Reverse transcriptase drug resistance mutations in HIV-1 subtype C infected patients on ART in Karonga District, Malawi

<p>Abstract</p> <p>Background</p> <p>Drug resistance testing before initiation of, or during, antiretroviral therapy (ART) is not routinely performed in resource-limited settings. High levels of viral resistance circulating within the population will have impact on treatment programs by increasing the chances of transmission of resistant strains and treatment failure. Here, we investigate Drug Resistance Mutations (DRMs) from blood samples obtained at regular intervals from patients on ART (Baseline-22 months) in Karonga District, Malawi. One hundred and forty nine reverse transcriptase (RT) consensus sequences were obtained via nested PCR and automated sequencing from blood samples collected at three-month intervals from 75 HIV-1 subtype C infected individuals in the ART programme.</p> <p>Results</p> <p>Fifteen individuals showed DRMs, and in ten individuals DRMs were seen from baseline samples (reported to be ART naïve). Three individuals in whom no DRMs were observed at baseline showed the emergence of DRMs during ART exposure. Four individuals who did show DRMs at baseline showed additional DRMs at subsequent time points, while two individuals showed evidence of DRMs at baseline and either no DRMs, or different DRMs, at later timepoints. Three individuals had immune failure but none appeared to be failing clinically.</p> <p>Conclusion</p> <p>Despite the presence of DRMs to drugs included in the current regimen in some individuals, and immune failure in three, no signs of clinical failure were seen during this study. This cohort will continue to be monitored as part of the Karonga Prevention Study so that the long-term impact of these mutations can be assessed. Documenting proviral population is also important in monitoring the emergence of drug resistance as selective pressure provided by ART compromises the current plasma population, archived viruses can re-emerge</p

QTrim : a novel tool for the quality trimming of sequence reads generated using the Roche/454 sequencing platform

Background Many high throughput sequencing (HTS) approaches, such as the Roche/454 platform, produce sequences in which the quality of the sequence (as measured by a Phred-like quality scores) decreases linearly across a sequence read. Undertaking quality trimming of this data is essential to enable confidence in the results of subsequent downstream analysis. Here, we have developed a novel, highly sensitive and accurate approach (QTrim) for the quality trimming of sequence reads generated using the Roche/454 sequencing platform (or any platform with long reads that outputs Phred-like quality scores). Results The performance of QTrim was evaluated against all other available quality trimming approaches on both poor and high quality 454 sequence data. In all cases, QTrim appears to perform equally as well as the best other approach (PRINSEQ) with these two methods significantly outperforming all other methods. Further analysis of the trimmed data revealed that the novel trimming approach implemented in QTrim ensures that the prevalence of low quality bases in the resulting trimmed data is substantially lower than PRINSEQ or any of the other approaches tested. Conclusions QTrim is a novel, highly sensitive and accurate algorithm for the quality trimming of Roche/454 sequence reads. It is implemented both as an executable program that can be integrated with standalone sequence analysis pipelines and as a web-based application to enable individuals with little or no bioinformatics experience to quality trim their sequence data

Characterization of chitinases from microorganisms isolated from Lonar lake

357-363Chitinases, the enzymes that breakdown chitin—the second most abundant polysaccharide in nature—are known to have numerous uses. Alkaline chitinases, in particular are considered to have a greater potential in this respect. Fifteen chitinolytic microbial strains were isolated from the alkaline soil of Lonar lake in Buldhana district of Maharashtra. In this paper, the characters of chitinases produced by the five most potent isolates are presented. All the enzymes exhibited maximum activity in the neutral to alkaline range of pH (from 7.0 to 9.6) with a wide stability range from pH 4 to 11. The temperature optima for all the enzymes were slightly on the higher side, between 35 and 60°C, with a stability range from 25 to 60°C. All the enzymes showed a typical response to substrate concentration. None of the enzymes had any specific requirement for any particular metal ion, though a considerable stimulatory effect of Ca2+ was observed on all the enzymes. Minor effects of Cu2+, Mn2+ and Na1+ were also observed on some of the enzymes

Qtrim: a novel tool for the quality trimming of sequence reads generated using the roche/454 sequencing platform

Background: Many high throughput sequencing (HTS) approaches, such as the Roche/454 platform, produce sequences in which the quality of the sequence (as measured by a Phred-like quality scores) decreases linearly across a sequence read. Undertaking quality trimming of this data is essential to enable confidence in the results of subsequent downstream analysis. Here, we have developed a novel, highly sensitive and accurate approach (QTrim) for the quality trimming of sequence reads generated using the Roche/454 sequencing platform (or any platform with long reads that outputs Phred-like quality scores). Results: The performance of QTrim was evaluated against all other available quality trimming approaches on both poor and high quality 454 sequence data. In all cases, QTrim appears to perform equally as well as the best other approach (PRINSEQ) with these two methods significantly outperforming all other methods. Further analysis of the trimmed data revealed that the novel trimming approach implemented in QTrim ensures that the prevalence of low quality bases in the resulting trimmed data is substantially lower than PRINSEQ or any of the other approaches tested. Conclusions: QTrim is a novel, highly sensitive and accurate algorithm for the quality trimming of Roche/454 sequence reads. It is implemented both as an executable program that can be integrated with standalone sequence analysis pipelines and as a web-based application to enable individuals with little or no bioinformatics experience to quality trim their sequence data

Modeling the ferrochelatase c.315-48C modifier mutation for erythropoietic protoporphyria (EPP) in mice

Erythropoietic protoporphyria (EPP) is caused by deficiency of ferrochelatase (FECH), which incorporates iron into protoporphyrin IX (PPIX) to form heme. Excitation of accumulated PPIX by light generates oxygen radicals that evoke excessive pain and, after longer light exposure, cause ulcerations in exposed skin areas of individuals with EPP. Moreover, ∼5% of the patients develop a liver dysfunction as a result of PPIX accumulation. Most patients (∼97%) have a severe FECH mutation (Mut) in trans to an intronic polymorphism (c.315-48C), which reduces ferrochelatase synthesis by stimulating the use of an aberrant 3' splice site 63 nt upstream of the normal site for exon 4. In contrast, with the predominant c.315-48T allele, the correct splice site is mostly used, and individuals with a T/Mut genotype do not develop EPP symptoms. Thus, the C allele is a potential target for therapeutic approaches that modify this splicing decision. To provide a model for pre-clinical studies of such approaches, we engineered a mouse containing a partly humanized Fech gene with the c.315-48C polymorphism. F1 hybrids obtained by crossing these mice with another inbred line carrying a severe Fech mutation (named m1Pas) show a very strong EPP phenotype that includes elevated PPIX in the blood, enlargement of liver and spleen, anemia, as well as strong pain reactions and skin lesions after a short period of light exposure. In addition to the expected use of the aberrant splice site, the mice also show a strong skipping of the partly humanized exon 3. This will limit the use of this model for certain applications and illustrates that engineering of a hybrid gene may have unforeseeable consequences on its splicing

Characterizing the emergence and persistence of drug resistant mutations in HIV-1 subtype C infections using 454 ultra deep pyrosequencing.

BACKGROUND: The role of HIV-1 RNA in the emergence of resistance to antiretroviral therapies (ARTs) is well documented while less is known about the role of historical viruses stored in the proviral DNA. The primary focus of this work was to characterize the genetic diversity and evolution of HIV drug resistant variants in an individual's provirus during antiretroviral therapy using next generation sequencing. METHODS: Blood samples were collected prior to antiretroviral therapy exposure and during the course of treatment from five patients in whom drug resistance mutations had previously been identified using consensus sequencing. The spectrum of viral variants present in the provirus at each sampling time-point were characterized using 454 pyrosequencing from multiple combined PCR products. The prevalence of viral variants containing drug resistant mutations (DRMs) was characterized at each time-point. RESULTS: Low abundance drug resistant viruses were identified in 14 of 15 sampling time-points from the five patients. In all individuals DRMs against current therapy were identified at one or more of the sampling time-points. In two of the five individuals studied these DRMs were present prior to treatment exposure and were present at high prevalence within the amplified and sequenced viral population. DRMs to drugs other than those being currently used were identified in four of the five individuals. CONCLUSION: The presence of DRMs in the provirus, regardless of their observed prevalence did not appear to have an effect on clinical outcomes in the short term suggesting that the drug resistant viral variants present in the proviral DNA do not appear to play a role in the short term in facilitating the emergence of drug resistance

Modeling the ferrochelatase c.315-48C modifier mutation for erythropoietic protoporphyria (EPP) in mice

Erythropoietic protoporphyria (EPP) is caused by deficiency of ferrochelatase (FECH), which incorporates iron into protoporphyrin IX (PPIX) to form heme. Excitation of accumulated PPIX by light generates oxygen radicals that evoke excessive pain and, after longer light exposure, cause ulcerations in exposed skin areas of individuals with EPP. Moreover, ∼5% of the patients develop a liver dysfunction as a result of PPIX accumulation. Most patients (∼97%) have a severe FECH mutation (Mut) in trans to an intronic polymorphism (c.315-48C), which reduces ferrochelatase synthesis by stimulating the use of an aberrant 3′ splice site 63 nt upstream of the normal site for exon 4. In contrast, with the predominant c.315-48T allele, the correct splice site is mostly used, and individuals with a T/Mut genotype do not develop EPP symptoms. Thus, the C allele is a potential target for therapeutic approaches that modify this splicing decision. To provide a model for pre-clinical studies of such approaches, we engineered a mouse containing a partly humanized Fech gene with the c.315-48C polymorphism. F1 hybrids obtained by crossing these mice with another inbred line carrying a severe Fech mutation (named m1Pas) show a very strong EPP phenotype that includes elevated PPIX in the blood, enlargement of liver and spleen, anemia, as well as strong pain reactions and skin lesions after a short period of light exposure. In addition to the expected use of the aberrant splice site, the mice also show a strong skipping of the partly humanized exon 3. This will limit the use of this model for certain applications and illustrates that engineering of a hybrid gene may have unforeseeable consequences on its splicing

Proceedings of National Conference on Relevance of Engineering and Science for Environment and Society

This conference proceedings contains articles on the various research ideas of the academic community and practitioners presented at the National Conference on Relevance of Engineering and Science for Environment and Society (R{ES}2 2021). R{ES}2 2021 was organized by Shri Pandurang Pratishthan’s, Karmayogi Engineering College, Shelve, Pandharpur, India on July 25th, 2021. Conference Title: National Conference on Relevance of Engineering and Science for Environment and SocietyConference Acronym: R{ES}2 2021Conference Date: 25 July 2021Conference Location: Online (Virtual Mode)Conference Organizers: Shri Pandurang Pratishthan’s, Karmayogi Engineering College, Shelve, Pandharpur, India