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Abstract Retrogenes are a class of functional genes derived from the mRNA of various intron-containing genes. PIPSL was created through a unique mechanism, whereby distinct genes were assembled at the RNA level, and the resulting chimera was then reverse transcribed and integrated into the genome by the L1 retrotransposon. Expression of PIPSL RNA via its transcription start sites (TSSs) has been confirmed in the testes of humans and chimpanzee. Here, we demonstrated that PIPSL RNA is expressed in the testis of the white-handed gibbon. The 5′-end positions of gibbon RNAs were confined to a narrow range upstream of the PIPSL start codon and overlapped with those of orangutan and human, suggesting that PIPSL TSSs are similar among hominoid species. Reporter assays using a luciferase gene and the flanking sequences of human PIPSL showed that an upstream sequence exhibits weak promoter activity in human cells. Our findings suggest that PIPSL might have acquired a promoter at an early stage of hominoid evolution before the divergence of gibbons and ultimately retained similar TSSs in all of the lineages. Moreover, the upstream sequence derived from the phosphatidylinositol-4-phosphate 5-kinase, type I, alpha 5′ untranslated region and/or neighboring repetitive sequences in the genome possibly exhibits promoter activity. Furthermore, we observed that a TATA-box-like sequence has emerged by nucleotide substitution in a lineage leading to humans, with this possibly responsible for a broader distribution of the human PIPSL TSSs. Highlights • We verified the expression of the PIPSL retrogene in white-handed gibbon. • The promoter is located upstream of the PIPSL TSS, similar to human PIPSL. • Promoter development occurred early in hominoid evolution before gibbon divergence. [ABSTRACT FROM AUTHOR]

Aims and Objectives: Lamivudine is a nucleoside analogue reverse transcriptase inhibitor used for treatment of hepatitis B and HIV virus infection in pregnant females and to prevent maternal to child transmission. The present study is aimed to investigate the teratogenicity of Lamivudine in growing embryo in swiss albino mice. Material and Method: Lamivudine was given to pregnant Swiss Albino mice by oral gavage in doses of 18mg/kg body weight and 30mg/kg body weight from 6-15th day of gestation. The control mice were fed distilled water by the same route on the same gestational days. The pregnant mice were sacrificed on 18th day of gestation by cervical dislocation. The foetuses and placentae were observed for any gross malformation. Result: On the gross examination it was observed that there was a significant reduction in the weight of foetuses and growth retardation. Also haemorrhagic patches were observed on the body. Reduction in weight and diameter of placenta size was not significant. Conclusion: Lamivudine causes teratological changes in mice foetus and should be used judiciously in pregnant females. [ABSTRACT FROM AUTHOR]

Here, we present a technique that performs on-chip picoliter real-time reverse transcriptase loop mediated isothermal amplification (RT-LAMP) reactions on a histological tissue section without any analyte purification while preserving the native spatial location of the nucleic acid molecules. We demonstrate this method by amplifying TOP2A messenger RNA (mRNA) in a prostate cancer xenograft with 100 μm spatial resolution and by visualizing the variation in threshold time of amplification across the tissue. The on-chip reaction was validated by mRNA fluorescence in situ hybridization (mFISH) from cells in the tissue section. The entire process, from tissue loading on microchip to results from RT-LAMP can be carried out in less than 2 h. We anticipate that this technique, with its ease of use, fast turnaround, and quantitative molecular outputs, would become an invaluable tissue analysis tool for researchers and clinicians in the biomedical arena. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the mutational patterns on the pol gene of the main HIV-1 strain archived in cell genome of 10 chronically infected men according to their clinical and therapeutic history. The genotyping resistance profiles were compared between the first blood plasma available at the time of HIV diagnosis and rectal biopsies and PBMC sampled 1-5 years after the initiation of combined antiretroviral therapy (cART). Methods: HIV-1 RNA and cell-associated HIV-1 DNA were quantified by Abbott Real-Time HIV-1 and Generic HIV ® DNA cell (Biocentric) assays. The mutations in protease and reverse transcriptase genes were assessed by the Trugene ® assay (Siemens). The C2V3 region was amplified to determine the viral tropism. Results: In 9 patients, slight or no differences were observed between the 3 resistance profiles. Those mostly detected were related to the resistance to nucleos(t)ide (D67N, L210W, T215A, T69D) and nonnucleoside (K103N, V106I, V179I) inhibitors. In 1 rilpivirine-treated patient, the M230I mutation was detected in PBMC. No change of viral tropism was observed between samples. Conclusion: These data suggest that resistance mutations harbored by the main HIV strain in plasma at the time of diagnosis are durably archived in DNA cells whatever the delay between infection and initiation of therapy in patients well controlled by cART. [ABSTRACT FROM AUTHOR]

Fitness is a key parameter in the measurement of transmission capacity of individual drug-resistant HIV. Drug-resistance related mutations (DRMs) T369V/I and A371V in the connection subdomain (CN) of reverse transcriptase (RT) occur at higher frequencies in the individuals experiencing antiretroviral therapy failure. Here, we evaluated the effects of T369V/I and A371V on viral fitness, in the presence or in the absence of thymidine analogue resistance-associated mutations (TAMs) and assessed the effect of potential RT structure-related mechanism on change in viral fitness. Mutations T369V/I, A371V, alone or in combination with TAMs were introduced into a modified HIV-1 infectious clone AT1 by site-directed mutagenesis. Then, experiments on mutant and wild-type virus AT2 were performed separately using a growth-competition assay, and then the relative fitness was calculated. Structural analysis of RT was conducted using Pymol software. Results showed that T369V/I severely impaired the relative virus fitness, and A371V compensated for the viral fitness reduction caused by TAMs. Structural modeling of RT suggests that T369V/I substitutions disrupt powerful hydrogen bonds formed by T369 and V365 in p51 and p66. This study indicates that the secondary DRMs within CN might efficiently damage viral fitness, and provides valuable information for clinical surveillance and prevention of HIV-1 strains carrying these DRMs. [ABSTRACT FROM AUTHOR]

The isatin scaffold is the constitutive fragment of several natural and synthetic bioactive molecules. Albeit several benzene sulphonamide-based carbonic anhydrase inhibitors (CAIs) have been reported, only recently isatin benzene sulphonamides have been studied and proposed as CAIs. In this study we have designed, synthesised, and evaluated the biological activity of a series of differently substituted isatin-based benzene sulphonamides which have been designed for the inhibition of carbonic anhydrase isoforms. The activity of all the synthesised compounds was evaluated towards human carbonic anhydrase I, II, IX, and XII isozymes. Our results indicate that the nature and position of substituents on the isatin ring can modulate both activity and isozyme selectivity. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the economic impact of the disease by using milk production records and to determine the serotypes circulating in the region during 2015. Sampling was done from different outbreaks initially on the basis of clinical signs and later reverse transcriptase-polymerase chain reaction (RT-PCR) was employed for the conformation of FMDV genome. Out of total 88 samples, 73 were found positive which were then serotyped into type O (n=44), Asia1 (n=18) and A (n=06). The economic impact was analyzed by recording milk loss at four affected farms. Their average milk yield was observed 9.2 liters before the onset of disease that decreased dramatically after the disease. Milk loss of 225 and 195 liters was recorded for buffalo and cattle respectively, during 70 days of the study period. [ABSTRACT FROM AUTHOR]

HIV-1-infected macrophages are long-lived and act as human immunodeficiency virus 1 (HIV-1) virus reservoirs. Lipopolysaccharide (LPS) has been demonstrated to suppress HIV-1 replication in macrophages, but the mechanism is not clear. Previous research suggested that downregulation of CD4 and CCR5 as well as blockage of the interaction of HIV-1 with cells are major causes of inhibition of HIV-1 replication in macrophages by LPS. In order to study whether LPS blocks the post-entry event of HIV-1 replication, we developed a macrophage HIV-1 infection model by using VSV-G pseudotyped HIV-1-luciferase virus to infect THP-1 differentiated macrophage-like cells. We found that LPS can suppress HIV-1 replication at post-entry steps. Further study suggested that HIV-1 reverse transcription was blocked by LPS, but addition of exogenous deoxyribonucleosides led to only partial recovery of HIV-1 replication. However, the inhibition of pro-inflammatory pathway completely rescued HIV-1 replication. Thus, our study shows that LPS can suppress the events of HIV-1 replication post-entry, including reverse transcription, and this restriction is mediated by more than one mechanism. [ABSTRACT FROM AUTHOR]

Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours. [ABSTRACT FROM AUTHOR]

HIV related mutations can be associated with decreased susceptibility to antiretrovirals and treatment failures. There is scarce information about HIV mutations in persons failing HIV treatment in North of Brazil. Our aim was to evaluate evolution of HIV subtypes and mutations patterns related to antiretroviral therapy in this region. We investigated HIV resistance profile in adults failing antiretroviral regimen in Northern Brazil from January, 2004, through December, 2013. Genotype data was evaluated through Stanford University algorithm. There were 377 genotypes from different individuals to evaluate. Resistance mutations were similar to worldwide reports and related to antiretroviral exposure. Most prevalent mutations in the reverse transcriptase gene were M184V (80.1%) and K130N (40.6%). Thymidine associated mutations were more frequent in multiexperienced patients. Most common protease mutations were M46I, V82A, I54V, L90M, I84V, M46L, and L76V. Subtype B was the most prevalent (90.7%). There were differences between subtypes B and non-B mutations. We documented for the first time subtypes and patterns of HIV associated mutations in Northern Brazil. A1 subtype was identified for the first time in this area. Depending on drug regimen and how experienced the patient is, an empirical switch of a failing antiretroviral treatment could be a reasonable option. [ABSTRACT FROM AUTHOR]

Pristimerin (PM) is a quinonemethide triterpenoid present in various plant species with strong antiprolifertive and proapoptotic activities in cancer cells. The effect of PM on telomerase which is reactivated in most cancers including carcinoma of the prostate (CaP) has not been studied. We investigated the effect of PM on the expression of human telomerase reverse transcriptase (hTERT) gene that codes for the catalytic subunit of the telomerase holoenzyme complex in prostate cancer cell lines LNCaP and PC-3 cells. The inhibition of cell proliferation and induction of apoptosis by PM in both cell lines was associated with the inhibition of hTERT mRNA expression, suppression of native and phosphorylated hTERT protein and hTERT telomerase activity. The ablation of hTERT expression increased the sensitivity of cancer cells to PM. In addition, results also revealed that the inhibition of hTERT expression is attributed to the inhibition of transcription factors SP1, c-Myc and STAT3 and protein kinase B/Akt which regulate hTERT transcriptionally and post-translationally, respectively. These data provide evidence that telomerase is a potential target of PM in prostate cancer. [ABSTRACT FROM AUTHOR]

Enhanced expression of the reverse transcriptase (RT) protein encoded by human endogenous retrovirus-K (ERVK) is a promising biomarker for several inflammatory and neurological diseases. However, unlike RT enzymes encoded by exogenous retroviruses, little work has been done to identify ERVK RT isoforms, their expression patterns, and cellular localization. Using Western blot, we showcase the ERVK gag-pro-pol polyprotein processing leading to the production of several ERVK RT isoforms in human neuronal (ReNcell CX) and astrocytic (SVGA) models of neuroinflammatory disease. Since the proinflammatory cytokine IFNγ plays a key role in the pathology of several ERVK-associated neurological diseases, we sought to determine if IFNγ can drive ERVK RT expression. IFNγ signalling markedly enhanced ERVK polyprotein and RT expression in both human astrocytes and neurons. RT isoforms were expressed in a cell-type specific pattern and the RT-RNase H form was significantly increased with IFNγ treatment. Fluorescent imaging revealed distinct cytoplasmic, perinuclear and nuclear ERVK RT staining patterns upon IFNγ stimulation of astrocytes and neurons. These findings indicate that ERVK expression is inducible under inflammatory conditions such as IFNγ exposure-and thus, these newly established in vitro models may be useful in exploring ERVK biology in the context of neuroinflammatory disease. [ABSTRACT FROM AUTHOR]

Wax gourd (2n = 2 x = 24) is an important vegetable species in Cucurbitaceae . Because it can be stored for a very long period of time, it plays an important role in ensuring the annual supply and regulating off-season supply of the vegetables. However, the availability of genetic information about wax gourd is limited. This study aimed to identify the useful genetic information for wax gourd. The conserved domains of reverse transcriptase (RT) genes of Ty1- copia retrotransposons were isolated from the genome of wax gourd using degenerate oligonucleotide primers. A total of twenty eight RT sequences were obtained, which showed high heterogeneity with the similarity ranging from 47.5% to 94.3%. Sixteen (57.1%) of them were found to be defective, being disrupted by stop codons and/or frameshift mutations. These 28 sequences were divided into five subfamilies. The comparative phylogenetic analysis with other Cucurbitaceae species from GenBank database showed that most retrotransposons derived from the same genus tended to cluster together, although there were a few exceptions. These results indicate that both vertical transmission and horizontal transmission are the sources of Ty1- copia retrotransposons in wax gourd. Fluorescent in situ hybridization (FISH) with Ty1- copia retrotransposon sequences as probes revealed that this kind of retrotransposons had a dispersed genomic organization, physically distributed among all the chromosomes of wax gourd, with clusters in the heterochromatin regions. This is the first report of Ty1- copia retrotransposons in wax gourd, which would be helpful for our understanding about the organization and evolutions of wax gourd genome and also provide valuable information for our utilization of wax gourd retrotransposons. [ABSTRACT FROM AUTHOR]

Abstract: The paper presents a real-time PCR method allowing the simultaneous detection of traces of black mustard (Brassica nigra) and brown mustard (Brassica juncea) in food. The primers and the probe target the B. nigra partial RT gene for reverse transcriptase from gypsy-like retroelement 13G42-26. The real-time PCR method does not show any cross-reactivity with other Brassicaceae species with the exception of white mustard. Low cross-reactivities with cinnamon, cumin, fenugreek, ginger, rye and turmeric can be ignored because in common mustard containing foodstuffs these biological species are present in very low amounts. By analysing serially diluted DNA extracts from black and brown mustard, the DNA of both mustard species could be detected down to 0.1pg. With 10ng DNA per PCR tube the real-time PCR method allows the detection of 5ppm black and brown mustard in brewed sausages. [Copyright &y& Elsevier]

Background Accurate and complete identification of mobile elements is a challenging task in the current era of sequencing, given their large numbers and frequent truncations. Group II intron retroelements, which consist of a ribozyme and an intron-encoded protein (IEP), are usually identified in bacterial genomes through their IEP; however, the RNA component that defines the intron boundaries is often difficult to identify because of a lack of strong sequence conservation corresponding to the RNA structure. Compounding the problem of boundary definition is the fact that a majority of group II intron copies in bacteria are truncated. Results Here we present a pipeline of 11 programs that collect and analyze group II intron sequences from GenBank. The pipeline begins with a BLAST search of GenBank using a set of representative group II IEPs as queries. Subsequent steps download the corresponding genomic sequences and flanks, filter out non-group II introns, assign introns to phylogenetic subclasses, filter out incomplete and/or non-functional introns, and assign IEP sequences and RNA boundaries to the full-length introns. In the final step, the redundancy in the data set is reduced by grouping introns into sets of ⩾95% identity, with one example sequence chosen to be the representative. Conclusions These programs should be useful for comprehensive identification of group II introns in sequence databases as data continue to rapidly accumulate. [ABSTRACT FROM AUTHOR]

Abstract: The increasing diversity of HIV-1 isolates makes virus quantitation challenging, especially when diverse isolates co-circulate in a geographical area. Measuring the HIV-1 DNA levels in cells has become a valuable practical tool for fundamental and clinical research. A quantitative HIV-1 DNA assay was developed based on TaqMan® technology. Primers that target the highly conserved LTR region were designed to detect a broad array of HIV-1 variants, including viral isolates from many subtypes, with high sensitivity. Introduction of a pre-amplification step prior to the TaqMan® reaction allowed the specific amplification of fully reverse transcribed viral DNA. Execution of the pre-amplification step with a second primer set enables for the exclusive quantitation of the 2-LTR circular HIV-1 DNA form. [Copyright &y& Elsevier]

Abstract: Simian–human immunodeficiency virus encoding both reverse transcriptase (RT) and envelope genes of HIV-1 (RT Env SHIV) is important for evaluating biomedical prevention modalities for HIV/AIDS. We describe virological characterization of a clade B RT Env SHIV following infection of macaques via multiple routes. In vivo passage of the RT Env SHIV through Indian rhesus macaque enhanced infectivity. Expanded virus had minimal envelope heterogeneity and was inhibited by NNRTIs and CCR5 antagonists. Infection of macaques with RT Env SHIV via mucosal or intravenous routes resulted in stable infection accompanied by peak plasma viremia of approximately 5×106 copies/ml that was controlled beyond set point. Molecular homogeneity of the virus was maintained following in vivo passage. Inhibition of RT Env SHIV by RT and entry inhibitors and ease of in vivo transmission make it a useful model for testing the efficacy of combinations of entry and RT inhibitors in nonhuman primates. [Copyright &y& Elsevier]

We aimed to determine the prevalence of the coexistence of HBsAg and anti-HBs and to analyze the clinical and virological features of infection, including amino acid (aa) patterns of the S gene and reverse transcriptase (RT) region in Chinese chronic hepatitis B (CHB) patients. Fifty-four (2.90%) CHB patients who were positive for both HBsAg and anti-HBs were tested, and sequences were obtained from 52 of them as well as 48 patients from a control group. S gene and RT region sequences were amplified and sequenced using in-house protocols. There was no significant difference between patients with and without anti-HBs with regard to age, gender, alanine aminotransferase level, and the proportion positive for HBeAg and HBcAb. The occurrence of genotype C (P = 0.001) and anti-HBeAb positivity (P = 0.027) was significantly higher in HBsAg+/anti-HBs+ individuals. In the S gene, the number of mutated residues in the HBsAg+/anti-HBs+ group was markedly higher than in control patients (1.88 versus 1.02 substitutions per 100 amino acids, P = 0.022). The amino acid exchange occurred mostly within the N-terminal region (2.15 versus 0.87 substitutions per 100 amino acids, P = 0.023) and the 'a' determinant (3.61 versus 1.56 substitutions per 100 amino acids, P = 0.049) in the two groups. In the RT region, the mean number of substitution per 100 aa showed a tendency to be significantly higher in HBsAg+/anti-HBs+ patients than in controls (2.34 versus 1.46, P = 0.040). This study showed a prevalence of coexistence of anti-HBs in HBsAg-positive patients and an increased frequency of genotype C and aa variability within both HBsAg and RT involving functionally important regions of those proteins. [ABSTRACT FROM AUTHOR]

A correction to the article related to human telomerase reverse transcriptase (hTERT) that was published in a 2003 issue of the periodical is presented.