Evaluation of Genotype MTBDRsl Assay to Detect Drug Resistance Associated with Fluoroquinolones,Aminoglycosides and Ethambutol on Clinical Sediments

Background

The emergence of resistant tuberculosis (TB) is a major setback to the global control of the disease as the treatment of such resistance is complex and expensive. Use of direct detection of mutations by molecular methods could facilitate rapid diagnosis of resistance to offset diagnostic delays. We evaluated the performance of the Genotype MTBDRsl (Hain Life Sciences) for the detection of second line resistant TB directly from stored smear positive sputum sediments.

Methodology/Principal Findings

The assay showed a diverse range of sensitivity and specificity, 91.26% [95% CI, 84–96] and 95.5% [95% CI, 87–99] for FQ (PPV ∼97% & NPV ∼ 87.67%), 56.19% [95%CI, 46–66] and 81% [95%CI, 66–91] for EMB (PPV ∼ 88.06% & NPV ∼ 43.21%) and 100% for SLD. Diagnostic accuracy for FQ, SLD and EMB was 94%, 100% and 63.51%, respectively. 1.17% (2/170) were heteroresistance strains, where the heteroresistance was linked to rrs gene. A varying rate of validity was observed 100% (170/170) for FQ, 94.11% (160/170) for EMB, 88.23% (150/170) for SLD.

Conclusions/Significance

Genotype MTBDRsl is simple, rapid, economical assay that can be used to detect commonly known resistance associated with Fluoroquinolone, second line injectable drugs and ethambutol. The assay detects the targeted resistance in less time as compared to phenotypic DST. But due to low NPV to FQ (88%) and EMB (43.21%), the assay results must be interpreted in coordination with the phenotypic DST.     

GenoType MTBDRplus Assay for Rapid Detection of Multidrug Resistance in Mycobacterium tuberculosis: A Meta-Analysis

Background

There is an urgent demand for rapid and accurate drug-susceptibility testing for the detection of multidrug-resistant tuberculosis. The GenoType MTBDRplus assay is a promising molecular kit designed for rapid identification of resistance to first-line anti-tuberculosis drugs, isoniazid and rifampicin. The aim of this meta-analysis was to evaluate the diagnostic accuracy of GenoType MTBDRplus in detecting drug resistance to isoniazid and rifampicin in comparison with the conventional drug susceptibility tests.

Methods

We searched PubMed, EMBASE, and Cochrane Library databases to identify studies according to predetermined criteria. A total of 40 studies were included in the meta-analysis. QUADAS-2 was used to assess the quality of included studies with RevMan 5.2. STATA 13.0 software was used to analyze the tests for sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and area under the summary receiver operating characteristic curves. Heterogeneity in accuracy measures was tested with Spearman correlation coefficient and Chi-square.

Results

Patient selection bias was observed in most studies. The pooled sensitivity (95% confidence intervals were 0.91 (0.88–0.94) for isoniazid, 0.96 (0.95–0.97) for rifampicin, and 0.91(0.86–0.94) for multidrug-resistance. The pooled specificity (95% CI) was 0.99 (0.98–0.99) for isoniazid, 0.98 (0.97–0.99) for rifampicin and 0.99 (0.99–1.00) for multidrug-resistance, respectively. The area under the summary receiver operating characteristic curves ranged from 0.99 to 1.00.

Conclusion

This meta-analysis determined that GenoType MTBDRplus had good accuracy for rapid detection of drug resistance to isoniazid and/or rifampicin of M. tuberculosis. MTBDRplus method might be a good alternative to conventional drug susceptibility tests in clinical practice.     

Identification of the Biosynthetic Gene Cluster and an Additional Gene for Resistance to the Antituberculosis Drug Capreomycin

Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance.     

Rapid,Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H) Strain Frequency in Genotype 1b HCV by Invader Assay

Daclatasvir and asunaprevir dual oral therapy is expected to achieve high sustained virological response (SVR) rates in patients with HCV genotype 1b infection. However, presence of the NS5A-Y93H substitution at baseline has been shown to be an independent predictor of treatment failure for this regimen. By using the Invader assay, we developed a system to rapidly and accurately detect the presence of mutant strains and evaluate the proportion of patients harboring a pre-treatment Y93H mutation. This assay system, consisting of nested PCR followed by Invader reaction with well-designed primers and probes, attained a high overall assay success rate of 98.9% among a total of 702 Japanese HCV genotype 1b patients. Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed. Our assay system showed a better lower detection limit of Y93H proportion than using direct sequencing, and Y93H frequencies obtained by this method correlated well with those of deep-sequencing analysis (r = 0.85, P <0.001). The proportion of the patients with the mutant strain estimated by this assay was 23.6% (164/694). Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10^-4), higher serum HCV RNA levels (p=4.3 x 10^-7), and lower HCC risk (p=6.9 x 10^-3) than those with the wild type strain. Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients.     

Rapid Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis Using a Multiplex Real-Time PCR Assay

Background

Arduous to differ clinically, extrapulmonary tuberculosis and focal complications of brucellosis remain important causes of morbidity and mortality in many countries. We developed and applied a multiplex real-time PCR assay (M RT-PCR) for the simultaneous detection of Mycobacterium tuberculosis complex and Brucella spp.

Methodology

Conventional microbiological techniques and M RT-PCR for M. tuberculosis complex and Brucella spp were performed on 45 clinical specimens from patients with focal complications of brucellosis or extrapulmonary tuberculosis and 26 control samples. Fragments of 207 bp and 164 bp from the conserved region of the genes coding for an immunogenic membrane protein of 31 kDa of B. abortus (BCSP31) and the intergenic region SenX3-RegX3 were used for the identification of Brucella and M. tuberculosis complex, respectively.

Conclusions

The detection limit of the M RT-PCR was 2 genomes per reaction for both pathogens and the intra- and inter-assay coefficients of variation were 0.44% and 0.93% for Brucella and 0.58% and 1.12% for Mycobacterium. M RT-PCR correctly identified 42 of the 45 samples from patients with tuberculosis or brucellosis and was negative in all the controls. Thus, the overall sensitivity, specificity, PPV and NPV values of the M RT PCR assay were 93.3%, 100%, 100% and 89.7%, respectively, with an accuracy of 95.8% (95% CI, 91.1%–100%). Since M RT-PCR is highly reproducible and more rapid and sensitive than conventional microbiological tests, this technique could be a promising and practical approach for the differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.     

Rapid Antigen Group A Streptococcus Test to Diagnose Pharyngitis: A Systematic Review and Meta-Analysis

Background

Pharyngitis management guidelines include estimates of the test characteristics of rapid antigen streptococcus tests (RAST) using a non-systematic approach.

Objective

To examine the sensitivity and specificity, and sources of variability, of RAST for diagnosing group A streptococcal (GAS) pharyngitis.

Data Sources

MEDLINE, Cochrane Reviews, Centre for Reviews and Dissemination, Scopus, SciELO, CINAHL, guidelines, 2000–2012.

Study Selection

Culture as reference standard, all languages.

Data Extraction and Synthesis

Study characteristics, quality.

Main Outcome(s) and Measure(s)

Sensitivity, specificity.

Results

We included 59 studies encompassing 55,766 patients. Forty three studies (18,464 patients) fulfilled the higher quality definition (at least 50 patients, prospective data collection, and no significant biases) and 16 (35,634 patients) did not. For the higher quality immunochromatographic methods in children (10,325 patients), heterogeneity was high for sensitivity (inconsistency [I²] 88%) and specificity (I² 86%). For enzyme immunoassay in children (342 patients), the pooled sensitivity was 86% (95% CI, 79–92%) and the pooled specificity was 92% (95% CI, 88–95%). For the higher quality immunochromatographic methods in the adult population (1,216 patients), the pooled sensitivity was 91% (95% CI, 87 to 94%) and the pooled specificity was 93% (95% CI, 92 to 95%); however, heterogeneity was modest for sensitivity (I² 61%) and specificity (I² 72%). For enzyme immunoassay in the adult population (333 patients), the pooled sensitivity was 86% (95% CI, 81–91%) and the pooled specificity was 97% (95% CI, 96 to 99%); however, heterogeneity was high for sensitivity and specificity (both, I² 88%).

Conclusions

RAST immunochromatographic methods appear to be very sensitive and highly specific to diagnose group A streptococcal pharyngitis among adults but not in children. We could not identify sources of variability among higher quality studies. The present systematic review provides the best evidence for the wide range of sensitivity included in current guidelines.     

A Bioluminescence Assay Using Nitrosomonas europaea for Rapid and Sensitive Detection of Nitrification Inhibitors

An expression vector for the luxAB genes, derived from Vibrio harveyi, was introduced into Nitrosomonas europaea. Although the recombinant strain produced bioluminescence due to the expression of the luxAB genes under normal growing conditions, the intensity of the light emission decreased immediately, in a time-and dose-dependent manner, with the addition of ammonia monooxygenase inhibitors, such as allylthiourea, phenol, and nitrapyrin. When whole cells were challenged with several nitrification inhibitors and toxic compounds, a close relationship was found between the change in the intensity of the light emission and the level of ammonia-oxidizing activity. The response of bioluminescence to the addition of allylthiourea was considerably faster than the change in the ammonia-oxidizing rate, measured as both the O₂ uptake and NO₂⁻ production rates. The bioluminescence of cells inactivated by ammonia monooxygenase inhibitor was recovered rapidly by the addition of certain substrates for hydroxylamine oxidoreductase. These results suggested that the inhibition of bioluminescence was caused by the immediate decrease of reducing power in the cell due to the inactivation of ammonia monooxygenase, as well as by the destruction of other cellular metabolic pathways. We conclude that the assay system using luminous Nitrosomonas can be applied as a rapid and sensitive detection test for nitrification inhibitors, and it will be used to monitor the nitrification process in wastewater treatment plants.     

A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals:A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis

The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species.Moreover,these novel tools have become easier to use and have resulted in a great increase of applications.Whilst gene knockout(KO) or knockin(KI) animal models are relatively easy to achieve,there is a bottleneck in the detection and analysis of these mutations.Although several methods exist to detect these targeted mutations,we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process.The HMA-CE method uses a simple PCR amplification of genomic DNA(gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes(HD) signature for each mutation.This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.     

GSTT1 Null Genotype Contributes to Lung Cancer Risk in Asian Populations: A Meta-Analysis of 23 Studies

Background

Genetic variation in glutathione S-transferases (GSTs) may contribute to lung cancer risk. Many studies have investigated the correlation between the Glutathione S-transferase T1 (GSTT1) null genotype and lung cancer risk in Asian population but yielded inconclusive results.

Methodology/Principal Findings

We performed a meta-analysis of 23 studies including 4065 cases and 5390 controls. We assessed the strength of the association of GSTT1 with lung cancer risk and performed sub-group analyses by source of controls, smoking status, histological types, and sample size. A statistically significant correlation between GSTT1 null genotype and lung cancer in Asian population was observed (OR = 1.28, 95% CI = 1.10, 1.49; P_{heterogeneity}<0.001 and I² = 62.0%). Sub-group analysis revealed there was a statistically increased lung cancer risk in ever-smokers who carried the GSTT1 null genotype (OR = 1.94, 95% CI = 1.27, 2.96; P heterogeneity = 0.02 and I² = 58.1%). It was also indicated that GSTT1 null genotype could increase lung cancer risk among population-based studies (OR = 1.25, 95% CI = 1.04, 1.50; P_{heterogeneity} = 0.003 and I² = 56.8%). The positive association was also found in studies of sample size (≤500 participants) (OR = 1.34, 95% CI = 1.10, 1.62; P_{heterogeneity}<0.001 and I² = 65.4%).

Conclusions

These meta-analysis results suggest that GSTT1 null genotype is associated with a significantly increased risk of lung cancer in Asian population.     

Efficacy and Tolerability of Telaprevir for Chronic Hepatitis Virus C Genotype 1 Infection: A Meta-Analysis

Background

Chronic hepatitis C virus (HCV) infection is one of the leading causes of hepatic cirrhosis and hepatocellular carcinoma, and HCV genotype 1 is the most prevalent genotype and is resistant to current standard therapy. We performed this meta-analysis to evaluate the efficacy and safety of telaprevir-based therapy for chronic HCV genotype 1 infection.

Methods

We included randomized controlled trials with no year or language restriction. All data were analyzed using a random-effects model by Review Manager v5.0. The primary outcome was the proportion of patients achieving sustained virologic response (SVR), and the secondary outcomes were HCV relapse rate, incidence of severe adverse events (SAEs), and discontinuation due to adverse events.

Results

The proportion of achieving SVR was significantly higher in the telaprevir group (odds ratio [OR] = 3.40 [1.92, 6.00], P<0.0001; I² = 87%) regardless of a patients’ previous treatment status. It was also significantly higher in the 24-week and 48-week treatment groups (OR = 4.52 [2.08, 9.81], P<0.001; I² = 85%, and OR = 4.05 [1.56, 10.56], P = 0.004; I² = 92%, respectively), while it was comparable in the 12-week treatment group (OR = 1.32 [0.63, 2.75], P = 0.46; I² = 35%). In addition, the HCV relapse rate was significantly reduced in the telaprevir group (OR = 0.28 [0.16, 0.49], P<0.001; I² = 76%). However, the incidence of SAE (OR = 1.56 [1.15, 2.10], P = 0.004; I² = 0%) and study discontinuation due to adverse events (OR = 2.24 [1.43, 3.50], P<0.001; I² = 37%) were significantly higher in the telaprevir group.

Conclusion

Despite its higher incidence of SAEs and discontinuation due to adverse events, telaprevir-based therapy can increase the proportion of achieving SVR in both previously treated and untreated chronic HCV-1 infected patients.     

A Rapid, High-Yield Purification of L-Alanine:4,5-Dioxovalerate Transaminase from Rat Kidney Mitochondria Using an Improved Enzyme Assay Method

The present report documents an improved enzyme assay method for the mammalian L-alanine:4,5-dioxovalerate transaminase which is of significant utility in work with crude tissue homogenates, cell cultures, or purified enzyme preparations. We also describe a new and rapid purification procedure for this enzyme from rat kidney mitochondria. The three-step procedure involves the use of digitonin and lubrol for mitochondrial matrix preparation and L-alanine-Sepharose 4B column chromatography followed by gel filtration on Sepharose 6B. By this procedure it is possible to obtain a highly purified enzyme preparation in a relatively short time with a 37.5% yield.     

Benefits of Therapeutic Drug Monitoring of Vancomycin: A Systematic Review and Meta-Analysis

Background and Objective

The necessity of therapeutic drug monitoring (TDM) for vancomycin is controversial. The objective of the current review was to evaluate the available evidence for the necessity of TDM in patients given vancomycin to treat Gram-positive infections.

Methods

Medline, Embase, Web of Sciences, the Cochrane Library and two Chinese literature databases (CNKI, CBM) were searched. Randomized controlled studies and observational studies that compared the clinical outcomes of TDM groups vs. non-TDM groups were included. Two reviewers independently extracted the data. The primary outcome was clinical efficacy of therapy. Secondary outcomes included vancomycin associated nephrotoxicity, duration of vancomycin therapy, length of hospital stay, and mortality. Meta-analysis was performed using the Mantel-Haenszel fixed effect method (FEM). Odds ratios (ORs) or weighted mean differences (WMD) with 95% confidence intervals (95%CIs) were calculated for categorical and continuous outcomes, respectively.

Results

One randomized controlled trial (RCT) and five cohort studies were included in the meta-analysis. Compared with non-TDM groups, TDM groups had significantly higher rates of clinical efficacy (OR = 2.62, 95%CI 1.34–5.11 P = 0.005) and decreased rates of nephrotoxicity (OR = 0.25, 95%CI 0.13–0.48 P<0.0001). Subgroup analyses showed that TDM group had significantly higher rates of clinical efficacy in both cohort studies subgroup (OR = 3.04, 95%CI 1.34–6.90) and in Asian population subgroup (OR = 3.04, 95%CI 1.34–6.90). TDM group had significantly decreased rates of nephrotoxicity in all subgroup. There was no significant difference in duration of vancomycin therapy (WMD = −0.40, 95%CI −2.83–2.02 P = 0.74) or length of stay (WMD = −1.01, 95%CI −7.51-5.49 P = 0.76) between TDM and non-TDM groups. Subgroup analyses showed there were no differences in duration of vancomycin therapy. Only one study reported mortality rates.

Conclusions

Studies to date show that TDM significantly increases the rate of clinical efficacy and decreases the rate of nephrotoxicity in patients treated with vancomycin.     

Association between Glutathione S-Transferase T1 Null Genotype and Gastric Cancer Risk: A Meta-Analysis of 48 Studies

Background

Glutathione S-transferases (GSTs) have proved to be involved in the detoxifying several carcinogens and may play an important role in carcinogenesis of cancer. Previous studies on the association between Glutathione S-transferase T1 (GSTT1) polymorphism and gastric cancer risk reported inconclusive results. To clarify the possible association, we conducted a meta-analysis of eligible studies.

Methods

We searched in the Pubmed, Embase, and Wangfang Medicine databases for studies assessing the association between GSTT1 null genotype and gastric cancer risk. The pooled odds ratio (OR) and its 95% confidence interval (95%CI) was calculated to assess the strength of the association. A total of 48 studies with a total of 24,440 individuals were ultimately eligible for meta-analysis.

Results

Overall, GSTT1 null genotype was significantly associated with increased risk of gastric cancer (Random-effect OR = 1.23, 95%CI 1.13–1.35, P _OR <0.001, I² = 45.5%). Significant association was also found in Caucasians, East Asians, and Indians (P _Caucasians = 0.010; P _{East Asians} = 0.003; P _Indians = 0.017). After adjusting for other confounding variables, GSTT1 null genotype was also significantly associated with increased risk of gastric cancer (Random-effect OR = 1.43, 95%CI 1.20–1.71, P _OR <0.001, I² = 48.1%).

Conclusion

The meta-analysis provides strong evidence for the significant association between GSTT1 null genotype and increased risk of gastric cancer.     

Increased Transmission of Mycobacterium tuberculosis Beijing Genotype Strains Associated with Resistance to Streptomycin: A Population-Based Study

Background

Studies have shown that the Mycobacterium tuberculosis Beijing genotype is an emerging pathogen that is frequently associated with drug resistance. This suggests that drug resistant Beijing strains have a relatively high transmission fitness compared to other drug-resistant strains.

Methods and Findings

We studied the relative transmission fitness of the Beijing genotype in relation to anti-tuberculosis drug resistance in a population-based study of smear-positive tuberculosis patients prospectively recruited and studied over a 4-year period in rural Vietnam. Transmission fitness was analyzed by clustering of cases on basis of three DNA typing methods. Of 2531 included patients, 2207 (87%) were eligible for analysis of whom 936 (42%) were in a DNA fingerprint cluster. The clustering rate varied by genotype with 292/786 (37%) for the Beijing genotype, 527/802 (67%) for the East-African Indian (EAI) genotype, and 117/619 (19%) for other genotypes. Clustering was associated with the EAI compared to the Beijing genotype (adjusted odds ratio (OR^adj) 3.4: 95% CI 2.8–4.4). Patients infected with streptomycin-resistant strains were less frequently clustered than patients infected with streptomycin-susceptible strains when these were of the EAI genotype (OR^adj 0.6, 95% CI 0.4–0.9), while this pattern was reversed for strains of the Beijing genotype (OR^adj 1.3, 95% CI 1.0–1.8, p for difference 0.002). The strong association between Beijing and MDR-TB (OR^adj 7.2; 95% CI 4.2–12.3) existed only if streptomycin resistance was present.

Conclusions

Beijing genotype strains showed less overall transmissibility than EAI strains, but when comparisons were made within genotypes, Beijing strains showed increased transmission fitness when streptomycin-resistant, while the reverse was observed for EAI strains. The association between MDR-TB and Beijing genotype in this population was strongly dependent on resistance to streptomycin. Streptomycin resistance may provide Beijing strains with a fitness advantage over other genotypes and predispose to multidrug resistance in patients infected with Beijing strains.     

Focus: Antimicrobial Resistance: The Status of Carbapenem Resistance in Cystic Fibrosis: A Systematic Review and Meta-Analysis

Background: Antibiotic resistance in cystic fibrosis (CF) is a well-known phenomenon. However, the comprehensive epidemiological impact of antibiotic resistance in CF is not clearly documented. So, this meta-analysis evaluated the proportion rates of carbapenem resistance (imipenem, meropenem, and doripenem) in CF based on publication date (1979-2000, 2001-2010, and 2011-2021), continents, pathogens, and antimicrobial susceptibility testing (AST). Methods: We searched studies in PubMed, Scopus, and Web of Science (until April 2021). Statistical analyses were conducted using STATA software (version 14.0). Results: The 110 studies included in the analysis were performed in 25 countries and investigated 13,324 pathogens associated with CF. The overall proportion of imipenem, meropenem, and doripenem resistance in CF were 43% (95% CI 36-49), 48% (95% CI 40-57), 28% (95% CI 23-33), and 45% (95% CI 32-59), respectively. Our meta-analysis showed that trends of imipenem, meropenem, and doripenem-resistance had gradual decreases over time (1979-2021). This could be due to the limited clinical effectiveness of these antibiotics to treat CF cases over time. Among the opportunistic pathogens associated with CF, the highest carbapenem resistance rates were shown in Stenotrophomonas maltophilia, Burkholderia spp., Pseudomonas aeruginosa, and Staphylococcus aureus. The highest and lowest carbapenem resistance rates among P. aeruginosa in CF patients were shown against meropenem (23%) and doripenem (39%). Conclusions: We showed that trends of carbapenem resistance had decreased over time (1979-2021). This could be due to the limited clinical effectiveness of these antibiotics to treat CF cases over time. Plans should be directed to fight biofilm-associated infections and prevent the emergence of mutational resistance. Systematic surveillance for carbapenemase-producing pathogens in CF by molecular surveillance is necessitated.     

Mitochondrial Contributions to Cancer Cell Physiology: Redox Balance, Cell Cycle, and Drug Resistance

Alterations in the biochemistry of mitochondria have been associated with cell transformation and the acquisition of drug resistance to certain chemotherapeutic agents, suggesting that mitochondria may play a supportive role for the cancer cell phenotype. Mitochondria are multifunctional organelles that contribute to the cellular adenosine triphosphate (ATP) pool and cellular redox balance through the production of reactive oxygen intermediates (ROI). Our laboratory has focused on these mitochondrial functions in the context of cancer cell physiology to evaluate the potential role of mitochondria as controllers of tumour cell proliferation. Low concentrations of ROI have been implicated as messengers in intracellular signal transduction mechanisms; thus an imbalance of ROI production from the mitochondria may support cancer cell growth. In addition, suppression of mitochondrial ATP production can halt cell cycle progression at two energetic checkpoints, suggesting that the use of tumor-selective agents to reduce ATP production may offer a therapeutic target for cancer growth control.     

Interferon Resistance of Hepatitis C Virus Genotype 1b: Relationship to Nonstructural 5A Gene Quasispecies Mutations

A 40-amino-acid sequence located in the nonstructural 5A (NS5A) protein of hepatitis C virus genotype 1b (HCV-1b) was recently suggested to be the interferon sensitivity-determining region (ISDR), because HCV-1b strains with an ISDR amino acid sequence identical to that of the prototype strain HCV-J were found to be resistant to alpha interferon (IFN-α) whereas strains with amino acid substitutions were found to be sensitive (N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, N. Izumi, F. Marumo, and C. Sato, J. Clin. Invest. 96:224–230, 1995; N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, Y. Ogura, N. Izumi, F. Marumo, and C. Sato, N. Engl. J. Med. 334:77–81, 1996). We used single-strand conformation polymorphism (SSCP) analysis, combined with cloning and sequencing strategies, to characterize NS5A quasispecies in HCV-1b-infected patients and determine the relationships between pre- and posttreatment NS5A quasispecies mutations and the IFN-α sensitivity of HCV-1b. The serine residues involved in phosphorylation of NS5A protein were highly conserved both in the various patients and in quasispecies in a given patient, suggesting that phosphorylation is important in NS5A protein function. A hot spot for amino acid substitutions was found at positions 2217 to 2218; it could be the result of either strong selection pressure or tolerance to these amino acid replacements. The proportion of synonymous mutations was significantly higher than the proportion of nonsynonymous mutations, suggesting that genetic variability in the region studied was the result of high mutation rates and viral replication kinetics rather than of positive selection. Sustained HCV RNA clearance was associated with low viral load and low nucleotide sequence entropy, suggesting (i) that the replication kinetics when treatment is started plays a critical role in HCV-1b sensitivity to IFN-α and (ii) that HCV-1b resistance to IFN-α could be conferred by numerous and/or related mutations that could be patient specific and located at different positions throughout the viral genome and could allow escape variants to be selected by IFN-α-stimulated immune responses. No NS5A sequence appeared to be intrinsically resistant or sensitive to IFN-α, but the HCV-J sequence was significantly more frequent in nonresponder quasispecies than in sustained virological responder quasispecies, suggesting that the balance between NS5A quasispecies sequences in infected patients could have a subtle regulatory influence on HCV replication.     

Thin Layer Chromatography of Radiolabeled Oligonucleotide Analogues: A Rapid and Sensitive Purity Assay

Abstract

Oligonucleotide analogues are being developed for use in cell culture, animals and for therapy. Prior to use it is important to have an indication of the oligomers' purity. Thin layer chromatography (TLC) has been applied to analyze hosphoromonothioate and phosphorodithioate oligonucleotides radiolabeled with either ³²P Or ¹⁴C. TLC coupled with radioactivity has been compared to Polyacrylamide Gel Electrophoresis (PAGE) and High Pressure Liquid Chromatography (HPLC). TLC is a rapid and sensitive alternative to these methods and is particularly suited for chemically modified oligonucleotides.