Reverse transcriptase recombinase polymerase amplification for detection of tomato spotted wilt orthotospovirus from crude plant extracts.

Virus detection in early stages of infection could prove useful for identification and isolation of foci of inoculum before its spread to the rest of susceptible individuals via vectoring insects. However, the low number of viruses present at the beginning of infection renders their detection and identification difficult and requires the use of highly sensitive laboratory techniques that are often incompatible with a field application. To obviate this challenge, utilized Recombinase Polymerase Amplification, an isothermal amplification technique that makes millions of copies of a predefined region in the genome, to detect tomato spotted wilt orthotospovirus in real time and at the end point. The reaction occurs isothermically and can be used directly from crude plant extracts without nucleic acid extraction. Notably, a positive result can be seen with the naked eye as a flocculus made of newly synthesized DNA and metallic beads. The objective of the procedure is to create a portable and affordable system that can isolate and identify viruses in the field, from infected plants and suspected insect vectors, and can be used by scientists and extension managers for making informed decisions for viral management. Results can be obtained in situ without the need of sending the samples to a specialized lab.

Effectiveness of COVID-19 vaccines on hospitalization and death in Guilan, Iran: a test-negative case-control study.

OBJECTIVES: The present study was conducted to estimate the effectiveness of (BBIBP)-CorV (Sinopharm), ChAdOx1-S/nCoV-19 (AZD1222, Oxford-AstraZeneca), rAd26-rAd5 (Gam-COVID-Vac, Sputnik V), and BIV1-CovIran (COVIran Barekat) and BBV152 COVAXIN (Bharat Biotech) vaccines against hospitalization and death of COVID-19 in Guilan Province of Iran from May 22 to December 21, 2021. METHODS: This test-negative case-control study was conducted on the population aged 5 years and above by extracting information from local databases (The Medical Care Monitoring Center and The Integrated Health System). A logistic regression analysis was performed to estimate the effectiveness of the vaccines against COVID-19 hospitalization and death. RESULTS: The total study population was 42,084, including 19,500 cases (with a positive Reverse Transcriptase-Polymerase Chain Reaction test admitted to hospitals in Guilan Province) and 22,586 controls (with a negative Reverse Transcriptase-Polymerase Chain Reaction test). Among the admitted patients, 1887 deaths occurred. The maximum effectiveness of BBIBP-CorV (Sinopharm) in preventing temporary hospitalization and regular hospitalization was observed 151 days after receiving the second dose, 95% (95% CI: 67-99.4%) and 85% (95% CI: 77-91%) respectively. The maximum effectiveness of the BBIBP-CorV (Sinopharm) vaccine 91-120 days after receiving the second dose against death was showed 56% (95% CI: 33-71%). The maximum effectiveness of ChAdOx1-S/nCoV-19 (AZD1222, Oxford-AstraZeneca) and BIV1-CovIran (COVIran Barekat) in preventing regular hospitalization and death was observed 121-150 and 61-90 days (respectively) after receiving the second dose, reaching 98% (95% CI: 94-99%) and 92% (95% CI: 48-99%), respectively for ChAdOx1-S/nCoV-19 and 95% (95% CI: 91-97%) and 89% (95% CI: 55-98%) respectively, for BIV1-CovIran. CONCLUSION: For almost all vaccines, the study observed an increase in effectiveness against hospitalization and death over time.

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.

Screening and Evaluation of Novel Compounds against Hepatitis B Virus Polymerase Using Highly Purified Reverse Transcriptase Domain.

Hepatitis B virus (HBV) polymerase seems to be very hard to express and purify sufficiently, which has long hampered the generation of anti-HBV drugs based on the nature of the polymerase. To date, there has been no useful system developed for drug screening against HBV polymerase. In this study, we successfully obtained a highly purified reverse transcriptase (RT) domain of the polymerase, which has a template/primer and substrate binding activity, and established a novel high-throughput screening (HTS) system using purified RT protein for finding novel polymerase inhibitors. To examine whether the assay system provides reliable results, we tested the small scale screening using pharmacologically active compounds. As a result, the pilot screening identified already-known anti-viral polymerase agents. Then, we screened 20,000 chemical compounds and newly identified four hits. Several of these compounds inhibited not only the HBV RT substrate and/ template/primer binding activity, but also Moloney murine leukemia virus RT activity, which has an elongation activity. Finally, these candidates did show to be effective even in the cell-based assay. Our screening system provides a useful tool for searching candidate inhibitors against HBV.

Establishment of a system for finding inhibitors of ε RNA binding with the HBV polymerase.

Although several nucleo(s)tide analogs are available for treatment of HBV infection, long-term treatment with these drugs can lead to the emergence of drug-resistant viruses. Recent HIV-1 studies suggest that combination therapies using nucleo(s)tide reverse transcriptase inhibitors (NRTIs) and non-nucleo(s)tide reverse transcriptase inhibitors (NNRTIs) could drastically inhibit the viral genome replication of NRTI-resistant viruses. In order to carry out such combinational therapy against HBV, several new NRTIs and NNRTIs should be developed. Here, we aimed to identify novel NNRTIs targeting the HBV polymerase terminal protein (TP)-reverse transcriptase (RT) (TP-RT) domain, which is a critical domain for HBV replication. We expressed and purified the HBV TP-RT with high purity using an Escherichia coli expression system and established an in vitro ε RNA-binding assay system. Then, we used TP-RT in cell-free assays to screen candidate inhibitors from a chemical compound library, and identified two compounds, 6-hydroxy-DL-DOPA and N-oleoyldopamine, which inhibited the binding of ε RNA with the HBV polymerase. Furthermore, these drugs reduced HBV DNA levels in cell-based assays as well by inhibiting packaging of pregenome RNA into capsids. The novel screening system developed herein should open a new pathway the discovery of drugs targeting the HBV TP-RT domain to treat HBV infection.

Non-nucleoside hepatitis B virus polymerase inhibitors identified by an in vitro polymerase elongation assay.

BACKGROUND: Hepatitis B virus (HBV) polymerase is the only virus-encoded enzyme essential for producing the HBV genome and is regarded as an attractive drug target. However, the difficulty of synthesizing and purifying recombinant HBV polymerase protein has hampered the development of new drugs targeting this enzyme, especially compounds unrelated to the nucleoside structure. We recently have developed a technique for the synthesis and purification of recombinant HBV polymerase containing the reverse transcriptase (RT) domain that carried DNA elongation activity in vitro. METHODS: We used the overproduced protein to establish an in vitro high-throughput screening system to identify compounds that inhibit the elongation activity of HBV polymerase. RESULTS: We screened 1120 compounds and identified a stilbene derivative, piceatannol, as a potential anti-HBV agent. Derivative analysis identified another stilbene derivative, PDM2, that was able to inhibit HBV replication with an IC50 of 14.4 ± 7.7 µM. An infection experiment suggested that the compounds inhibit the replication of HBV rather than the entry process, as expected. Surface plasmon resonance analysis demonstrated a specific interaction between PDM2 and the RT domain. Importantly, PDM2 showed similar inhibitory activity against the replication of both wild-type HBV and a lamivudine/entecavir-resistant HBV variant. Furthermore, PDM2 showed an additive effect in combination with clinically used nucleos(t)ide analogs. CONCLUSIONS: We report the development of a screening system that is useful for identifying non-nucleos(t)ide RT inhibitors.

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.

Limited reverse transcriptase activity of phi29 DNA polymerase.

Phi29 (Φ29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real time (SMRT) sequencing. Here, we report the ability of phi29 DNA polymerase to amplify RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions are amplified at a similar amplification rate as non-chimeric DNA substrates, and that consecutive RNA pyrimidines were generally preferred over purines. We observed RCA suppression with higher number of ribonucleotide substitutions, which was partially restored by interspacing RNA bases with DNA. We show that supplementing manganese ions as cofactor supports replication of RNAs during RCA. Sequencing of the RCA products demonstrated accurate base incorporation at the RNA base with both Mn2+ and Mg2+ as cofactors during replication, proving reverse transcriptase activity of the phi29 DNA polymerase. In summary, the ability of phi29 DNA polymerase to accept RNA-containing substrates broadens the spectrum of applications for phi29 DNA polymerase-mediated RCA. These include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.

Histopathological and molecular classification of colorectal cancer and corresponding peritoneal metastases.

BACKGROUND: Patients with colorectal peritoneal carcinomatosis have a very poor prognosis. The recently developed consensus molecular subtype (CMS) classification of primary colorectal cancer categorizes tumours into four robust subtypes, which could guide subtype-targeted therapy. CMS4, also known as the mesenchymal subtype, has the greatest propensity to form distant metastases. CMS4 status and histopathological features of colorectal peritoneal carcinomatosis were investigated in this study. METHODS: Fresh-frozen tissue samples from primary colorectal cancer and paired peritoneal metastases from patients who underwent cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy were collected. Histopathological features were analysed, and a reverse transcriptase-quantitative PCR test was used to assess CMS4 status of all collected lesions. RESULTS: Colorectal peritoneal carcinomatosis was associated with adverse histopathological characteristics, including a high percentage of stroma in both primary tumours and metastases, and poor differentiation grade and high-grade tumour budding in primary tumours. Furthermore, CMS4 was significantly enriched in primary tumours with peritoneal metastases, compared with unselected stage I-IV tumours (60 per cent (12 of 20) versus 23 per cent; P = 0.002). The majority of peritoneal metastases (75 per cent, 21 of 28) were also classified as CMS4. Considerable intrapatient subtype heterogeneity was observed. Notably, 15 of 16 patients with paired tumours had at least one CMS4-positive tumour location. CONCLUSION: Significant enrichment for CMS4 was observed in colorectal peritoneal carcinomatosis. Surgical relevance Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) improves survival of selected patients with colorectal peritoneal carcinomatosis, but recurrence is common. Histopathological and molecular analysis of colorectal peritoneal carcinomatosis could provide clues for development of novel therapies. In this study, colorectal peritoneal carcinomatosis was found to be enriched for tumours with high stromal content and CMS4-positive status. To further improve prognosis for patients with colorectal peritoneal carcinomatosis, therapies that target tumour-stroma interaction could be added to CRS-HIPEC.

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 occurrence of mutant rtI233V of HBV in patients receiving adefovir therapy.

BACKGROUND: The study aimed to clarify whether the rtI233V substitution affects adefovir (ADV) resistance. METHODS: A total of 18,419 patients from Beijing 302 Hospital were investigated. HBV complete reverse transcriptase region of the polymerase was screened by direct sequencing and verified by clonal sequencing if necessary. Replication-competent wild-type and mutant HBV genomic amplicons were transfected into HepG2 cells for phenotypic analysis of viral replication capacity and drug susceptibility. RESULTS: The rtI233V substitution was detected in 38/5,344 (0.71%) ADV-treated patients and in 8/13,075 patients without receiving ADV (P<0.001). Eight patients with rtI233V ± rtA181V/rtN236T had virological breakthrough in the clinical course of ADV treatment. Phenotypic analysis showed that rtI233V mutants from patient 1 and patient 2 exhibited 1.57-fold and 1.51-fold decreased susceptibility to ADV, respectively, compared to wild-type virus; by contrast, rtN236T and rtI233V+N236T mutants from patient 1 had 6.82-fold and 5.28-fold decreased susceptibility to ADV. rtI233V, rtN236T and rtI233V+N236T mutants had 97.5%, 30.2% and 69.7% of replication capacity compared to wild-type virus in the absence of antivirals and all remained susceptible to lamivudine, entecavir and tenofovir. Viral replication capacity correspondingly decreased after eliminating rtI233V from rtI233V+N236T mutant and was restored after introducing rtI233V into rtN236T mutant. In clinical practice, switching to entecavir rescue therapy suppressed HBV DNA to an undetectable level for both patients. CONCLUSIONS: rtI233V usually emerged in ADV-treated patients with little impact on ADV susceptibility but it effectively restored replication capacity of the rtN236T mutant, suggesting that rtI233V may partly serve as a compensatory mutation associated with ADV resistance.

[Effect and mechanism of danshensu on hepatitis B virus reverse transcriptase and antigen expression].

MTT assay was used in this study to investigate the inhibitory effect of danshensu on the activity of 2.2.15 cells among human hepatoma cell line (HepG2); indirect fluorescence labeling method was used to measure the changes of reactive oxygen levels in the cells; ELISA method was used to determine hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels in cellular supernatants; HBV DNA level was measured with fluorogenic quantitative PCR method. The inhibitory effect of danshensu on HBV RT(hepatitis B virus reverse transcriptase) was studied by using enzyme inhibition dynamics, and the effect of danshensu on secondary structure of HBV reverse transcriptase was monitored by using circular dichroism. The results showed that danshensu had a good inhibitory effect on the growth of HepG2.2.15 cells, with a half inhibitory concentration (IC50) of (15.35±2.43) µmol•L⁻¹; danshensu could significantly inhibit HBsAg and HBeAg expressions, and showed an inhibitory effect on HBV DNA replication. In addition, danshensu was an effective inhibitor for HBV reverse transcriptase [IC50 (21.32±2.43) µmol•L⁻¹]. The fluorescence labeling results showed that the reactive oxygen levels in the cells were increased with the increase of danshensu concentration. Circular dichroism analysis showed that danshensu could induce partial change of conformation of HBV reverse transcriptase and gradually increased α-helical content. These results indicated that danshensu could make the structure of the enzyme become closer by binding to HBV reverse transcriptase, which was not conducive to the formation of the active center, so it could finally decrease the activity of HBV reverse transcriptase. Such decrease in enzyme activity would directly affect the HBV DNA replication, and combined with the decrease of the antigen levels, the effect of danshensu on HBV was increased.

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.

[Cloning and analysis of reverse transcriptase(RT) of Ty1-copia retrotransposons in Dendrobium officinale].

Using universal primer Ty1-copia retrotransposon RT,43 Ty1-copia like retrotransposon RT with high heterogeneity, stop codon mutation and frameshift mutation were amplified by PCR from genomic DNA of Zhejiang Lin'an (C15) and Yunnan Guangnan (A39) of Dendrobium officinale. The length of these sequences varied from 260 to 266 bp, and was rich in AT and consistency ranged from 47.1% to 97.7%. Different c/s-acting regulatory elements induced by low temperature, heat, light, all kinds of plant growth regulating substances and the starting transcription signals, corresponding to CAAT box, TATA box conserved sequences and some other regulatory elements. When being translated into amino acids, ten sequences presented stop codon mutation, five sequences presented frameshift mutation, and thirty-seven sequences presented conserved sequence "SLYGKQ" mutation. Six categories were identified through phylogenic analysis after alignment analyses of their amino acid sequences, and with other plants (eg. Triticum aestivum, Eleocharis quinqueflora) having high homology, which indicated that horizontal transmission of retrotransposon occurred among the plants in the past.

Characterization and analysis of a transcriptome from the boreal spider crab Hyas araneus.

Research investigating the genetic basis of physiological responses has significantly broadened our understanding of the mechanisms underlying organismic response to environmental change. However, genomic data are currently available for few taxa only, thus excluding physiological model species from this approach. In this study we report the transcriptome of the model organism Hyas araneus from Spitsbergen (Arctic). We generated 20,479 transcripts, using the 454 GS FLX sequencing technology in combination with an Illumina HiSeq sequencing approach. Annotation by Blastx revealed 7159 blast hits in the NCBI non-redundant protein database. The comparison between the spider crab H. araneus transcriptome and EST libraries of the European lobster Homarus americanus and the porcelain crab Petrolisthes cinctipes yielded 3229/2581 sequences with a significant hit, respectively. The clustering by the Markov Clustering Algorithm (MCL) revealed a common core of 1710 clusters present in all three species and 5903 unique clusters for H. araneus. The combined sequencing approaches generated transcripts that will greatly expand the limited genomic data available for crustaceans. We introduce the MCL clustering for transcriptome comparisons as a simple approach to estimate similarities between transcriptomic libraries of different size and quality and to analyze homologies within the selected group of species. In particular, we identified a large variety of reverse transcriptase (RT) sequences not only in the H. araneus transcriptome and other decapod crustaceans, but also sea urchin, supporting the hypothesis of a heritable, anti-viral immunity and the proposed viral fragment integration by host-derived RTs in marine invertebrates.