Enhancement of cytochrome c catalytic behaviour by affecting the heme environment using functionalized carbon-based nanomaterials

The effect of carbon-based nanomaterials (CBNs), such as multi-wall carbon nanotubes (CNTs) and graphene oxide (GO) nanomaterials functionalized with carboxyl, alkyl and amine groups, on the peroxidase-like activity and structure of cytochrome c (cyt c) was investigated. The catalytic efficiency of cyt c increases up to 78-fold in the presence of graphene oxide and up to 2.5-fold in the presence of other functionalized CBNs. Moreover, the use of functionalized CBNs enhances the thermal stability of the protein as well as its tolerance against hydrogen peroxide up to 2.5-fold. UV–vis and circular dichroism spectroscopy studies suggest that the increase in the peroxidase activity of cyt c in the presence of some functionalized GO nanomaterials, correlates to perturbations of the heme microenvironment, while the secondary structure of the enzyme remains intact. These results indicate that the beneficial effect the functionalized CBNs have on the activity and on the stability of cyt c depends on CBNs geometry and surface functionalization.     

A phylogenomic resolution for the taxonomy of Aegean green lizards

Lacerta pamphylica and Lacerta trilineata are two currently recognized green lizard species with a historically problematic taxonomy. In cases of tangled phylogenies, next-generation sequencing and double-digest restriction-site-associated DNA protocols can provide a wealth of genomic data and resolve difficult taxonomic issues. Here, we generated genome-wide SNPs and mitochondrial sequences, and applied molecular species delimitation approaches to provide a stable taxonomy for the Aegean green lizards. Mitochondrial gene trees, genetic cluster delimitation and population structure analyses converged into recognizing the populations of (a) L. pamphylica, (b) east Aegean islands, Anatolia and Thrace (diplochondrodes lineage), (c) central Aegean islands (citrovittata), and (d) remaining Balkan populations and islands (trilineata), as separate clusters. Phylogenomic analyses revealed a split into two major clades, east and west of the Aegean Barrier, unambiguously showing a sister–clade relationship between pamphylica and diplochondrodes, rendering L. trilineata paraphyletic. Species delimitation models were tested in a Bayesian framework using the genomic SNPs: lumping all populations into a single ‘species’ had the lowest likelihood but the current taxonomy was also outperformed by all other models. All lines of evidence support the Pamphylian green lizard as a valid species; thus, east Aegean L. trilineata should also be considered a distinct species under the name Lacerta diplochondrodes. Finally, evidence from the mitochondrial and nuclear genomes is overwhelmingly in favour of recognizing the morphologically distinct Cycladian green lizards as a distinct species. We propose their elevation to full species under the name Lacerta citrovittata. All remaining insular and continental populations of the Balkan Peninsula represent the species L. trilineata.     

Energy,Nitrogen, and Farm Surplus Transitions in Agriculture from Historical Data Modeling. France, 1882–2013.

This article addresses agricultural metabolism and transitions for energy, nitrogen, farm production, self‐sufficiency, and surplus from historical data since the nineteenth century. It builds on an empirical data set on agricultural production and production means in France covering 130 consecutive years (1882–2013). Agricultural transitions have increased the net production and surplus of farms by a factor of 4 and have zeroed self‐sufficiency. The energy consumption remained quasi‐stable since 1882, but the energy and nitrogen structure of agriculture fully changed. With an EROI (energy return to energy invested) of 2 until 1950, preindustrial agriculture consumed as much energy to function as it provided in exportable surplus to sustain the nonagricultural population. The EROI doubled to 4 over the last 60 years, driven, on the one hand, by efficiency improvements in traction through the replacement of draft animals by motors and, on the other hand, by the joint increase in crop yields and efficiency in nitrogen use. Agricultural energy and nitrogen transitions shifted France from a self‐sufficiency agri‐food‐energy regime to a fossil‐dependent food export regime. Knowledge of resource conversion mechanisms over the long duration highlights the effects of changing agricultural metabolism on the system's feeding capacity. Farm self‐sufficiency is an asset against fossil fuel constraints, price volatility, and greenhouse gas emissions, but it equates to lower farm surplus in support of urbanization.     

Synthesis of Novel Thiopurine Pyranonucleosides: Evaluation of Their Bioactivity

We report the synthesis of novel thiopurine pyranonucleosides. Direct coupling of silylated 6-mercaptopurine and 6-thioguanine with the appropriate pyranoses 1a–e via Vorbrüggen nucleosidation, gave the N-9 linked mercaptopurine 2a–e and thioguanine 4a–e nucleosides, while their N-7 substituted congeners 10a–e and 7a–e, were obtained through condensation of the same acetates with 6-chloro and 2-amino-6-chloropurines, followed by subsequent thionation. Nucleosides 3a–e, 5a–e, 8a–e, and 11a–e were evaluated for their cytostatic activity in three different tumor cell proliferative assays.     

The Multi-Drug Resistant Tuberculosis Diagnosis and Treatment Cascade in Bangladesh

Objectives

To determine, in areas supported by BRAC, Bangladesh i) the pre-diagnosis and pre-treatment attrition among presumptive and confirmed Multi-Drug Resistant Tuberculosis (MDR-TB) patients and ii) factors associated with attrition.

Methods

This was a retrospective cohort study involving record review. Presumptive MDR-TB patients from peripheral microscopy centres serving 60% of the total population of Bangladesh were included in the study. Attrition and turnaround time for MDR-TB diagnosis by Xpert MTB/RIF and treatment initiation were calculated between July 2012 and June 2014.

Results

Of 836 presumptive MDR-TB patients referred from 398 peripheral microscopy centres, 161 MDR-TB patients were diagnosed. The number of diagnosed MDR-TB patients was less than country estimates of MDR-TB patients (2000 cases) during the study period. Among those referred, pre-diagnosis and pre-treatment attrition was 17% and 21% respectively. Median turnaround time for MDR-TB testing, result receipt and treatment initiation was four, zero and five days respectively. Farmers (RR=2.3, p=0.01) and daily wage laborers (RR=2.1, p=0.04) had twice the risk of having pre-diagnosis attrition. Poor record-keeping and unreliable upkeep of presumptive MDR-TB patient databases were identified as challenges at the peripheral microscopy centres.

Conclusion

There was a low proportion of pre-diagnosis and pre-treatment attrition in patients with presumptive and confirmed MDR-TB under programmatic conditions. However, the recording and reporting system did not detect all presumptive MDR-TB patients, highlighting the need to improve the system in order to prevent morbidity, mortality and transmission of MDR-TB in the community.     

“They Know,They Agree,but They Don’t Do”- The Paradox of Tuberculosis Case Notification by Private Practitioners in Alappuzha District,Kerala, India

Background

Despite being a recognized standard of tuberculosis (TB) care internationally, mandatory TB case notification brings forth challenges from the private sector. Only three TB cases were notified in 2013 by private practitioners compared to 2000 TB cases notified yearly from the public sector in Alappuzha district. The study objective was to explore the knowledge, opinion and barriers regarding TB Notification among private practitioners offering TB services in Alappuzha, Kerala state, India.

Methods & Findings

This was a mixed-methods study with quantitative (survey) and qualitative components conducted between December 2013 and July 2014. The survey, using a structured questionnaire, among 169 private practitioners revealed that 88% were aware of mandatory notification. All patient-related details requested in the notification form (except government-issued identification number) were perceived to be important and easy to provide by more than 80% of practitioners. While more than 95% felt that notification should be mandatory, punitive action in case of failure to notify was considered unnecessary by almost two third. General practitioners (98%) were more likely to be aware of notification than specialists (84 %). (P<0.01). Qualitative purposive personal interviews (n=34) were carried out among private practitioners and public health providers. On thematic framework analysis of the responses, barriers to TB notification were grouped into three themes: ‘private provider misconceptions about notification’, ‘patient confidentiality, and stigma and discrimination ’and ‘lack of cohesion and coordination between public and private sector’. Private practitioners did not consider it necessary to notify TB cases treated with daily regimen.

Conclusion

Communication strategies like training, timely dissemination of information of policy changes and one-to-one dialogue with private practitioners to dispel misconceptions may enhance TB notification. Trust building strategies like providing feedback about referred cases from private sector, health personnel visit or a liaison private doctor may ensure compliance to public health activities.     

Effects of particulate matter exposure on blood 5-hydroxymethylation: results from the Beijing truck driver air pollution study

Previous studies have reported epigenetic changes induced by environmental exposures. However, previous investigations did not distinguish 5-methylcytosine (5mC) from a similar oxidative form with opposite functions, 5-hydroxymethylcytosine (5hmC). Here, we measured blood DNA global 5mC and 5hmC by ELISA and used adjusted mixed-effects regression models to evaluate the effects of ambient PM₁₀ and personal PM_2.5 and its elemental components—black carbon (BC), aluminum (Al), calcium (Ca), potassium (K), iron (Fe), sulfur (S), silicon (Si), titanium (Ti), and zinc (Zn)—on blood global 5mC and 5hmC levels. The study was conducted in 60 truck drivers and 60 office workers in Beijing, China from The Beijing Truck Driver Air Pollution Study at 2 exams separated by one to 2 weeks. Blood 5hmC level (0.08%) was ∼83-fold lower than 5mC (6.61%). An inter-quartile range (IQR) increase in same-day PM₁₀ was associated with increases in 5hmC of 26.1% in office workers (P = 0.004), 20.2% in truck drivers (P = 0.014), and 21.9% in all participants combined (P < 0.001). PM₁₀ effects on 5hmC were increasingly stronger when averaged over 4, 7, and 14 d preceding assessment (up to 132.6% for the 14-d average in all participants, P < 0.001). PM₁₀ effects were also significant after controlling for multiple testing (family-wise error rate; FWER < 0.05). 5hmC was not correlated with personal measures of PM_2.5 and elemental components (FWER > 0.05). 5mC showed no correlations with PM₁₀, PM_2.5, and elemental components measures (FWER > 0.05). Our study suggests that exposure to ambient PM₁₀ affects 5hmC over time, but not 5mC. This finding demonstrates the need to differentiate 5hmC and 5mC in environmental studies of DNA methylation.     

Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative,Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing^TM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.     

Doing No Harm? Adverse Events in a Nation-Wide Cohort of Patients with Multidrug-Resistant Tuberculosis in Nigeria

Background

Adverse events (AEs) of second line anti-tuberculosis drugs (SLDs) are relatively well documented. However, the actual burden has rarely been described in detail in programmatic settings. We investigated the occurrence of these events in the national cohort of multidrug-resistant tuberculosis (MDR-TB) patients in Nigeria.

Method

This was a retrospective, observational cohort study, using pharmacovigilance data systematically collected at all MDR-TB treatment centers in Nigeria. Characteristics of AEs during the intensive phase treatment were documented, and risk factors for development of AEs were assessed.

Results

Four hundred and sixty patients were included in the analysis: 62% were male; median age was 33 years [Interquartile Range (IQR):28–42] and median weight was 51 kg (IQR: 45–59). Two hundred and three (44%) patients experienced AEs; four died of conditions associated with SLD AEs. Gastro-intestinal (n = 100), neurological (n = 75), ototoxic (n = 72) and psychiatric (n = 60) AEs were the most commonly reported, whereas ototoxic and psychiatric AEs were the most debilitating. Majority of AEs developed after 1–2 months of therapy, and resolved in less than a month after treatment. Some treatment centers were twice as likely to report AEs compared with others, highlighting significant inconsistencies in reporting at different treatment centers. Patients with a higher body weight had an increased risk of experiencing AEs. No differences were observed in risk of AEs between HIV-infected and uninfected patients. Similarly, age was not significantly associated with AEs.

Conclusion

Patients in the Nigerian MDR-TB cohort experienced a wide range of AEs, some of which were disabling and fatal. Early identification and prompt management as well as standardized reporting of AEs at all levels of healthcare, including the community is urgently needed. Safer regimens for drug-resistant TB with the shortest duration are advocated.     

Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

Introduction

Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity.

Methods

Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood.

Results

The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood.

Conclusion

Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.