymerization. Restriction to DGR RNA may protect the host genome from damage. These results define the early steps in a novel pathway for massive sequence diversification.

Uvaria angolensis as a promising source of inhibitors of HIV-1 RT-associated RNA-dependent DNA polymerase and RNase H functions.

Reverse transcriptase (RT)-associated DNA polymerase (RDDP) and ribonucleaser H (RNase H) functions are both essential for HIV-1 genome replication, and the identification of new inhibitors to block both of them is a goal actively pursued by the scientific community. In this field, natural extracts have shown a great potential as source of new antivirals. In the present work, we investigated the effect of Uvaria angolensis extracts on the HIV-1 reverse transcriptase-associated DNA polymerase and ribonuclease H activities. The U. angolensis stem bark methanol extract inhibit both HIV-1 RNase H function and RDDP activity with IC50 values of 1.0 ± 0.2 and 0.62 ± 0.15 µg/mL, respectively and, after been fractionated with different solvents, its solid residue showed an IC50 of 0.10 ± 0.03 and of 0.23 ± 0.04 µg/mL against RNase H and RDDP, respectively, hence laying the bases for further studies for identification of single active components.

Increased transcriptome sequencing efficiency with modified Mint-2 digestion-ligation protocol.

The standard digestion-ligation cloning method enables synthesis of large amounts of complementary DNA (cDNA) from a model organism facilitating study of the transcriptome. Here, we used cDNA amplification of the dimorphic yeast Taphrina betulina as an example of how a library construction protocol can significantly increase sequencing throughput. Two modification steps were introduced to the Evrogen standard Mint-2 protocol to improve its suitability for next-generation sequencing projects. We performed two partial Illumina MiSeq sequencing runs with the modified protocol: one with and one without biotin-purified primers. The results demonstrated that biotinylated libraries increased both accuracy and throughput of the modified protocol. Moreover, our sequencing results indicate that a sequence-specific miscall may affect the output of Illumina's MiSeq platform.

Investigation into drug-resistant mutations of HBV from 845 nucleoside/nucleotide analogue-naive Chinese patients with chronic HBV infection.

BACKGROUND: This study aimed to clarify the clinical significance of drug-resistant HBV in nucleoside/nucleotide analogue (NA)-naive Chinese patients with chronic HBV infection in real clinical practice. METHODS: A total of 845 NA-naive patients who were admitted to Beijing 302 Hospital between July 2007 and March 2012 were included in the study. HBV drug-resistant mutations were examined by direct sequencing of the viral reverse transcriptase gene and verified by clonal sequencing. Phenotypic analysis of viral replication capacity and drug susceptibility were performed by measuring viral replicative intermediate level in 1.1-mer mutant or wild-type HBV amplicon-transfected HepG2 cells in absence or presence of serially diluted drugs. RESULTS: Drug-resistant mutations were detected in 2.01% (17/845) of the patients by direct sequencing, including 15 with lamivudine-resistant mutations (rtM204V, rtM204I), one with adefovir-resistant mutation (rtA181V), and one with both lamivudine- and adefovir-resistant mutations (rtA181V, rtM204I). Clonal sequencing identified 13 drug-resistant HBV strains: rtL80I+M204I, rtL80I+M204V, rtL180M+M204I, rtL180M+M204V, rtM204I, rtM204V, rtL80I+L180M+M204I, rtL80I+L180M+M204V, rtA181V, rtA181V+M204I, rtA181T+N236T, rtA181V+N236T and rtN236T. Phenotypic analysis showed that two pre-existing lamivudine-resistant strains (rtL80I+M204I, rtL180M+M204V) had >1,000-fold resistance to lamivudine, and one pre-existing adefovir-resistant strain (rtA181V+N236T) had 15.4-fold resistance to adefovir compared with the wild-type strain. A follow-up study showed that the presence of pre-existing rtM204I strain in one patient increased from 20% at baseline to 85% after 13 months of entecavir treatment with corresponding recession of wild-type strain in the viral pool. CONCLUSIONS: The incidence of drug-resistant HBV mutations was low in NA-naive Chinese HBV-infected patients. Pre-existing mutants had similar resistance characteristics to those from NA refractory patients.

Medicinal attributes of Solanum xanthocarpum fruit consumed by several tribal communities as food: an in vitro antioxidant, anticancer and anti HIV perspective.

BACKGROUND: Solanum xanthocarpum (Solanaceae) has been used for treatment of many infectious and degenerative diseases in traditional medicine. Present study reports the medicinal efficacy of S. xanthocarpum fruit as antioxidant, anticancer and anti HIV agents. METHODS: Extracts were prepared using Soxhlet apparatus and partially characterized by thin layer chromatography (TLC). Total flavonoid content was determined spectrophotometrically. Reducing power, DPPH radical scavenging activity and lipid peroxidation inhibition assays were used for measurement of antioxidant potential. Cytotoxic (SRB assay) and anti-HIV RT inhibition (RT assay kit, Roche) activities were determined using ELISA. RESULTS: TLC revealed the diversity of phytoconstituents in various sequential extracts of S. xanthocarpum fruit. Total flavonoid contents in extracts ranged between 10.22-162.49 µg quercetin equivalent/mg. Spectroscopic scanning of water soluble phenolics showed maximum absorbance at 250 and 280 nm. Polar extracts displayed potent radical scavenging activity (>80%). Several sub-fractions (spots) of extracts separated on TLC plates also exhibited powerful radical scavenging activity. Considerable reducing power was observed in extracts. Hexane fraction provided 55% lipoprotection in rat kidney homogenate. Non-polar extracts exhibited appreciable cytotoxic activity (70-91%) against leukemia (THP-1) and lung cancer (HOP-62) cell lines. Lower inhibitory activity was observed in extracts against HIV Reverse Transcriptase enzyme. CONCLUSION: The study demonstrated considerable antioxidant and anticancer activities in S. xanthocarpum fruit.

Identification of a novel sulfonamide non-nucleoside reverse transcriptase inhibitor by a phenotypic HIV-1 full replication assay.

Classical target-based, high-throughput screening has been useful for the identification of inhibitors for known molecular mechanisms involved in the HIV life cycle. In this study, the development of a cell-based assay that uses a phenotypic drug discovery approach based on automated high-content screening is described. Using this screening approach, the antiviral activity of 26,500 small molecules from a relevant chemical scaffold library was evaluated. Among the selected hits, one sulfonamide compound showed strong anti-HIV activity against wild-type and clinically relevant multidrug resistant HIV strains. The biochemical inhibition, point resistance mutations and the activity of structural analogs allowed us to understand the mode of action and propose a binding model for this compound with HIV-1 reverse transcriptase.

H2O2 signals via iron induction of VL30 retrotransposition correlated with cytotoxicity.

The impact of oxidative stress on mobilization of endogenous retroviruses and their effects on cell fate is unknown. We investigated the action of H2O2 on retrotransposition of an EGFP-tagged mouse LTR-retrotransposon, VL30, in an NIH3T3 cell-retrotransposition assay. H2O2 treatment of assay cells caused specific retrotranspositions documented by UV microscopy and PCR analysis. Flow cytometric analysis revealed an unusually high dose- and time-dependent retrotransposition frequency induced, ∼420,000-fold at 40 µM H2O2 compared to the natural frequency, which was reduced by ectopic expression of catalase. Remarkably, H2O2 moderately induced the RNA expression of retrotransposon B2 without affecting the basal expression of VL30s and L1 and significantly induced the expression of various endogenous reverse transcriptase genes. Further, whereas treatment with 50 µM FeCl2 alone was ineffective, cotreatment with 10 µM H2O2 and 50 µM FeCl2 caused a 6-fold higher retrotransposition induction than H2O2 alone, which was associated with cytotoxicity. H2O2- or H2O2/FeCl2-induced retrotransposition was significantly reduced by the iron chelator DFO or the antioxidant NAC, respectively. Furthermore, both H2O2-induced retrotransposition and associated cytotoxicity were inhibited after pretreatment of cells with DFO or the reverse transcriptase inhibitors efavirenz and etravirine. Our data show for the first time that H2O2, acting via iron, is a potent stimulus of retrotransposition contributing to oxidative stress-induced cell damage.

Inhibition of HIV-1 reverse transcriptase associated activities by the hydroalcoholic extract of Casimiroa edulis seeds.

The hydroalcoholic extract obtained from the seeds of Casimiroa edulis cultivated in Sardinia (Italy) have been assayed on the two enzymatic-associated activities of the HIV-1 reverse transcriptase (RT), the RNA-dependent DNA polymerase (RDDP) and the ribonuclease H. In biochemical assays, the extract inhibited both activities in a dose-dependent manner, showing a 10-fold more potent inhibition of the HIV-1 RT RDDP activity. Furthermore, the extract was cytotoxic on K562 cell replication.

Inhibition of HIV-1 and M-MLV reverse transcriptases by a major polyphenol (3,4,5 tri-O-galloylquinic acid) present in the leaves of the South African resurrection plant, Myrothamnus flabellifolia.

A polyphenol-rich extract of the medicinal resurrection plant Myrothamnus flabellifolia was shown to inhibit viral (M-MLV and HIV-1) reverse transcriptases. Fractionation and purification of this extract yielded the major polyphenol, 3,4,5 tri-O-galloylquinic acid, as the main active compound. A sensitive, ethidium bromide based fluorescent assay, was developed and used to monitor the kinetics of M-MLV and HIV-1 reverse transcriptases in the presence and absence of 3,4,5 tri-O-galloylquinic acid. Kinetic monitoring of these enzymes in the presence of 3,4,5 tri-O-galloylquinic acid revealed non-competitive inhibition with IC(50) values of 5 µM and 34 µM for the M-MLV and HIV-1 enzymes, respectively. We propose that 3,4,5 tri-O-galloylquinic acid and related polymers have potential as indigenous drugs for anti-viral therapy.

Anti-HIV-1 and anti-inflammatory lupanes from the leaves, twigs, and resin of Garcinia hanburyi.

Two new lupanes, 2 alpha-acetoxy-3 beta-hydroxy-19 beta-hydrogen-lup-20(29)-en-28-oic acid (2-acetoxyalphitolic acid) ( 1) and 2 alpha-hydroxy-3 beta-acetoxy-19 beta-hydrogen-lup-20(29)-en-28-oic acid (3-acetoxyalphitolic acid) ( 2), together with the known betulinic acid ( 3), betulin ( 4), and stimasterol-3- O- beta- D-glucopyranoside ( 5), were isolated from the leaves and twigs of GARCINIA HANBURYI. Compounds 1- 3 were also isolated from the resin of this plant. The structure of 2 was confirmed by single-crystal X-ray diffraction analysis. All of the lupanes ( 1- 4) displayed anti-HIV-1 activities in the anti-HIV-1 reverse transcriptase (IC (50) values 16.3-116.9 microg/mL) and syncytium assays (EC (50) 5.6-73.6 microg/mL, SI 1.7-3.3). Moreover compounds 1- 4 exhibited anti-inflammatory activity in an ethyl phenylpropiolate (EPP)-induced ear edema model.

Comparative in vitro effects of AZT and extracts of Ocimum gratissimum, Ficus polita, Clausena anisata, Alchornea cordifolia, and Elaeophorbia drupifera against HIV-1 and HIV-2 infections.

The effects of Ocimum gratissimum (GHX-2), Ficus polita (GHX-6), Clausena anisata (GHX-7), Alchornea cordifolia (GHX-26), Elaeophorbia drupifera (GHX-27), and AZT on in vitro HIV-1 and HIV-2 replication and cytopathicity were compared. All plant extracts inhibited HIV-1 strain HTLVIII(B) cytopathicity, the leaves of GHX-2 and the seeds of GHX-26 having high antiviral indices (110 and 90, respectively). Against HIV-2 strain GH1, the EC(50) values ranged from <0.005 to 0.075 mg/ml when treatment was started at 40min after virus adsorption, except for GHX-7 which showed only moderate activity and GHX-26 which had no activity. When treatment was delayed for 2h, the plant extracts, unlike AZT, were still very effective against HIV-2. Likewise, only the plant extracts were able to attain EC(90) values when high multiplicity of infection (MOI) with HIV-1 strain GH3 was used when treatment was delayed for 2h. In Molt-4 cocultures with Molt-4/HIV, early cytopathic effect (CPE) of cell fusion was unaffected by AZT but was completely inhibited by all plants at noncytotoxic concentrations. In addition, GHX-27 was selectively toxic to Molt-4/HIV cells. The plant extracts also inhibited HIV-1 reverse transcriptase (RT) activity at EC(50) values of <0.01-0.03 mg/ml. HIV-1 proviral DNA copying as determined in a polymerase chain reaction, was completely inhibited by GHX-2 and GHX-6 at 0.011 and 0.015 mg/ml, respectively. GHX-26 and GHX-27 showed only very moderate activity.

Inhibition of reverse transcription in vivo by elevated manganese ion concentration.

Mutations in PMR1, a yeast gene encoding a calcium/manganese exporter, dramatically decrease Ty1 retrotransposition. Ty1 cDNA is reduced in pmr1 mutant cells, despite normal levels of Ty1 RNA and proteins. The transposition defect results from Mn(2+) accumulation that inhibits reverse transcription. Cytoplasmic accumulation of Mn(2+) in pmr1 cells may directly affect reverse transcriptase (RT) activity. Trace amounts of Mn(2+) potently inhibit Ty1 RT and HIV-1 RT in vitro when the preferred cation, Mg(2+), is present. Both Mn(2+) and Mg(2+) alone activate Ty1 RT cooperatively with Hill coefficients of 2, providing kinetic evidence for a dual divalent cation requirement at the RT active site. We propose that occupancy of the B site is the major determinant of catalytic activity and that Mn(2+) at this site greatly reduces catalytic activity.

Advantages and disadvantages of using PCR techniques to characterize transgenic plants.

The polymerase chain reaction (PCR) revolutionized molecular biology to a similar extent as the discovery of plasmids and restriction endonucleases. However, there are some limitations to the use of PCR. Transgenic plants containing potato spindle tuber viroid (PSTVd) cDNA constructs, demonstrated to become de novo methylated upon PSTVd infection, represent a good example to illustrate the advantages of PCR. PSTVd is a 359 nt long autonomously replicating plant pathogenic RNA where all of its enzymatic requirements are entirely provided by the host cell. In addition, viroids that propagate without a DNA intermediate barely tolerate nucleotide substitutions of their RNA genome without losing infectivity. PCR is the method of choice to characterize the sequence context of genome-integrated viroid cDNA or of reverse transcribed PSTVd RNA, and can hardly be replaced by any alternative procedure. Furthermore, the precise examination of DNA methylation patterns (genomic sequencing) is entirely dependent on PCR. In contrast, the use of PCR is critical for the determination of copy number and arrangement of transgene constructs. Here, the advantages and disadvantages of PCR are discussed and protocols for PCR amplification of cDNA, genomic DNA, and bisulfite-treated DNA from transgenic plants are presented.

A novel, non-nested reverse-transcriptase polymerase chain reaction (RT-PCR) test for the detection of the t(15;17) translocation: a comparative study of RT-PCR cytogenetics, and fluorescence In situ hybridization.

BACKGROUND: The development of a rapid and simple reverse-transcription polymerase chain reaction (RT-PCR) assay is described that identifies the promyelocytic leukemia- retinoic acid receptor alpha (PML-RARa) hybrid messenger RNA (mRNA), a characteristic feature of acute promyelocytic leukemia (APL). METHODS AND RESULTS: Randomly primed complementary (cDNA) is synthesized from leukocyte RNA and amplified in the presence of Taq Gold in 2 separate reaction tubes containing primer pairs specific for intron 3 (bcr 3, long [L] form mRNA transcript) and intron 6 (bcr 1, short [S] form)/exon 6 (bcr 2, variant [V] form) breakpoints in PML, respectively. The different sized products generated from each RNA transcript (S, L, or V forms) are readily and unambiguously distinguishable after agarose gel electrophoresis without the need for either nested PCR or hybridization. The sensitivity of the assay is 1 in 10,000 to 1 in 100,000. The separate amplification of a b2-microglobulin transcript controls for adequate RNA and cDNA preparation. The newly developed assay was used clinically for the evaluation of 78 patients with APL. It was rapid and more sensitive than cytogenetic karyotyping, both for the diagnosis of APL and the assessment of minimal residual disease (MRD) after therapy. RT-PCR detected PML-RARa mRNA in all cases positive for the t(15;17) translocation by cytogenetics. However, as many as 50% and 80% of the diagnostic specimens and the specimens for MRD assessment, respectively, that were positive by RT-PCR were negative by cytogenetics. The ratio of cases with L-form to S-form PML-RARa fusion transcript was 2:1, whereas 3 cases (10%) had fusion sites in exon 6 of the PML gene (V forms). In addition, approximately 50% of the patients were diagnosed morphologically with microgranular M3V-type leukemia, but no significant correlation with PML breakpoints was found. CONCLUSION: The current assay is rapid, sensitive, and specific without using nested PCR or hybridization.