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Background Depression is common amongst patients receiving haemodialysis (HD). Assessment and intervention when faced with language and cultural barriers is challenging. To support clinician decisions, we conducted a cross-sectional study to assess the use of culturally adapted and translated versions of commonly-used depression screening questionnaires with South Asian patients receiving HD in England. Methods Patients completed adapted versions of the Patient Health Questionnaire (PHQ-9), the Centre for Epidemiological Studies Depression Scale Revised (CESD-R), and the Beck Depression Inventory II (BDI-II). All questionnaires were available in Gujarati, Punjabi, Urdu, and Bengali. A comparative sample of white-Europeans completed the questionnaires in English. The research was based across 9 National Health Service (NHS) Trusts in England. Structural validity of translated questionnaires was assessed using confirmatory factor analysis. Diagnostic accuracy was explored in a subgroup of South Asians against ICD-10 categories using the Clinical Interview Schedule Revised (CIS-R) with receiver operating curve (ROC) analysis. Results 229 South Asian and 120 white-European HD patients participated. A single latent depression factor largely accounted for the correlations between items of the PHQ-9, CESD-R and BDI-II. Issues with measurement equivalence implied that scores on the translations may not be comparable with the English language versions. Against CIS-R based ICD-10 diagnosis of depression, sensitivity was modest across scales (50–66.7%). Specificity was higher (81.3–93.8%). Alternative screening cut-offs did not improve positive predictive values. Conclusions Culturally adapted translations of depression screening questionnaires are useful to explore symptom endorsement amongst South Asian patients. However, data indicate that standard cut-off scores may not be appropriate to classify symptom severity. Use of the CIS-R algorithms for optimal case identification requires further exploration in this setting. Strategies to encourage recruitment of under-represented groups in renal research are also warranted, especially for in-depth discussions related to psychological care needs.

In our search for bioactive mushrooms native to British Columbia, we determined that the ethanol extracts from fruiting bodies of the terrestrial polypore Albatrellus flettii had potent anti-cell viability activity. Using bioassay-guided fractionation, mass spectrometry and nuclear magnetic resonance, we successfully isolated three known compounds (grifolin, neogrifolin and confluentin). These compounds represent the major anti-cell viability components from the ethanol extracts of A. flettii. We also identified a novel biological activity for these compounds, specifically in down-regulating KRAS expression in two human colon cancer cell lines. Relatively little is known about the anti-cell viability activity and mechanism of action of confluentin. For the first time, we show the ability of confluentin to induce apoptosis and arrest the cell cycle at the G2/M phase in SW480 human colon cancer cells. The oncogenic insulin-like growth factor 2 mRNA-binding protein 1 (IMP1) has been previously shown to regulate KRAS mRNA expression in colon cancer cells, possibly through its ability to bind to the KRAS transcript. Using a fluorescence polarization assay, we show that confluentin dose-dependently inhibits the physical interaction between KRAS RNA and full-length IMP1. The inhibition also occurs with truncated IMP1 containing the KH1 to KH4 domain (KH1to4 IMP1), but not with the di-domain KH3 and KH4 (KH3&4 IMP1). In addition, unlike the control antibiotic neomycin, grifolin, neogrifolin and confluentin do not bind to KRAS RNA. These results suggest that confluentin inhibits IMP1-KRAS RNA interaction by binding to the KH1&2 di-domains of IMP1. Since the molecular interaction between IMP1 and its target RNAs is a pre-requisite for the oncogenic function of IMP1, confluentin should be further explored as a potential inhibitor of IMP1 in vivo.

Promoters are specified segments of DNA that lead to the initiation of transcription of a specific gene. The designing of a gene cassette for plant transformation is significantly dependent upon the specificity of a promoter. Constitutive Cauliflower mosaic virus promoter, CaMV35S, due to its developmental role, is the most commonly used promoter in plant transformation. While Gossypium hirsutum (Gh) being fiber-specific promoter (GhSCFP) specifically activates transcription in seed coat and fiber associated genes. The Expansin genes are renowned for their versatile roles in plant growth. The overexpression of Expansin genes has been reported to enhance fiber length and fineness. Thus, in this study, a local Cotton variety was transformed with Expansin (CpEXPA1) gene, in the form of two separate cassettes, each with a different promoter, named as 35SEXPA1 and FSEXPA1 expressed under CaMV35S and GhSCFP promoters respectively. Integration and Spatiotemporal relative expression of the transgene were studied in an advanced generation. GhSCFP bearing transgene expression was significantly higher in Cotton fiber than other plant parts. While transgene with CaMV35S promoter was found to be continually expressing in all tissues but the expression was lower in fiber than that expressed under GhSCFP. The temporal expression profile was quite interesting with a gradual increasing pattern of both constructs from 1DPA (days post anthesis) to 18DPA and decreased expression from 24 to 30 DPA. Besides the relative expression of promoters, fiber cellulose quantification and fluorescence intensity were also observed. The study significantly compared the two most commonly used promoters and it is deduced from the results that the GhSCFP promoter could be used more efficiently in fiber when compared with CaMV35S which being constitutive in nature preferred for expression in all parts of the plant.

IntroductionTuberculosis in children may be difficult to diagnose and is often not reported to routine surveillance systems. Understanding and addressing the tuberculosis (TB) case detection and reporting gaps strengthens national routine TB surveillance systems.ObjectiveThe present study aimed to measure the percentage of childhood TB cases that are diagnosed but not reported to the national surveillance system in Pakistan.DesignThe study design was cross sectional. The study was nationwide in 12 selected districts across Pakistan, each representing a cluster. Health facilities that diagnose and treat childhood TB from all sectors were mapped and invited to participate. Lists of child TB cases were created for the study period (April-June 2016) from all study facilities and compared against the list of child TB cases notified to the national TB surveillance system for the same districts and the same period.ResultsAll public and private health facilities were mapped across 12 sampled districts in Pakistan and those providing health services to child TB cases were included in the study. From all private health facilities, 7,125 children were found with presumptive TB during the study period. Of them, 5,258 were diagnosed with tuberculosis: 11% were bacteriologically-confirmed and 89% clinically-diagnosed; only 4% were notified to National TB Control Program. An additional 1,267 children with TB were also registered in the National TB Control Program. Underreporting was measured to be 78%.ConclusionThis is the first nationwide childhood TB inventory study globally and confirmed that childhood TB underreporting is very high in Pakistan. TB surveillance in the country must be strengthened to address this, with particular attention to guiding and supporting general practitioners and pediatricians to notify their TB cases.

BackgroundInfluenza is known to have a specific pattern of seasonality the reasons for which are yet to be fully ascertained. Temperate zones show influenza epidemic during the winter months. The tropical and subtropical regions show more diverse influenza outbreak patterns. This study explores the seasonality of influenza activity and predicts influenza peak based on historical surveillance time series data in Islamabad and Multan, Pakistan.MethodsThis is a descriptive study of routinely collected monthly influenza sentinel surveillance data and meteorological data from 2012-16 in two sentinel sites of Pakistan: Islamabad (North) and Multan (Central).ResultsMean number of cases of influenza and levels of precipitation were higher in Islamabad compared to Multan. Mean temperature and humidity levels were similar in both the cities. The number of influenza cases rose with decrease in precipitation and temperature in Islamabad during 2012-16, although the same cannot be said about humidity. The relationship between meteorological parameters and influenza incidence was not pronounced in case of Multan. The forecasted values in both the cities showed a significant peak during the month of January.ConclusionThe influenza surveillance system gave a better understanding of the disease trend and could accurately forecast influenza activity in Pakistan.

The burden of hepatitis C (HCV) infection in Pakistan is among the highest in the world, with a reported national HCV prevalence of 6.7% in 2014. In specific populations, such as in urban communities in Karachi, the prevalence is suspected to be higher. Interferon-free treatment for chronic HCV infection (CHC) could allow scale up, simplification and decentralization of treatment to such communities. We present an interim analysis over the course of February-December 2015 of an interferon-free, decentralised CHC programme in the community clinic in Machar Colony, Karachi, Pakistan.A retrospective analysis of a treatment cohort.There were 1,089 patients included in this analysis. Aspartate to platelet ratio index score was used to prioritize patients in terms of treatment initiation, with 242 patients placed in high priority for treatment and 202 starting treatment as scheduled. 169 patients started HCV treatment with Sofosbuvir-Ribavirin regimen according to HCV genotype over the course of 2015: of these, 35% had Hemoglobin reductions below 11.0 g/dl during the treatment course. Among the 153 patients (85%) with genotype 3 HCV infection, 84% of patients achieved sustained virologic response at 12 weeks following treatment completion (SVR 12).Outcomes of HCV treatment with all oral combination in an integrated, decentralized model of care for CHC in a primary care setting, using simplified diagnostic and treatment algorithms, are comparable to the outcomes achieved in clinical trial settings for Sofosbuvir-based regimens. Our results suggest the feasibility and the pertinence if including interferon-free treatment regimens in the national programme, at both provincial and national levels.

Background Currently, only 62% of incident tuberculosis (TB) cases are reported to the national programme in Pakistan. Several innovative interventions are being recommended to detect the remaining ‘missed’ TB cases. One such intervention involved expanding contact investigation to the community using the Xpert MTB/RIF test. Methods This was a before and after intervention study involving retrospective record review. Passive case finding and household contact investigation was routinely done in the pre-intervention period July 2011-June 2013. Four districts with a high concentration of slums were selected as intervention areas; Lahore, Rawalpindi, Faisalabad and Islamabad. Here, in the intervention period, July 2013-June 2015, contact investigation beyond household was conducted: all people staying within a radius of 50 metres (using Geographical Information System) from the household of smear positive TB patients were screened for tuberculosis. Those with presumptive TB were investigated using smear microscopy and the Xpert MTB/RIF test was performed on smear negative patients. All the diagnosed TB patients were linked to TB treatment and care. Results A total of 783043 contacts were screened for tuberculosis: 23741(3.0%) presumptive TB patients were identified of whom, 4710 (19.8%) all forms and 4084(17.2%) bacteriologically confirmed TB patients were detected. The contribution of Xpert MTB/RIF to bacteriologically confirmed TB patients was 7.6%. The yield among investigated presumptive child TB patients was 5.1%. The overall yield of all forms TB patients among investigated was 22.3% among household and 19.1% in close community. The intervention contributed an increase of case detection of bacteriologically confirmed tuberculosis by 6.8% and all forms TB patients by 7.9%. Conclusion Community contact investigation beyond household not only detected additional TB patients but also increased TB case detection. However, further long term assessments and cost-effectiveness studies are required before national scale-up.

Background & aims Hepatitis B virus (HBV) quantification is essential in the management of chronic hepatitis B, both to determine treatment eligibility and in the monitoring of treatment effect. This test, however, is rarely available in resource-limited settings due to high costs and stringent requirements for shipment and storage of plasma. Dried Blood Spots (DBS) can be a convenient alternative to plasma, but its use for HBV monitoring has not been investigated under real-life conditions in Africa. Methods The performance of DBS in HBV quantification was investigated using a modified commercial test (Abbott RealTime HBV assay). Paired DBS and plasma samples were collected from an HBV positive cohort in Addis Ababa, Ethiopia. DBS were stored at ambient temperature for 4-39 days before shipment to the laboratory. Results Twenty-six paired samples were selected covering the total range of quantification, from 2.14 log IU/ml to >7 log IU/ml. HBV was detected in 21 of 21 (100%) DBS from patients with a corresponding plasma viral load above 2.70 log IU/ml. The mean difference between plasma and DBS was 0.59 log IU/ml, and the correlation was strong (R2 = 0.92). In stability studies there was no significant change in DBS viral load after storage at room temperature for up to 12 weeks. Conclusions This study suggests that DBS can be a feasible and reliable alternative to plasma for quantification of HBV in resource-limited settings. DBS can expand access to antiviral treatment for patients in low- and middle-income countries.

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress.

A highly convenient method has been developed for the synthesis of (prop-2-ynyloxy) benzene and its derivatives. Differently substituted phenol and aniline derivatives were allowed to react with propargyl bromide in the presence of K2CO3 base and acetone as solvent. The compounds were synthesized in good yields (53–85%). Low cost, high yields and easy availability of compounds helped in the synthesis. Electron withdrawing groups favor the formation of stable phenoxide ion thus in turn favors the formation of product while electron donating groups do not favor the reaction. Phenol derivatives gave good yields as compared to that of aniline. As aprotic polar solvents favor SN2 type reactions so acetone provided best solvation for the reactions. K2CO3 was proved to be good for the synthesis. Antibacterial, Antiurease and NO scavenging activity of synthesized compounds were also examined. 4-bromo-2-chloro-1-(prop-2-ynyloxy)benzene 2a was found most active compound against urease enzyme with a percentage inhibition of 82.00±0.09 at 100 µg/mL with IC50 value of 60.2. 2-bromo-4-methyl-1-(prop-2-ynyloxy)benzene 2d was found potent antibacterial against Bacillus subtillus showing excellent inhibitory action with percentage inhibition of 55.67±0.26 at 100 µg/ml wih IC50 value of 79.9. Based on results, it can be concluded that some of the synthesized compounds may have potential antiurease and antibacterial effects against several harmful substances.

Background and objectivesVitamin D deficiency and endothelial dysfunction are non-traditional risk factors for cardiovascular events in chronic kidney disease. Previous studies in chronic kidney disease have failed to demonstrate a beneficial effect of vitamin D on arterial stiffness, left ventricular mass and inflammation but none have assessed the effect of vitamin D on microcirculatory endothelial function.Study designWe conducted a randomised controlled trial of 38 patients with non diabetic chronic kidney disease stage 3-4 and concomitant vitamin D deficiency (ResultsTwenty patients received ergocalciferol and 18 patients received placebo. After 6 months, there was a significant improvement in the ergocalciferol group in both endothelium dependent microcirculatory vasodilatation after iontophoresis of acetylcholine (p = 0.03) and a reduction in tissue advanced glycation end products (p = 0.03). There were no changes in sublingual microcirculatory parameters. Pulse pressure (p = 0.01) but not aortic pulse wave velocity was reduced. There were no significant changes in bone mineral parameters, blood pressure or left ventricular mass index suggesting that ergocalciferol improved endothelial function independently of these parameters. In parallel experiments, expression of endothelial nitric oxide synthase and activity were increased in human endothelial cells in a dose dependent manner.ConclusionsErgocalciferol improved microcirculatory endothelial function in patients with chronic kidney disease and concomitant vitamin D deficiency. This process may be mediated through enhanced expression and activity of endothelial nitric oxide synthase.Trial registrationClinical trials.gov NCT00882401.

Background We aimed to determine the prevalence of pulmonary tuberculosis (TB) amongst the adult population in 2010–2011 in Pakistan. Method A nationwide cross-sectional survey with multistage cluster sampling was conducted among adults (≥15 years) in 95 clusters in 2010–2011. All consenting participants were screened for cough and by chest X-ray. Participants with presumptive TB submitted two sputum samples for smear microscopy, culture, and molecular testing if needed. The TB prevalence estimates were adjusted for missing data and the cluster design. Result Of 131,329 eligible individuals, 105,913 (81%) participated in the survey, of whom 10,471 (9.9%) were eligible for sputum examination. We found 341 bacteriologically positive TB cases of whom 233 had sputum smear-positive TB. The adjusted prevalence estimates for smear and bacteriologically positive TB were 270/100,000 (95% confidence interval (CI) 217–323), and 398/100,000 (95% CI 333–463), respectively. Only 61% of the diagnosed TB cases screened positive on symptoms (cough >2wks), whereas the other TB cases were detected based on X-ray abnormalities. The TB prevalence increased with age and was 1.8 times higher among men than women. The prevalence-to-notification ratio of smear-positive TB was 3.1 (95% CI 2.5–3.7), was higher among men than women, and increased with age. Conclusion Our data suggest that there is under-detection and/or -notification of TB, especially among men and elderly. TB control should be strengthened specifically in these risk groups. X-ray examination should be combined with symptom screening to enhance case detection.

Refractive index imaging is a label-free technique that enables long-term monitoring of the internal structures and molecular composition in living cells with minimal perturbation. Existing tomographic methods for the refractive index imaging lack 3-D resolution and result in artifacts that prevent accurate refractive index quantification. To overcome these limitations without compromising the capability to observe a sample in its most native condition, we have developed a regularized tomographic phase microscope (RTPM) enabling accurate refractive index imaging of organelles inside intact cells. With the enhanced accuracy, we quantify the mass of chromosomes in intact living cells, and differentiate two human colon cancer lines, HT-29 and T84 cells, solely based on the non-aqueous (dry) mass of chromosomes. In addition, we demonstrate chromosomal imaging using a dual-wavelength RTPM, which shows its potential to determine the molecular composition of cellular organelles in live cells., National Institute of Biomedical Imaging and Bioengineering (U.S.) (9P41EB015871-26A1)

Binding of angiogenic molecules with cognate receptor tyrosine kinases (RTK) is required for angiogenesis however the precise link between RTK binding, endocytosis, and signaling requires further investigation. Here, we use FGFR1 as a model to test the effects of the large GTPase and endocytosis regulatory molecule dynamin-2 on angiogenic signaling in context of distinct FGF ligands. In vitro, overexpression of dominant negative dynamin-2 (DynK44A) attenuates FGFR1 activation of Erk and tubulogenesis by FGF2. Furthermore, we identify FGF21, a non-classical, FGF ligand implicated in diverse human pathologies as an angiogenic molecule acting through FGFR1 and β-Klotho coreceptor. Disruption of FGFR1 activation of ERK by FGF21 is achieved by perturbation of the function of both dynamin-2 and Rab5 GTPase. In vivo, mice harboring endothelial selective overexpression of DynK44A, show impaired angiogenesis in response to FGF21. In conclusion, dynamin dependent endocytosis of FGFR1 is required for in vitro and in vivo angiogenesis in response to FGF2 and the non-classical FGF ligand, FGF21. These studies extend our understanding of the relationships between RTK binding, internalization, endosomal targeting, and angiogenic signaling.

BACKGROUND: In 2010, WHO expanded previously-recommended indications for anti-retroviral treatment to include all HIV-infected TB patients irrespective of CD4 count. India, however, still limits ART to those TB patients with CD4 counts