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N Ashwanikumar,1,* Nisha Asok Kumar,2,* S Asha Nair,2 GS Vinod Kumar11Chemical Biology, 2Cancer Research Programme, Rajiv Gandhi Center for Biotechnology, Poojappura, Thiruvananthapuram, Kerala, India *These authors contributed equally to this workAbstract: Methacrylic-based copolymers in drug-delivery systems demonstrate a pH-sensitive drug-releasing behavior in the colon. In this study, copolymers of methacrylic acid and 2-ethyl hexyl acrylate were prepared using a microemulsion polymerization technique. The purified copolymer was characterized by Fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. 5-Fluorouracil (5-FU) was entrapped within methacrylic-based copolymers by a solvent evaporation method. The size of the nanogels formed was characterized by transmission electron microscopy and atomic force microscopy. In vitro drug-release studies using phosphate-buffered saline at different pH levels demonstrated the sustained release of 5-FU and its pH dependence. Cell proliferation assay of a human colon tumor colon cancer cell line (HCT-116) was performed and showed that the nanogels containing 5-FU exhibited considerable cytotoxicity in comparison with free 5-FU. Cell uptake of the nanogels was also monitored using confocal microscopy. Western blot analysis and flow cytometry studies confirmed that the nanogels could be successfully used as an efficient vector for pH-sensitive and controlled delivery of drugs specifically targeted to the colon.Keywords: 5-FU, methacrylic polymer, colon cancer, nanogel

K Lekha Nair1, Sankar Jagadeeshan2, S Asha Nair2, GS Vinod Kumar11Chemical Biology, Molecular Medicine Division, 2Cancer Research, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thiruvananthapuram, Kerala, IndiaAbstract: Nanoscaled devices have great potential for drug delivery applications due to their small size. In the present study, we report for the first time the preparation and evaluation of antitumor efficacy of 5-fluorouracil (5-FU)-entrapped poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles with dependence on the lactide/glycolide combination of PLGA. 5-FU-loaded PLGA nanoparticles with two different monomer combinations, 50-50 and 90-10 were synthesized using a modified double emulsion method, and their biological evaluation was done in glioma (U87MG) and breast adenocarcinoma (MCF7) cell lines. 5-FU-entrapped PLGA 50-50 nanoparticles showed smaller size with a high encapsulation efficiency of 66%, which was equivalent to that of PLGA 90-10 nanoparticles. Physicochemical characterization of nanoparticles using differential scanning calorimetry and X-ray diffraction suggested the presence of 5-FU in molecular dispersion form. In vitro release studies showed the prolonged and sustained release of 5-FU from nanoparticles with both the PLGA combinations, where PLGA 50-50 nanoparticles showed faster release. Nanoparticles with PLGA 50-50 combination exhibited better cytotoxicity than free drug in a dose- and time-dependent manner against both the tumor cell lines. The enhanced efficiency of PLGA 50-50 nanoparticles to induce apoptosis was indicated by acridine orange/ethidium bromide staining. Cell cycle perturbations studied using flow cytometer showed better S-phase arrest by nanoparticles in comparison with free 5-FU. All the results indicate that PLGA 50-50 nanoparticles possess better antitumor efficacy than PLGA 90-10 nanoparticles and free 5-FU. Since, studies have shown that long-term exposure of ailing tissues to moderate drug concentrations is more favorable than regular administration of higher concentration of the drug; our results clearly indicate the potential of 5-FU-loaded PLGA nanoparticles with dependence on carrier combination as controlled release formulation to multiplex the therapeutic effect of cancer chemotherapy.Keywords: 5-FU, poly (D, L-lactic-co-glycolic acid), controlled release

Currently, nanoparticles have gained considerable attention for the treatment of bacterial infectious diseases. The possibility for using this technology as an alternative therapeutic strategy for controlling microbial biofilms, colonizations and infections has been the subject of intense investigations. Even though, the potential toxicity and disadvantage of using nanoparticles, researchers focused on their high penetrability into bacterial membranes, capabilities to disrupt biofilm formation and the role of chemotaxis in this interaction. Face to this significant debate, we discuss the link between metal resistance, bacterial chemotaxis and the promising use of nanoparticles (NP). P. aeruginosa has emerged as a model organism for biofilm studies, the aim of this review is to provide a concise and comprehensive survey of certain relevant aspects related to the research on nanoparticles and these bacteria.

O Festival das Tribos Indígenas ou Festribal é um evento que ocorre anualmente desde o ano de 1995 na cidade de Juruti (Pará), na Amazônia brasileira, e compreende à competição entre as “tribos” Munduruku e Muirapinima, concorrentes dessa manifestação da cultura popular. A Amazônia sintetiza simultaneamente a singularidade e a complexidade de um território que em tempo muito antigo apresentava um cenário abundante, habitado por diversos grupamentos humanos (tribos), além de que, em tempos modernos, agrega marca e produto. A partir do século XVI, com a colonização e a chegada do europeu, houve um tensionamento entre culturas que resultou na dupla consciência, efeito da ampla violência que transmutouse, evidenciando-se nas festas populares. Com foco na diversidade cultural da região e da comercialização de seus produtos, a publicidade criou a “marca” Amazônia, nas festas, na música, na dança, na artesania regional etc., e, a partir da origem e projeção da festa, este artigo buscou identificar nas letras das músicas da Festa das tribos, em 2019, modos de resistência representados nessa manifestação da cultura popular e o modo como esse sentimento é expresso. A partir de uma abordagem qualitativa e objetivo exploratório, fez-se uma análise semiótica do evento, por meio do audiovisual na Plataforma YouTube - pela percepção e apreensão dos sentimentos desses povos. Embora ressignificadas, as festas indígenas se perenizaram com base na oralidade, na contação de histórias de seus povos, dos mitos, crenças e das festas vivenciadas por seus antepassados. Essa análise encontra abrigo na teoria da folkcomunicação.

Among the various green synthesis methods for nanoparticle synthesis, the honey-mediated green synthesis of nanoparticles is a fast, safe, biocompatible, and cost-effective method. In the present work, we demonstrate the sunlight-induced honey-mediated synthesis of silver nanoparticles and report the effect of light intensity, its color, and exposure time on the formation of nanoparticles. The visual inspection followed by UV-Vis spectral studies was performed to confirm the formation of silver nanoparticles. The HRTEM measurement confirms the formation of polydispersed silver particles. We further report the excellent antimicrobial activity of the synthesized nanoparticles against various strains of bacteria, which is found to be comparable to that of the antibiotic drug of choice. Our study points to further research on the possibility of considering these green synthesized silver nanoparticles as an alternative to antibiotics.

The performance characteristics of a commercially available rotary heat exchanger or were determined using CFD. A rotary air pre-heater of a thermal power plant (fuelled by natural gas), situated in Ramin, Iran, was numerically modelled with full scale dimensions using 3D approach. This model was then validated against the experimentally measured values. After a satisfactory validation of the CFD model, a design modification was applied to the model by modelling the heat exchanger with four ducts, with hot and cold fluid ducts arranged alternatively. This was done to improve the heat the heat transfer from hot gases to cold air. The medium of heat exchange (core) is modelled as porous media, since the core in the actual equipment has many number of fine passages arranged in complex manner, which would make the modelling difficult. The model was subjected to same boundary conditions as the validation model. The temperature of the outlets and effectiveness of both the models were compared and was found out that the 4-ducts model is around 12% more effective than the base model.

Abstract It is well established that CD4+ T cells play an important role in immunity to infections with retroviruses such as HIV. However, in recent years CD4+ T cells have been subdivided into several distinct populations that are differentially regulated and perform widely varying functions. Thus, it is important to delineate the separate roles of these subsets, which range from direct antiviral activities to potent immunosuppression. In this review, we discuss contributions from the major CD4+ T cell subpopulations to retroviral immunity. Fundamental concepts obtained from studies on numerous viral infections are presented along with a more detailed analysis of studies on murine Friend virus. The relevance of these studies to HIV immunology and immunotherapy is reviewed.

Abstract Background The human hepatitis B virus (HBV), a member of the hepadna viridae, causes acute or chronic hepatitis B, and hepatocellular carcinoma (HCC). The duck hepatitis B virus (DHBV) infection, a dependable and reproducible model for hepadna viral studies, does not result in HCC unlike chronic HBV infection. Information on differential gene expression in DHBV infection might help to compare corresponding changes during HBV infection, and to delineate the reasons for this difference. Findings A subtractive hybridization cDNA library screening of in vitro DHBV infected, cultured primary duck hepatocytes (PDH) identified cDNAs of 42 up-regulated and 36 down-regulated genes coding for proteins associated with signal transduction, cellular respiration, transcription, translation, ubiquitin/proteasome pathway, apoptosis, and membrane and cytoskeletal organization. Those coding for both novel as well as previously reported proteins in HBV/DHBV infection were present in the library. An inverse modulation of the cDNAs of ten proteins, reported to play role in human HCC, such as that of Y-box binding protein1, Platelet-activating factor acetylhydrolase isoform 1B, ribosomal protein L35a, Ferritin, α-enolase, Acid α-glucosidase and Caspase 3, copper-zinc superoxide dismutase (CuZnSOD), Filamin and Pyruvate dehydrogenase, was also observed in this in vitro study. Conclusions The present study identified cDNAs of a number of genes that are differentially modulated in in vitro DHBV infection of primary duck hepatocytes. Further correlation of this differential gene expression in in vivo infection models would be valuable to understand the little known aspects of the hepadnavirus biology.

Abstract Chikungunya virus (CHIKV), an arthritogenic alphavirus, is transmitted to humans by infected Aedes (Ae.) aegypti and Ae.albopictus mosquitoes. In the study, reverse-transcription PCR (RT PCR) and virus isolation detected CHIKV in patient samples and also in adult Ae.albopictus mosquitoes that was derived from larvae collected during a chikungunya (CHIK) outbreak in Kerala in 2009. The CHIKV strains involved in the outbreak were the East, Central and South African (ECSA) genotype that had the E1 A226V mutation. The viral strains from the mosquitoes and CHIK patients from the same area showed a close relationship based on phylogenetic analysis. Genetic characterization by partial sequencing of non-structural protein 2 (nsP2; 378 bp), envelope E1 (505 bp) and E2 (428 bp) identified one critical mutation in the E2 protein coding region of these CHIKV strains. This novel, non-conservative mutation, L210Q, consistently present in both human and mosquito-derived samples studied, was within the region of the E2 protein (amino acids E2 200-220) that determines mosquito cell infectivity in many alpha viruses. Our results show the involvement of Ae. albopictus in this outbreak in Kerala and appearance of CHIKV with novel genetic changes. Detection of virus in adult mosquitoes, emerged in the laboratory from larvae, also points to the possibility of transovarial transmission (TOT) of mutant CHIKV strains in mosquitoes.

The text analyzes a set of expeditions and research trips s and studies carried out simultaneously with the process of colonization and development of the Southwest region of the State of Paraná, between the decades of 1940 to 1970. It tries to map the diverse expeditions and its work of Diagnosis and recording of the physical and human characteristics of colonization and intensified territorial reoccupation after the 1940s with the governmental incentive for the movement of population cams from the extreme south of the country (Santa Catarina and Rio Grande do Sul) to the Southwest region of Paraná. The history of these geographical expeditions reveals a new look at the process of occupation and exploitation of the natural resources of the region, while they served to construct a set of spatial knowledge disseminated through the main scientific geographic magazine of that context: the Brazilian Journal of Geography , Linked to the Brazilian Institute of Geography and Statistics, a governmental body of extreme importance in the formulation of strategic planning and diagnostics focused on territorial sovereignty and proposing solutions to national problems.

Early-onset colorectal cancer (EOCRC), recognized as a distinct subgroup with an increased incidence over the past two decades, characterized by its aggressive nature and potentially unique molecular factors that differentiate it from traditional colorectal cancer (CRC). In this study, we investigated differentially expressed genes in a young-CRC patient using paired-end mRNA-sequencing. Validation of target genes through qRT-PCR highlighted a significant increase in SERPINA3 levels in EOCRC, representing a novel finding. Epithelial expression of SERPINA3 demonstrated a strong association with disease progression, whereas stromal expression showed a negative correlation. Our findings reveal the distinct expression patterns and potential involvement of SERPINA3 in both the initiation and progression of CRC, suggesting that SERPINA3 could serve as a marker for distinguishing early-onset from late-onset cases., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Published by Elsevier Ltd.)

Colorectal cancer (CRC) is a heterogeneous disease and classified into various subtypes, among which transcriptional alterations result in CRC progression, metastasis, and drug resistance. Forkhead-box M1 (FOXM1) is a proliferation-associated transcription factor which is overexpressed in CRC and the mechanisms of FOXM1 regulation have been under investigation. Previously, we showed that FOXM1 binds to promoters of certain microRNAs. Database mining led to several microRNAs that might interact with FOXM1 3'UTR. The interactions between shortlisted microRNAs and FOXM1 3'UTR were quantitated by a dual-luciferase reporter assay. MicroRNA-532-3p interacted with the 3'UTR of the FOXM1 mRNA transcript most efficiently. MicroRNA-532-3p was ectopically overexpressed in colorectal cancer (CRC) cell lines, leading to reduced transcript and protein levels of FOXM1 and cyclin B1 , a direct transcriptional target of FOXM1. Further, a clonogenic assay was conducted in overexpressed miR-532-3p CRC cells that revealed a decline in the ability of cells to form colonies and a reduction in migratory and invading potential. These alterations were reinforced at molecular levels by the altered transcript and protein levels of the conventional EMT markers E-cadherin and vimentin. Overall, this study identifies the regulation of FOXM1 by microRNA-532-3p via its interaction with FOXM1 3'UTR, resulting in the suppression of proliferation, migration, and invasion, suggesting its role as a tumor suppressor in CRC.

Gum arabic/acacia (GA), derived from Acacia trees, is a versatile natural product offering a broad spectrum of applications. Its rich content of soluble dietary fibers, coupled with a low caloric profile, renders GA a valuable dietary component associated with numerous health benefits. Furthermore, its fermentation by gut microbiota yields short-chain fatty acids, renowned for their positive impact on health. Immunomodulation, a crucially regulated mechanism in the body, serves to fend off pathogenic infections by releasing pro-inflammatory cytokines. However, prolonged synthesis of these cytokines can lead to chronic inflammation, tissue damage, and potentially contribute to the development of autoimmune diseases and cancer. Hence, there is an urgent need to identify plant-based biomolecules that can effectively reduce inflammation and inhibit inflammation-induced complications or disorders. In this context, edible biomolecules like GA are gaining prominence for their noteworthy immunomodulatory properties. Therefore, in the present review we have explored the role of GA in immunomodulation, inflammation, and inflammation-associated metabolic diseases, and cancer., (© 2024 John Wiley & Sons Ltd.)

Two BODIPY-biotin conjugates KDP1 and KDP2 are designed and synthesized for targeted PDT applications. Both have good absorption with a high molar absorption coefficient and decent singlet oxygen generation quantum yields. The photosensitizers KDP1 and KDP2 were found to be localized in the mitochondria with excellent photocytotoxicity of up to 18.7 nM in MDA-MB-231 breast cancer cells. The cell death predominantly proceeded through the apoptosis pathway via ROS production., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

With an objective to develop efficient photosensitizers to cancerous tissues, we synthesized two novel biocompatible sensitizers based on aza-BODIPYs incorporated with heavy atoms and biotin moieties. The bioconjugates DPR2a and DPR2b exhibited a favorable absorption range (600-750 nm) with excellent triplet-state quantum yields (up to 79%) and singlet oxygen generation yields (up to 75%). In vitro photobiological investigations employing MDA-MB-231 breast cancer cell lines exhibited rapid cellular uptake, negligible dark toxicity, and high photocytotoxicity. The mechanism of cell death of these systems was predominantly due to the mitochondrial damage, leading to apoptosis mediated via the generation of singlet oxygen-triggered reactive oxygen species. The in vivo studies with the representative conjugate DPR2a employing female NOD/SCID mice models showed inhibition in tumor growth and significantly decreased tumor volume post photodynamic therapy (PDT) treatment. Our results validate that both DPR2a and DPR2b with iodine incorporation exhibit favorable and superior photophysical and photobiological aspects and demonstrate thereby their potential applications in imaging and PDT of cancer., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Community-based active TB case finding (ACF) has become an essential part of TB elimination efforts in high-burden settings. In settings such as the state of Kerala in India, which has reported an annual decline of 7.5% in the estimated TB incidence since 2015, if ACF is not well targeted, it may end up with a less-than-desired yield, the wastage of scarce resources, and the burdening of health systems. Program managers have recognized the need to optimize resources and workloads, while maximizing the yield, when implementing ACF. We developed and implemented the concept of 'individuals'-vulnerability-based active surveillance' as a substitute for the blanket approach for population/geography-based ACF for TB. Weighted scores, based on an estimate of relative risk, were assigned to reflect the TB vulnerabilities of individuals. Vulnerability data for 22,042,168 individuals were available to the primary healthcare team. Individuals with higher cumulative vulnerability scores were targeted for serial ACF from 2019 onwards. In 2018, when a population-based ACF was conducted, the number needed to screen to diagnose one microbiologically confirmed pulmonary TB case was 3772 and the number needed to test to obtain one microbiologically confirmed pulmonary TB case was 112. The corresponding figures in 2019 for individuals'-vulnerability-based ACF were 881 and 39, respectively. Individuals'-vulnerability-based active surveillance is proposed here as a practical solution to improve health system efficiency in settings where the population is relatively stationary, the TB disease burden is low, and the health system is strong.

Background: The control of tuberculosis (TB) in India is complicated by the presence of a large, disorganised private sector where most patients first seek care. Following pilots in Mumbai and Patna (two major cities in India), an initiative known as the 'Public-Private Interface Agency' (PPIA) is now being expanded across the country. We aimed to estimate the cost-effectiveness of scaling up PPIA operations, in line with India's National Strategic Plan for TB control., Methods: Focusing on Mumbai and Patna, we collected cost data from implementing organisations in both cities and combined this data with models of TB transmission dynamics. Estimating the cost per disability adjusted life years (DALY) averted between 2014 (the start of PPIA scale-up) and 2025, we assessed cost-effectiveness using two willingness-to-pay approaches: a WHO-CHOICE threshold based on per-capita economic productivity, and a more stringent threshold incorporating opportunity costs in the health system., Findings: A PPIA scaled up to ultimately reach 50% of privately treated TB patients in Mumbai and Patna would cost, respectively, US$228 (95% uncertainty interval (UI): 159 to 320) per DALY averted and US$564 (95% uncertainty interval (UI): 409 to 775) per DALY averted. In Mumbai, the PPIA would be cost-effective relative to all thresholds considered. In Patna, if focusing on adherence support, rather than on improved diagnosis, the PPIA would be cost-effective relative to all thresholds considered. These differences between sites arise from variations in the burden of drug resistance: among the services of a PPIA, improved diagnosis (including rapid tests with genotypic drug sensitivity testing) has greatest value in settings such as Mumbai, with a high burden of drug-resistant TB., Conclusions: To accelerate decline in TB incidence, it is critical first to engage effectively with the private sector in India. Mechanisms such as the PPIA offer cost-effective ways of doing so, particularly when tailored to local settings., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

The discovery of a potent gene regulating tumorigenesis and drug resistance is of high clinical importance. STIL is an oncogene; however, its molecular associations and role in colorectal oncogenesis are unknown. In this study, we have explored the role of STIL gene in tumorigenesis and studied its molecular targets in colorectal cancer (CRC). STIL silencing reduced proliferation and tumor growth in CRC. Further, STIL was found to regulate stemness markers CD133 and CD44 and drug resistant markers thymidylate synthase , ABCB1 , and ABCG2 both in in-vitro and in-vivo CRC models. In addition, high expression of STIL mRNA was found to be associated with reduced disease-free survival in CRC cases. Interestingly, we observed that STIL -mediated regulation of stemness and drug resistant genes is not exclusively governed by Sonic hedgehog (Shh) signaling. Remarkably, we found STIL regulate β-catenin levels through p-AKT, independent of Shh pathway. This partially answers Shh independent regulatory mechanism of cancer stem cell (CSC) markers by STIL . Our study suggests an instrumental role of STIL in molecular manifestation of CRC and progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pradhan, Kumar, Surya H, Krishna, John, Jissa, Anjana, Chandramohan and Nair.)

Background: A significant proportion of pediatric tuberculosis (TB) patients go unnotified due to the challenges in diagnosis of TB among children. The experiences of this vulnerable group while going through the TB care cascade remain largely undocumented. The aim of this study was to explore the experiences of pediatric TB patients and families along the pathway to TB diagnosis and appropriate treatment in four cities of India., Methods: The study used a mixed methods, single phased, embedded design. The primary qualitative and secondary quantitative data were collected simultaneously by interviewing families of 100 randomly selected Xpert MTB/RIF positive pediatric TB patients, under the pediatric TB project, in 4 Indian cities using a semi-structured questionnaire. The qualitative component was analyzed to deduce patterns and themes on the patient and family experiences. Descriptive statistics were used to quantify various events along the TB care pathway including various delays (patient, diagnosis and total) and number of providers visited by patients during the diagnostic process., Results: The median patient, diagnostic and total delays were 3 (IQR: 2,5), 39 (IQR: 23, 91) and 43 days (IQR: 28.5, 98.5), respectively. Patients visited a median of 3 (IQR: 2,4) providers before accessing Xpert MTB/RIF testing. On an average, 68.4% of physicians ordered any test most of them being irrelevant for TB diagnosis. Qualitative data showed considerable suffering for children and their families before and after TB diagnosis including serious concerns of stigma, disruption in education and social life and recurrence of the disease., Conclusion: Our study highlights the significant physical and social distress that the children with TB and their families undergo along the TB care pathway. It also shows diagnostic delay in excess of a month during which multiple providers were met and the patients underwent several diagnostic tests, most of them being inappropriate. Efforts to make Xpert MTB/RIF testing more accessible and part of physicians' toolkit will be of considerable value to ease the complexity of TB diagnosis in children. In addition, communication strategy needs to be developed and implemented to generate awareness among general population around pediatric TB and its management., Competing Interests: Conflicts of interest The authors have none to declare, (Copyright © 2021 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.)

Background: Routine services for tuberculosis (TB) are being disrupted by stringent lockdowns against the novel SARS-CoV-2 virus. We sought to estimate the potential long-term epidemiological impact of such disruptions on TB burden in high-burden countries, and how this negative impact could be mitigated., Methods: We adapted mathematical models of TB transmission in three high-burden countries (India, Kenya and Ukraine) to incorporate lockdown-associated disruptions in the TB care cascade. The anticipated level of disruption reflected consensus from a rapid expert consultation. We modelled the impact of these disruptions on TB incidence and mortality over the next five years, and also considered potential interventions to curtail this impact., Findings: Even temporary disruptions can cause long-term increases in TB incidence and mortality. If lockdown-related disruptions cause a temporary 50% reduction in TB transmission, we estimated that a 3-month suspension of TB services, followed by 10 months to restore to normal, would cause, over the next 5 years, an additional 1⋅19 million TB cases (Crl 1⋅06-1⋅33) and 361,000 TB deaths (CrI 333-394 thousand) in India, 24,700 (16,100-44,700) TB cases and 12,500 deaths (8.8-17.8 thousand) in Kenya, and 4,350 (826-6,540) cases and 1,340 deaths (815-1,980) in Ukraine. The principal driver of these adverse impacts is the accumulation of undetected TB during a lockdown. We demonstrate how long term increases in TB burden could be averted in the short term through supplementary "catch-up" TB case detection and treatment, once restrictions are eased., Interpretation: Lockdown-related disruptions can cause long-lasting increases in TB burden, but these negative effects can be mitigated with rapid restoration of TB services, and targeted interventions that are implemented as soon as restrictions are lifted., Funding: USAID and Stop TB Partnership., Competing Interests: SA is employed by USAID and SAN, AM, EM, and SS are employed by the Stop TB Partnership. The other authors declare no conflicts of interest., (© 2020 The Authors.)

Background: Diagnosis of TB in pediatric population poses several challenges. A novel initiative was implemented in several major cities of India aimed at providing upfront access to free-of-cost Xpert MTB/RIF to presumptive pediatric TB cases. This paper aims to describe the experience of implementing this large initiative and assess feasibility of the intervention in high TB burden settings., Methods: Data were drawn from the pediatric TB project implemented in 10 major cities of India between April 2014 and March 2018. In each city, providers, both public and private, were engaged and linked with a high throughput Xpert MTB/RIF lab (established in that city) through rapid specimen transportation and electronic reporting system. Rates and proportions were estimated to describe the characteristics of this cohort., Results: Of the total 94,415 presumptive pediatric TB cases tested in the project, 6,270 were diagnosed positive for MTB (6.6%) on Xpert MTB/RIF (vs 2% on smear microscopy). Among MTB positives, 545 cases were rifampicin resistant (8.7%). The median duration between collection of specimens and reporting of results was 0 days (same day) and >89% cases were initiated on treatment. Approximately 50% of the specimens tested were non-sputum. The number of providers/facilities engaged under the project increased >10-fold (from 124 in Q2'14 to 1416 in Q1'18)., Conclusion: This project, which was one of the largest initiatives globally among pediatric population, demonstrated the feasibility of sustaining rapid and upfront access to free-of-cost Xpert MTB/RIF testing. The project underscores the efficiency of this rapid diagnostic assay in tackling several challenges in pediatric TB diagnosis, identifies opportunities for further interventions as well as brings to light scope for effective engagement with healthcare providers. The findings have facilitated a policy decision by National TB Programme mandating the use of Xpert MTB/RIF as a primary diagnostic tool for TB diagnosis in children, which is being scaled-up., Competing Interests: The authors [AK, DP, NR, SM, RS, SC, SS, CMD, CB] are affiliated with Foundation for Innovative New Diagnostics, a non-profit international organization. However, this does not alter our adherence to PLOS ONE policies on sharing data and materials.

BACKGROUND: For patients taking standard first-line tuberculosis treatment, missing 10% or more of their doses increases the risk of relapse six-fold. Digital technologies offer new approaches to adherence support for TB patients. We estimated the potential impact of new adherence technologies in India. METHOD: We developed a mathematical model of TB transmission dynamics in India, capturing the independent effects of missed doses and treatment default on post-treatment recurrence. We simulated the impact of interventions to address both missed doses and treatment default in the public and private healthcare sector. RESULTS: Adherence interventions, if deployed optimally in the public sector alone, would reduce cumulative TB incidence by 7.3% (95% credible intervals [CrI] 4.7-11) between 2020 and 2030, and by 16% (95% CrI 11-23) if also deployed in the private sector. This impact is roughly proportional to the effectiveness of the interventions. Reducing missed doses reduced incidence by 12% (95% CrI 7.0-18), while reducing treatment default reduced incidence by 7.9% (95% CrI 3.2-13). CONCLUSION: Minimising missed doses is at least as important as promoting treatment completion. Our results suggest that emerging technologies to improve treatment adherence could have a substantial impact on TB incidence and mortality in India.

There is increasing interest in future, highly-potent 'pan-TB' regimens against tuberculosis (TB), that may be equally effective in both drug-susceptible and rifampicin-resistant (RR) forms of TB. Taking the example of India, the country with the world's largest burden of TB, we show that adoption of these regimens could be: (i) epidemiologically impactful, and (ii) cost-saving to the national TB programme, even if the regimen itself is more costly than current TB treatment. Mathematical modelling suggests that deployment of a pan-TB regimen in 2022 would reduce the annual incidence of TB in 2030 by 23.9% [95% Bayesian credible intervals [CrI] 17.6-30.8%] if used to treat all TB cases, and by 2.30% [95% CrI 1.57-3.48%] if used to treat only RR-TB. Notably, with a regimen costing less than USD 359 (95% CrI 287-441), treating all diagnosed TB cases with the pan-TB regimen yielded greater cost-savings than treating just those diagnosed with RR-TB. One limitation of our approach is that it does not capture the risk of resistance to the new regimen. We discuss ways in which this risk could be mitigated using modern adherence support mechanisms, as well as drug sensitivity testing at the point of TB diagnosis, to prevent new resistant forms from becoming established. A combination of such approaches would be important for maximising the useful lifetime of any future regimen., Competing Interests: The authors have declared that no competing interests exist.

The major challenge in photodynamic therapy (PDT) is to discover versatile photosensitizers (PSs) that possess good solubility in biological media, enhanced singlet oxygen generation efficacy, and photodynamic activity. Working in this direction, we synthesized a picolylamine-functionalized porphyrin conjugate, compound 1 , and its zinc complex compound 2 . Compound 1 forms spherical structures in methanol, whereas compound 2 exhibited vesicular structures. Compared to the existing PSs like foscan and photofrin, compound 2 exhibited a high singlet oxygen generation efficiency and triplet quantum yield. The complex also showed good water solubility, and its PDT activity was demonstrated through in vitro studies using MDA-MB 231 breast cancer cells. The mechanism of biological activity evaluated using various techniques proved that the active compound 2 induced predominantly singlet oxygen-triggered apoptosis-mediated cancerous cell death. Our results demonstrate that zinc insertion in the picolyl porphyrin induces an enhanced triplet excited state, and the singlet oxygen yields quantitatively and imparts excellent in vitro photodynamic activity, thereby demonstrating their pertinence as a nanodrug in future photobiological applications., Competing Interests: The authors declare no competing financial interest.

Background: There is a pressing need for systematic approaches for monitoring how much TB treatment is ongoing in the private sector in India: both to cast light on the true scale of the problem, and to help monitor the progress of interventions currently being planned to address this problem., Methods: We used commercially available data on the sales of rifampicin-containing drugs in the private sector, adjusted for data coverage and indication of use. We examined temporal, statewise trends in volumes (patient-months) of TB treatment from 2013 to 2016. We additionally analysed the proportion of drugs that were sold in combination packaging (designed to simplify TB treatment), or as loose pills., Results: Drug sales suggest a steady trend of TB treatment dispensed by the private sector, from 18.4 million patient-months (95% CI 17.3-20.5) in 2013 to 16.8 patient-months (95% CI 15.5-19.0) in 2016. Overall, seven of 29 states in India accounted for more than 70% of national-level TB treatment volumes, including Uttar Pradesh, Maharashtra and Bihar. The overwhelming majority of TB treatment was dispensed not as loose pills, but in combination packaging with other TB drugs, accounting for over 96% of private sector TB treatment in 2017., Conclusions: Our findings suggest consistent levels of TB treatment in the private sector over the past 4 years, while highlighting specific states that should be prioritized for intervention. Drug sales data can be helpful for monitoring a system as large, disorganised and opaque as India's private sector.

Pathological assessment of excised tumour and surgical margins in colorectal cancer (CRC) play crucial role in prognosis after surgery. Molecular assessment of margins could be more sensitive and informative than conventional histopathological analysis. Considering this view, we evaluated the distal surgical margins for expression of cancer stem cell (CSC) markers. Cellular and molecular assessment of normal, tumour and distal margin tissues were performed by flow cytometry, real-time q-PCR and immuno-histochemical analysis for CRC patients after tumour excision. CRC patients were evaluated for expression of CSC markers in their normal, tumour and distal tissues. Flow cytometry assay revealed CD133 and CD44 enriched cells in distal margin and tumour compared to normal colorectal tissues, which was further confirmed by immunohistochemistry. Most importantly, immunohistochemistry also revealed the enrichment of CSC markers expression in pathologically negative distal margins. Patients with distal margin enriched for CD133 expression showed an increased recurrence rate and decreased disease-free survival. This study proposes that although distal margin seems to be tumour free in conventional histopathological analysis, it could harbour cells enriched for CSC markers. Further CD133 could be a promising molecule to be used in molecular pathology for disease prognosis after surgery in CRC patients., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Herein, we synthesised four aza-BODIPY dyes (1-4) with the singlet oxygen generation quantum yield values of ca. 65-85%. Furthermore, we formulated a nanomedicine by encapsulating these dyes into an amphiphilic micelle, DSPE. The spherical nanomicelles formed were characterized using photophysical and morphological analyses, and their in vitro and in vivo photodynamic efficacies were investigated. One of the conjugates, DSPE-1, showed the lowest IC 50 value of 2 μM against a human breast cancer cell line (MDA MB 231). The mechanism of photodynamic activity has been evaluated by employing different biophysical and morphological assays, which confirmed apoptotic cell death (ca. 80-90%) predominantly through the involvement of reactive oxygen species. Interestingly, we observed that 2 mg kg -1 DSPE-1 induced enhanced apoptosis and efficient inhibition of the growth of breast tumor xenografts in NOD/SCID mice models. Herein, we demonstrated the application of aza-BODIPY nanomicelles in photodynamic therapy for the first time, and our results revealed that the DSPE-BODIPY nanomicelles enhanced the cellular uptake as well as the photodynamic activity, thereby demonstrating the use of these nanomicelles as efficient sensitizers in biological applications.

In India, the country with the world's largest burden of tuberculosis (TB), most patients first seek care in the private healthcare sector, which is fragmented and unregulated. Ongoing initiatives are demonstrating effective approaches for engaging with this sector, and form a central part of India's recent National Strategic Plan: here we aimed to address their potential impact on TB transmission in urban settings, when taken to scale. We developed a mathematical model of TB transmission dynamics, calibrated to urban populations in Mumbai and Patna, two major cities in India where pilot interventions are currently ongoing. We found that, when taken to sufficient scale to capture 75% of patient-provider interactions, the intervention could reduce incidence by upto 21.3% (95% Bayesian credible interval (CrI) 13.0-32.5%) and 15.8% (95% CrI 7.8-28.2%) in Mumbai and Patna respectively, between 2018 and 2025. There is a stronger impact on TB mortality, with a reduction of up to 38.1% (95% CrI 20.0-55.1%) in the example of Mumbai. The incidence impact of this intervention alone may be limited by the amount of transmission that has already occurred by the time a patient first presents for care: model estimates suggest an initial patient delay of 4-5 months before first seeking care, followed by a diagnostic delay of 1-2 months before ultimately initiating TB treatment. Our results suggest that the transmission impact of such interventions could be maximised by additional measures to encourage early uptake of TB services.

Introduction: Factors associated with tuberculosis (TB) in Kerala, the southern Indian state that notifies approximately 33 microbiologically confirmed new cases per 100,000 population every year for the past two decades, are still unclear. We did a community-based case-control study in Kollam district, Kerala, to identify the individual-level risk factors for TB., Methods: Structured questionnaire was applied to 101 microbiologically confirmed new TB cases registered under Revised National Tuberculosis Control Program and 202 age- and gender-matched neighborhood controls without present or past TB. Information was sought on socioeconomic status (SES), smoking, consumption of alcohol, close contact with active TB during childhood or recent past, diabetes mellitus (DM), and other comorbid conditions., Results: Close contact with TB during childhood [odds ratio (OR) 15.88, 95% confidence interval (CI) 3.21-78.55], recent close contact with TB (OR 4.81, 95% CI 2.09-11.07), DM (OR 1.64, 95% CI 1.04-3.06), SES (OR 2.16, 95% CI 1.16-4.03), smoking more than 10 cigarettes/beedis per day (OR 3.32, 95% CI 1.27-8.96), consuming more than 10 standard drinks per week (OR 2.91, 95% CI 1.33-6.37), and the interaction term of having close contact with TB during childhood and DM at present (OR 7.37, 95% CI 1.18-50.29) were found to be associated with TB., Conclusion: Close contact with a case of TB, presence of DM, lower SES, smoking, and alcohol consumption were associated with active TB in Kollam. Having close contact with a case of TB during childhood and development of DM in later life together are significantly associated with active TB in the study population. The findings also direct further studies to confirm and explore mechanisms of interaction of diabetes with childhood exposure to TB., Competing Interests: There are no conflicts of interest.

The End TB Strategy envisions a world free of tuberculosis-zero deaths, disease and suffering due to tuberculosis by 2035. This requires reducing the global tuberculosis incidence from >1250 cases per million people to <100 cases per million people within the next two decades. Expanding testing and treatment of tuberculosis infection is critical to achieving this goal. In high-burden countries, like India, the implementation of tuberculosis preventive treatment (TPT) remains a low priority. In this analysis article, we explore potential challenges and solutions of implementing TPT in India. The next chapter in tuberculosis elimination in India will require cost-effective and sustainable interventions aimed at tuberculosis infection. This will require constant innovation, locally driven solutions to address the diverse and dynamic tuberculosis epidemiology and persistent programme monitoring and evaluation. As new tools, regimens and approaches emerge, midcourse adjustments to policy and practice must be adopted. The development and implementation of new tools and strategies will call for close collaboration between local, national and international partners-both public and private-national health authorities, non-governmental organisations, research community and the diagnostic and pharmaceutical industry. Leading by example, India can contribute to global knowledge through operational research and programmatic implementation for combating tuberculosis infection., Competing Interests: Competing interests: None declared.

Background: Tuberculosis (TB) is one of world's oldest infectious disease and ranks alongside HIV as leading infectious killer. Tuberculosis infection control especially in HIV and TB care facilities has warranted attention after the recent health care-associated outbreaks in South Africa. The aim of this study was to describe the tuberculosis infection control measures implemented by HIV and TB care facilities in five high HIV burden provinces in India., Methods: Baseline assessment of 30 high burden Antiretroviral centers and TB facilities was conducted during Oct 2015-Dec 2015 by AIC trained staff using a structured format., Results: Thirty HIV and TB care facilities in five high HIV burden provinces were enrolled. Facility infrastructure and airborne infection control practices were highly varied between facilities. TB screening and fast tracking at ART centers is happening at majority of centers however inadequate TB infection control training, poor compliance to administrative and personal protective measures and lack of mechanism for health care workers surveillance need attention., Conclusions: Local specific TB infection control interventions to be designed and implemented at HIV and TB care facilities including implementation of administrative, environmental and use of personal protective equipment's with the training of staff members. Health care workers surveillance needs to be prioritized considering the rising instances of tuberculosis among Health care workers., (Copyright © 2018 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.)

Background: Diagnosis of tuberculosis (TB) in infants is challenging due to non-specific clinical presentations of the disease in this age-group and low sensitivity of widely available TB diagnostic tools, which in turn delays prompt access to TB treatment. Upfront access to Xpert/MTB RIF (Xpert) testing, a highly sensitive and specific rapid diagnostic tool, could potentially address some of these challenges. Under the current project, we assessed the utility and feasibility of applying upfront Xpert for diagnosis of tuberculosis in infants, including for testing of non-sputum specimens., Methods: A high throughput lab was established in each of the four project cities, and linked to various health care providers across the city, through rapid specimen transportation and electronic reporting linkages. Free Xpert testing was offered to all infant (<2 years of age) presumptive TB cases (both pulmonary and extra-pulmonary) seeking care at public and private health facilities., Results: A total of 7,994 presumptive infant TB cases were enrolled in the project from April 2014 to October 2016, detecting 465 (5.8%, CI: 5.3-6.4) TB cases. The majority (93.9%; CI: 93.4-94.4) of patient specimens were non-sputum and TB positivity was higher amongst non-sputum specimens. Further, a high proportion (5.6% CI 3.8-8.1) of infant TB cases were found to be rifampicin resistant. Covering large cities with a single lab per city over more than two years, the project demonstrated the feasibility of same-day diagnosis with upfront Xpert testing. This in turn led to prompt treatment initiation, with a two-day median turnaround time to treatment initiation. Case mortality observed in the project cohort of diagnosed TB cases was 11.0% (CI 8.4-14.1), the majority of which was pre- or early treatment mortality, in spite of prompt access to treatment for most diagnosed cases., Conclusion: The current project demonstrated the feasibility of applying rapid and upfront Xpert testing for presumptive infant TB cases. Rapid TB diagnosis in turn facilitates prompt and appropriate treatment initiation. Further, levels of rifampicin resistance observed in infants TB cases highlight the additional benefit of upfront resistance testing. However, high rates of early case mortality, in spite of prompt diagnosis and treatment initiation, highlight the need for further research in infant patient pathways for overall improvement in TB care for infant populations., Competing Interests: The authors have declared that no competing interests exist.

Background: India has the world's highest estimated burden of multi-drug-resistant tuberculosis (MDR-TB). While prevalence of MDR-TB is known to be 2-3% among new TB patients and 12-17% in previously treated patients, programmatic information on the extent of transmission of TB and MDR-TB among household contacts of known MDR-TB patients is scarce. Systematic screening of household contacts of all MDR-TB patients on treatment was implemented as an intervention in the states of Andhra Pradesh and Telangana states of India. We undertook this prospective interventional study to measure the extent of TB symptoms developed among the household contacts of the known MDR-TB patients treated under Revised National TB Control Programme (RNTCP). The extent of rifampicin sensitive or resistance TB, bacteriologically confirmed using Xpert MTB-RIF, was examined among the symptomatic household contacts., Methods: All MDR-TB patients registered and on treatment under RNTCP between July 2011 and Sep 2013 in Andhra Pradesh and Telangana States were selected for the study. They were contacted through home visit by the trained RNTCP teams during 11th Dec 2013 and 7th Jan 2014. All household contacts of MDR-TB patients were screened once for TB symptoms such as cough, fever, weight loss, night sweats, and haemoptysis and extra pulmonary site specific symptoms if any. If found symptomatic, two sputum specimen were collected (spot-morning) from each of the contact and transported for testing on Xpert MTB-RIF for detection of pulmonary TB with or without RR-TB., Results: A total of 1750 MDR-TB patients were registered between July 2011 and Sep 2013. Of these, 1602 (91.5%) MDR-TB patients were included in the study. A total of 4858 household contacts of these 1602 patients were identified with an average of 3 contacts per MDR-TB patient. Of these, after excluding 87 (1.8%) contacts with past history of diagnosis and/or treatment for TB, 4771 (98.2%) contacts were screened for current signs and symptoms suggestive of TB. Their mean age was 28.5 years and 2151 (45%) were females. Of the 4771 contacts screened, 793 (16.6%) had at least one of the symptoms suggestive of TB of whom 781 (98.5%) had two sputum specimen transported and tested on Xpert MTB-Rif. Specimen could not be collected during the study period in 12 symptomatic patients including 4 with symptoms of extra pulmonary TB. Among 781 symptomatic contacts examined, 34 (4.4%) were bacteriologically confirmed with TB and 15 (44%) also had Rif resistance (RR)., Conclusions: High extent of TB, particularly RR-TB was observed among household contacts of known MDR-TB patients with symptom screening and early diagnosis using Xpert-MTB-Rif. Regular systematic active screening for TB and MDR-TB among this highly vulnerable group using Xpert-MTB-Rif is useful in India for early diagnosis among close contacts of known MDR-TB patients., (Copyright © 2018 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.)

Background: Globally, India has the world's highest burden of multidrug-resistant tuberculosis (MDR-TB). Programmatic Management of Drug Resistant TB (PMDT) in India began in 2007 and nationwide coverage was achieved in early 2013. Poor initial microbiological outcomes under the Revised National Tuberculosis Control Programme (RNTCP) prompted detailed analysis. This is the first study on factors significantly associated with poor outcomes in MDR-TB patients treated under the RNTCP., Objective: To evaluate initial sputum culture conversion, culture reversion and final treatment outcomes among MDR-TB patients registered in India from 2007 to early 2011 who were treated with a standard 24-month regimen under daily-observed treatment., Methods: This is a retrospective cohort study. Clinical and microbiological data were abstracted from PMDT records. Initial sputum culture conversion, culture reversion and treatment outcomes were defined by country adaptation of the standard WHO definitions (2008). Cox proportional hazards modeling with logistic regression, multinomial logistic regression and adjusted odds ratio was used to evaluate factors associated with interim and final outcomes respectively, controlling for demographic and clinical characteristics., Results: In the cohort of 3712 MDR-TB patients, 2735 (73.6%) had initial sputum culture conversion at 100 median days (IQR 92-125), of which 506 (18.5%) had culture reversion at 279 median days (IQR 202-381). Treatment outcomes were available for 2264 (60.9%) patients while 1448 (39.0%) patients were still on treatment or yet to have a definite outcome at the time of analysis. Of 2264 patients, 781 (34.5%) had treatment success, 644 (28.4%) died, 670 (29.6%) were lost to follow up, 169 (7.5%) experienced treatment failure or were changed to XDR-TB treatment. Factors significantly associated with either culture non-conversion, culture reversion and/or unfavorable treatment outcomes were baseline BMI < 18; ≥ seven missed doses in intensive phase (IP) and continuation phase (CP); cavitary disease; prior treatment episodes characterized by re-treatment regimen taken twice, longer duration and more episodes of treatment; any weight loss during treatment; males and additional resistance to first line drugs (Ethambutol, Streptomycin). In a subgroup of 104 MDR-TB patients, 62 (59.6%) had Ofloxacin resistance among whom only 25.8% had treatment success, half of the success (54.8%) seen in Ofloxacin sensitive patients. Baseline susceptibility to Ofloxacin (HR 2.04) and Kanamycin (HR 4.55) significantly doubled and quadrupled the chances for culture conversion respectively while baseline susceptibility to Ofloxacin (AOR 0.37) also significantly reduced the odds of unfavorable treatment outcomes (p value ≤0.05) in multinomial logistic regression model., Conclusion: India's initial MDR-TB patients' cohort treated under the RNTCP experienced poor treatment outcomes. To address the factors associated with poor treatment outcomes revealed in our study, a systematic multi-pronged approach would be needed. A series of policies and interventions have been developed to address these factors to improve DR-TB treatment outcomes and are being scaled-up in India.

Background: Diagnosing tuberculosis (TB) in children presents considerable challenges. Upfront testing on Xpert® MTB/RIF ('Xpert')-a rapid molecular assay with high sensitivity and specificity-for pediatric presumptive TB patients, as recommended by India's Revised National Tuberculosis Control Program (RNTCP), can pave the way for early TB diagnosis. As part of an ongoing project implemented by Foundation for Innovative New Diagnostics (FIND) dedicated to providing upfront free-of-cost (FOC) Xpert testing to children seeking care in the public and private sectors, a qualitative assessment was designed to understand how national guidelines on TB diagnosis and Xpert technology have been integrated into the pediatric TB care practices of different health providers., Methods: We conducted semi-structured interviews with a sample of health providers from public and private sectors engaged in the ongoing pediatric project in 4 major cities of India. Providers were sampled from intervention data based on sector of practice, number of Xpert referrals, and TB detection rates amongst referrals. A total of 55 providers were interviewed with different levels of FOC Xpert testing uptake. Data were transcribed and analyzed inductively by a medical anthropologist using thematic content analysis and narrative analysis., Results: It was observed that despite guidance from RNTCP on the use of Xpert and significant efforts by FIND and state authorities to disseminate these guidelines, there was notable diversity in their implementation by different health care providers. Xpert, apart from being utilized as intended, i.e. as a first diagnostic test for children, was utilized variably-as an initial screening test (to rule out TB), confirmatory test (once TB diagnosis is established based on antibiotic trial or clinically) and/or only for drug susceptibility testing after TB diagnosis was confirmed. Most providers who used Xpert frequently reported that Xpert was an important tool for managing pediatric TB cases, by reducing the proportion of cases diagnosed only on clinical suspicion and by providing upfront information on drug resistance, which is seldom suspected in children. Despite non-standard use, these results showed that Xpert access helped raise awareness, aided in antibiotic stewardship, and reduced dependence on clinical diagnosis among those who diagnose and treat TB in children., Conclusion: Access to free and rapid Xpert testing for all presumptive pediatric TB patients has had multiple positive effects on pediatricians' diagnosis and treatment of TB. It has important effects on speed of diagnosis, empirical treatment, and awareness of drug resistance among TB treatment naive children. In addition, our study shows that access to public sector Xpert machines may be an important way to encourage Public-Private integration and facilitate the movement of patients from the private to public sector for anti-TB treatment. Despite availability of rapid and free Xpert testing, our study showed an alarming diversity of Xpert utilization strategies across different providers who may be moving toward suggested practice over time. The degree of diversity in TB diagnostic approaches in children reported here highlights the urgent need for concerted efforts to place Xpert early in diagnostic algorithms to positively impact the pediatric TB care pathway. A positive change in diagnostic algorithms may be possible with continued advocacy, time, and increased access.

Background: Unlike in adults, diagnosis of TB can be challenging in children, as signs and symptoms of paediatric TB can be very non-specific and similar to other common childhood chest infections, which may lead to under or delayed diagnosis of TB disease. In spite of the increasing availability of rapid high-sensitivity diagnostics in public and private sectors, majority of paediatric TB cases are empirically diagnosed, without laboratory confirmation. To address these diagnostic challenges, World Health Organization (WHO) has recommended upfront Xpert MTB/RIF (Xpert) testing for the diagnosis of TB in paediatric presumptive pulmonary and extra-pulmonary TB (EPTB) cases. However, in spite of the increasing availability of rapid high-sensitivity diagnostics, a significant gap exists in its application with Xpert being rarely used as an upfront diagnostic among patients presumed to have TB. Under an ongoing paediatric project since April 2014, which provided free-of-cost upfront Xpert testing, several low-cost outreach and education interventions were undertaken to increase the diagnostic uptake by different providers catering to the paediatric population, thereby increasing adherence to global guidance., Methods: Providers catering to paediatric population in the project cities were systematically mapped and contacted using different outreach strategies. The focus of outreach efforts was to increase provider literacy and increase their awareness of the availability of free rapid diagnostic services with the goal of changing their diagnostic approaches., Results: From April 2014 to June 2016, more than 5,700 providers/facilities were mapped and 3,670 of them were approached. The number of providers/facilities engaged under the project increased more than 10-fold (43 in April, 2014 to 466 in June, 2016), with significant increase in project uptake, both from public and private sector. Overall 42,238 paediatric presumptive TB cases were enrolled in the project, across the four cities. Over the project period, quarterly diagnostic uptake and paediatric TB cases detection rates increased more than two-fold. TB detection rates were similar in patients from public and private sectors., Conclusions: Ongoing efforts in scaling up new rapid diagnostics involves significant investments. These efforts need to be complemented with proactive provider engagement to ensure provider-literacy and awareness, for maximizing impact of this scale-up. The current project demonstrated the usefulness of outreach and education interventions for the effective uptake of newer diagnostics.

Background: Diagnosis of TB in children is challenging, and is largely based on positive history of contact with a TB case, clinical and radiological findings, often without microbiological confirmation. Diagnostic efforts are also undermined by challenges in specimen collection and the limited availability of high sensitivity, rapid diagnostic tests that can be applied with a quick turnaround time. The current project was undertaken in four major cities of India to address TB diagnostic challenges in pediatric population, by offering free of cost Xpert testing to pediatric presumptive TB cases, thereby paving the way for better TB care., Methods: A high throughput lab was established in each of the four project cities, and linked to various health care providers across the city through rapid specimen transportation and electronic reporting linkages. Free Xpert testing was offered to all pediatric (0-14 years) presumptive TB cases (both pulmonary and extra-pulmonary) seeking care at public and private health facilities., Results: The current project enrolled 42,238 pediatric presumptive TB cases from April, 2014 to June, 2016. A total of 3,340 (7.91%, CI 7.65-8.17) bacteriologically confirmed TB cases were detected, of which 295 (8.83%, CI 7.9-9.86) were rifampicin-resistant. The level of rifampicin resistance in the project cohort was high. Overall Xpert yielded a high proportion of valid results and TB detection rates were more than three-fold higher than smear microscopy. The project provided same-day testing and early availability of results led to rapid treatment initiation and success rates and very low rates of treatment failure and loss to follow-up., Conclusion: The current project demonstrated the feasibility of rolling out rapid and upfront Xpert testing for pediatric presumptive TB cases through a single Xpert lab per city in an efficient manner. Rapid turnaround testing time facilitated prompt and appropriate treatment initiation. These results suggest that the upfront Xpert assay is a promising solution to address TB diagnosis in children. The high levels of rifampicin resistance detected in presumptive pediatric TB patients tested under the project are a major cause of concern from a public health perspective which underscores the need to further prioritize upfront Xpert access to this vulnerable population.

Background: The Global Fund encourages operational research (OR) in all its grants; however very few reports describe this aspect. In India, Project Axshya was supported by a Global Fund grant to improve the reach and visibility of the government Tuberculosis (TB) services among marginalised and vulnerable communities. OR was incorporated to build research capacity of professionals working with the national TB programme and to generate evidence to inform policies and practices., Objectives: To describe how Project Axshya facilitated building OR capacity within the country, helped in addressing several TB control priority research questions, documented project activities and their outcomes, and influenced policy and practice., Methods: From September 2010 to September 2016, three key OR-related activities were implemented. First, practical output-oriented modular training courses were conducted (n = 3) to build research capacity of personnel involved in the TB programme, co-facilitated by The Union, in collaboration with the national TB programme, WHO country office and CDC, Atlanta. Second, two large-scale Knowledge, Attitude and Practice (KAP) surveys were conducted at baseline and mid-project to assess the changes pertaining to TB knowledge, attitudes and practices among the general population, TB patients and health care providers over the project period. Third, studies were conducted to describe the project's core activities and outcomes., Results: In the training courses, 44 participant teams were supported to develop research protocols on topics of national priority, resulting in 28 peer-reviewed scientific publications. The KAP surveys and description of project activities resulted in 14 peer-reviewed publications. Of the published papers at least 12 have influenced change in policy or practice., Conclusions: OR within a Global Fund supported TB project has resulted in building OR capacity, facilitating research in areas of national priority and influencing policy and practice. We believe this experience will provide guidance for undertaking OR in Global Fund projects.

Background: India is considering the scale-up of the Xpert MTB/RIF assay for detection of tuberculosis (TB) and rifampicin resistance. We conducted an economic analysis to estimate the costs of different strategies of Xpert implementation in India., Methods: Using a decision analytical model, we compared four diagnostic strategies for TB patients: (i) sputum smear microscopy (SSM) only; (ii) Xpert as a replacement for the rapid diagnostic test currently used for SSM-positive patients at risk of drug resistance (i.e. line probe assay (LPA)); (iii) Upfront Xpert testing for patients at risk of drug resistance; and (iv) Xpert as a replacement for SSM for all patients., Results: The total costs associated with diagnosis for 100,000 presumptive TB cases were: (i) US$ 619,042 for SSM-only; (ii) US$ 575,377 in the LPA replacement scenario; (iii) US$ 720,523 in the SSM replacement scenario; and (iv) US$ 1,639,643 in the Xpert-for-all scenario. Total cohort costs, including treatment costs, increased by 46% from the SSM-only to the Xpert-for-all strategy, largely due to the costs associated with second-line treatment of a higher number of rifampicin-resistant patients due to increased drug-resistant TB (DR-TB) case detection. The diagnostic costs for an estimated 7.64 million presumptive TB patients would comprise (i) 19%, (ii) 17%, (iii) 22% and (iv) 50% of the annual TB control budget. Mean total costs, expressed per DR-TB case initiated on treatment, were lowest in the Xpert-for-all scenario (US$ 11,099)., Conclusions: The Xpert-for-all strategy would result in the greatest increase of TB and DR-TB case detection, but would also have the highest associated costs. The strategy of using Xpert only for patients at risk for DR-TB would be more affordable, but would miss DR-TB cases and the cost per true DR-TB case detected would be higher compared to the Xpert-for-all strategy. As such expanded Xpert strategy would require significant increased TB control budget to ensure that increased case detection is followed by appropriate care.

Setting: Patients with multidrug-resistant tuberculosis (MDR-TB) registered for treatment (2011-2012 cohort) using the standard 24-month regimen, under the Revised National TB Control Programme's programmatic management of drug-resistant TB (PMDT), Maharashtra, India. Objectives: To assess the treatment outcomes and the timing and risk factors for unfavourable treatment outcomes, with a focus on death and loss to follow-up (LTFU). Method: This was a retrospective cohort study involving a review of PMDT records. Treatment outcomes were reported on 31 December 2014. Results: Of 4024 patients, treatment success was recorded in 1168 (29%). Unfavourable outcomes occurred in 2242 (56%), of whom 857 (21%) died and 768 (19%) were lost to follow-up. Treatment outcomes were missing on record review for 375 (9%) patients, and 239 (6%) were still undergoing treatment. Half of LTFU occurred within 3 months, and more than four fifths of deaths occurred after 6 months of treatment. Human immunodeficiency virus infection, being underweight, age ⩾ 15 years, male sex and pulmonary TB were the main risk factors for death, LTFU or other unfavourable treatment outcomes. Conclusion: The study found poor treatment outcomes in patients with MDR-TB registered for treatment in Maharashtra, India. Interventions are required to address the high rates of LTFU and death., Competing Interests: Conflicts of interest: none declared.

Cyclin-dependent kinases (cdks) are central catalytic units of cell division cycle. Among the cdk family members, cdk1 has critical roles in multiple phases of the cell cycle. Aberrant expression or hyper-actions of cdk1 are tumorigenic and yet the complex oncogenic network that regulates its turnover is poorly understood. We found a hitherto unexplored functional connection between skp2 and cdk1 turn over. In vitro knockdown or overexpression of skp2 in cultured cells reduced or induced cdk1 expression indicating skp2 as a positive driver for cdk1. A partial inhibitory role for p27 was identified in this context. Interestingly, concurrent overexpression of skp2 and p27 favored cdk1 upregulation in vitro, which correlated well with similar observations in clinical tumor samples. We found that the transcription factor FOXM1 may play a central role in the skp2-cdk1 loop. Additional molecular involvement in the skp2-cdk1 loop was also explored. In conclusion, our results revealed hitherto unexplored p27 independent molecular mechanisms for skp2 driven tumor progression. Our results support the previous findings that skp2 may be a potential therapeutic target for the management of tumors. J. Cell. Biochem. 118: 797-807, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Setting: Of 18 sites that participated in an implementation study of the Xpert® MTB/RIF assay in India, we selected five microscopy centres and two reference laboratories., Objective: To obtain unit costs of diagnostic tests for tuberculosis (TB) and drug-resistant TB., Design: Laboratories were purposely selected to capture regional variations and different laboratory types. Both bottom-up and the top-down methods were used to estimate unit costs., Results: At the microscopy centres, mean bottom-up unit costs were respectively US$0.83 (range US$0.60-US$1.10) and US$12.29 (US$11.61-US$12.89) for sputum smear microscopy and Xpert. At the reference laboratories, mean unit costs were US$1.69 for the decontamination procedure, US$9.83 for a solid culture, US$11.06 for a liquid culture, US$29.88 for a drug susceptibility test, and US$18.18 for a line-probe assay. Top-down mean unit cost estimates were higher for all tests, and for sputum smear microscopy and Xpert these increased to respectively US$1.51 and US$13.58. The difference between bottom-up and top-down estimates was greatest for tests performed at the reference laboratories., Conclusion: These unit costs for TB diagnostics can be used to estimate resource requirements and cost-effectiveness in India, taking into account geographical location, laboratory type and capacity utilisation.

The proportion of tuberculosis (TB) patients tested for the human immunodeficiency virus (HIV) in the state of Ra-jasthan, India, is limited by the availability of HIV testing facilities. Rajasthan implemented a policy of initiating TB-HIV diagnosis at all health institutions in July 2013. The number of TB diagnostic facilities increased from 33 to 63 in Banswara District and from 22 to 68 in Jhunjhunu District, while the number of HIV testing facilities in these districts increased from 1 to 53 and from 10 to 81, respectively, after the policy implementation. The proportion of TB patients tested for HIV increased by respectively 27% and 19%.

Dimethyl Oxalate (DMO) has recently gained prominence as a valuable intermediate for the production of compounds of commercial importance. The stability of DMO is poor and hence this can result in the decomposition of DMO under reaction conditions. The mechanism of DMO decomposition is however not reported and more so on catalytic surfaces. Insights into the mechanism of decomposition would help in designing catalysts for its effective molecular transformation. It is well known that DMO is sensitive to moisture, which can also be a factor contributing to its decomposition. The present work reports the results of decomposition of DMO on various catalytic materials. The materials studied consist of acidic (γ-Al 2 O 3 ), basic (MgO), weakly acidic (ZnAl 2 O 4 ) and neutral surfaces such as α-Al 2 O 3 and mesoporous precipitated SiO 2 . Infrared spectroscopy is used to identify the nature of adsorption of the molecule on the various surfaces. The spectroscopy study is done at a temperature of 200 °C, which is the onset of gas phase decomposition of DMO. The results indicate that the stability of DMO is lower than the corresponding acid, i.e. oxalic acid. It is also one of the products of decomposition. Spectroscopic data suggest that DMO decomposition is related to surface acidity and the extent of decomposition depends on the number of surface hydroxyl groups. Decomposition was also observed on α-Al 2 O 3 , which was attributed to the residual surface hydroxyl groups. DMO decomposition to oxalic acid was not observed on the basic surface (MgO).

Photodynamic therapy (PDT) is a clinically established and highly evolving treatment modality for cancer. PDT utilizes a light responsive drug called photosensitizer that selectively destroys tumor cells upon light irradiation. Squaraines are a class of dyes possessing all favorable characteristics of a photosensitizer and have been considered to be a potent candidate for next generation PDT. In this study we chose an iodo derivative of squaraine called diiodo-squaraine (bis(3, 5-diiodo-2,4,6-trihydroxyphenyl)squaraine) which has been reported for its tumor specificity but least studied for its cellular and molecular functions. Our studies revealed that the iodo derivative of squaraine possess maximum photodynamic activity in human breast cancer cells MDA- MB- 231 and had very little cytotoxicity in normal breast cells MCF-10A. We analyzed its pro and anti-apoptotic events initiated by oxidative stress exploring a proteomic approach and delineated other critical molecular pathways and key proteins involved in regulating the complex network of cellular response upon PDT. Our study showed that, diiodo- squaraines predominantly accumulate in mitochondria and induce mitochondria-mediated apoptosis. Our study also reveals the novel mechanistic role of diiodo-squaraines to induce oxidative stress there by activating both protective and death inducing pathways post PDT., Competing Interests: The authors declare no competing financial interests.

Setting: Nine district-level microscopy centres in Assam and Tripura, India. Objective: Same-day sputum microscopy is now recommended for tuberculosis (TB) diagnosis. We compared this method against the conventional 2-day approach in routine programmatic settings. Methods: During October-December 2012, all adult presumptive TB patients were requested to provide three sputum samples (one at the initial visit, the second 1 h after the first sample, and the third the next morning) for examination by Ziehl-Neelsen smear microscopy. Detection of acid-fast bacilli with any sample was diagnostic. The first and second spot sample comprised the same-day approach, and the first spot sample and next-day sample comprised the 2-day approach. Results: Of 2168 presumptive TB patients, 403 (18.6%) were smear-positive according to the same-day method compared to 427 (19.7%) by the 2-day method (McNemar's test, P < 0.001). Of the total 429 TB patients, 26 (6.1%) were missed by the same-day method and 2 (0.5%) by the 2-day method. Conclusion: Same-day specimen collection for microscopy missed more TB than 2-day collection. In India, missing cases by using same-day microscopy would translate into a considerable absolute number, hindering TB control efforts. We question the indiscriminate switch to same-day diagnosis in settings where patients reliably return for testing the next day.

Background: Understanding the amount of tuberculosis managed by the private sector in India is crucial to understanding the true burden of the disease in the country, and thus globally. In the absence of quality surveillance data on privately treated patients, commercial drug sales data offer an empirical foundation for disease burden estimation., Methods: We used a large, nationally representative commercial dataset on sales of 189 anti-tuberculosis products available in India to calculate the amount of anti-tuberculosis treatment in the private sector in 2013-14. We corrected estimates using validation studies that audited prescriptions against tuberculosis diagnosis, and estimated uncertainty using Monte Carlo simulation. To address implications for numbers of patients with tuberculosis, we explored varying assumptions for average duration of tuberculosis treatment and accuracy of private diagnosis., Findings: There were 17·793 million patient-months (95% credible interval 16·709 million to 19·841 million) of anti-tuberculosis treatment in the private sector in 2014, twice as many as the public sector. If 40-60% of private-sector tuberculosis diagnoses are correct, and if private-sector tuberculosis treatment lasts on average 2-6 months, this implies that 1·19-5·34 million tuberculosis cases were treated in the private sector in 2014 alone. The midpoint of these ranges yields an estimate of 2·2 million cases, two to three times higher than currently assumed., Interpretation: India's private sector is treating an enormous number of patients for tuberculosis, appreciably higher than has been previously recognised. Accordingly, there is a re-doubled need to address this burden and to strengthen surveillance. Tuberculosis burden estimates in India and worldwide require revision., Funding: Bill & Melinda Gates Foundation., (Copyright © 2016 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY license. Published by Elsevier Ltd.. All rights reserved.)