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Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a rare, X-linked disorder caused by deficient activity of the enzyme iduronate-2-sulfatase. Signs and symptoms typically emerge at 1.5-4 years of age and may include cognitive impairment, depending on whether patients have the neuronopathic or non-neuronopathic form of the disease. Treatment is available in the form of enzyme replacement therapy (ERT) with recombinant iduronate-2-sulfatase (idursulfase). A systematic literature review was conducted to assess the evidence regarding efficacy, effectiveness, and safety of ERT with intravenous idursulfase for MPS II. Electronic databases were searched in January 2023, and 33 eligible articles were found. These were analyzed to evaluate the effects of intravenous idursulfase and the overall benefits and disadvantages in patient subgroups. Studies showed that intravenous idursulfase treatment resulted in improved short- and long-term clinical and patient-centered outcomes, accompanied by a favorable safety profile. Patients with non-neuronopathic MPS II had more pronounced improvements in clinical outcomes than those with neuronopathic MPS II. In addition, the review identified that improvements in clinical outcomes are particularly apparent if intravenous idursulfase is started early in life, strengthening previous recommendations for early ERT initiation to maximally benefit patients. This review provides a comprehensive summary of our current knowledge on the efficacy of ERT in different populations of patients with MPS II and will help to inform the overall management of the disease in an evolving treatment landscape.

Background: Undernutrition is a common comorbidity of tuberculosis in countries with a high tuberculosis burden, such as India. RATIONS is a field-based, cluster-randomised controlled trial evaluating the effect of providing nutritional support to household contacts of adult patients with microbiologically confirmed pulmonary tuberculosis in Jharkhand, India, on tuberculosis incidence. The patient cohort in both groups of the trial was provided with nutritional support. In this study, we assessed the effects of nutritional support on tuberculosis mortality, treatment success, and other outcomes in the RATIONS patient cohort., Methods: We enrolled patients (aged 18 years or older) with microbiologically confirmed pulmonary tuberculosis across 28 tuberculosis units. Patients received nutritional support in the form of food rations (1200 kcal and 52 g of protein per day) and micronutrient pills. Nutritional support was for 6 months for drug-susceptible tuberculosis and 12 months for multidrug-resistant tuberculosis; patients with drug-susceptible tuberculosis could receive an extension of up to 6 months if their BMI was less than 18·5 kg/m 2 at the end of treatment. We recorded BMI, diabetes status, and modified Eastern Cooperative Oncology Group (ECOG) performance status at baseline. Clinical outcomes (treatment success, tuberculosis mortality, loss to follow-up, and change in performance status) and weight gain were recorded at 6 months. We assessed the predictors of tuberculosis mortality with Poisson and Cox regression using adjusted incidence rate ratios (IRRs) and adjusted hazard ratios (HRs). The RATIONS trial is registered with the Clinical Trials Registry of India (CTRI/2019/08/020490)., Findings: Between Aug 16, 2019, and Jan 31, 2021, 2800 patients (mean age 41·5 years [SD 14·5]; 1979 [70·7%] men and 821 [29·3%] women) were enrolled. At enrolment, 2291 (82·4%) patients were underweight (BMI <18·5 kg/m 2 ), and 480 (17·3%) had a BMI of less than 14 kg/m 2 . The mean weight and BMI were 42·6 kg (SD 7·8) and 16·4 kg/m 2 (2·6) in men and 36·1 kg (7·3) and 16·2 kg/m 2 (2·9) in women. During the 6-month follow-up, treatment was successful in 2623 (93·7%) patients, 108 (3·9%) tuberculosis deaths occurred, 28 (1·0%) patients were lost to follow-up, and treatment failure was experienced by five (0·2%) patients. The median weight gain was 4·6 kg (IQR 2·8-6·8), but 1441 (54·8%) of 2630 patients remained underweight. At 2 months, 1444 (54·0%) of 2676 patients gained at least 5% of baseline weight. Baseline weight (adjusted IRR 0·95, 95% CI 0·90-0·99), BMI (0·88, 0·76-1·01), poor performance status (ECOG categories 3-4; 5·33, 2·90-9·79), diabetes (3·30, 1·65-6·72), and haemoglobin (0·85, 0·71-1·00) were predictors of tuberculosis mortality. A reduced hazard of death (adjusted HR 0·39, 95% CI 0·18-0·86) was associated with a 5% weight gain at 2 months., Interpretation: In this study, nutritional support was provided to a cohort with a high prevalence of severe undernutrition. Weight gain, particularly in the first 2 months, was associated with a substantially decreased hazard of tuberculosis mortality. Nutritional support needs to be an integral component of patient-centred care to improve treatment outcomes in such settings., Funding: India Tuberculosis Research Consortium, Indian Council of Medical Research., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: In India, tuberculosis and undernutrition are syndemics with a high burden of tuberculosis coexisting with a high burden of undernutrition in patients and in the population. The aim of this study was to determine the effect of nutritional supplementation on tuberculosis incidence in household contacts of adults with microbiologically confirmed pulmonary tuberculosis., Methods: In this field-based, open-label, cluster-randomised controlled trial, we enrolled household contacts of 2800 patients with microbiologically confirmed pulmonary tuberculosis across 28 tuberculosis units of the National Tuberculosis Elimination Programme in four districts of Jharkhand, India. The tuberculosis units were randomly allocated 1:1 by block randomisation to the control group or the intervention group, by a statistician using computer-generated random numbers. Although microbiologically confirmed pulmonary tuberculosis patients in both groups received food rations (1200 kcal, 52 grams of protein per day with micronutrients) for 6 months, only household contacts in the intervention group received monthly food rations and micronutrients (750 kcal, 23 grams of protein per day with micronutrients). After screening all household contacts for co-prevalent tuberculosis at baseline, all participants were followed up actively until July 31, 2022, for the primary outcome of incident tuberculosis (all forms). The ascertainment of the outcome was by independent medical staff in health services. We used Cox proportional hazards model and Poisson regression via the generalised estimating equation approach to estimate unadjusted hazard ratios, adjusted hazard ratios (aHRs), and incidence rate ratios (IRRs). This study is registered with CTRI-India, CTRI/2019/08/020490., Findings: Between Aug 16, 2019, and Jan 31, 2021, there were 10 345 household contacts, of whom 5328 (94·8%) of 5621 household contacts in the intervention group and 4283 (90·7%) of 4724 household contacts in the control group completed the primary outcome assessment. Almost two-thirds of the population belonged to Indigenous communities (eg, Santhals, Ho, Munda, Oraon, and Bhumij) and 34% (3543 of 10 345) had undernutrition. We detected 31 (0·3%) of 10 345 household contact patients with co-prevalent tuberculosis disease in both groups at baseline and 218 (2·1%) people were diagnosed with incident tuberculosis (all forms) over 21 869 person-years of follow-up, with 122 of 218 incident cases in the control group (2·6% [122 of 4712 contacts at risk], 95% CI 2·2-3·1; incidence rate 1·27 per 100 person-years) and 96 incident cases in the intervention group (1·7% [96 of 5602], 1·4-2·1; 0·78 per 100 person-years), of whom 152 (69·7%) of 218 were patients with microbiologically confirmed pulmonary tuberculosis. Tuberculosis incidence (all forms) in the intervention group had an adjusted IRR of 0·61 (95% CI 0·43-0·85; aHR 0·59 [0·42-0·83]), with an even greater decline in incidence of microbiologically confirmed pulmonary tuberculosis (0·52 [0·35-0·79]; 0·51 [0·34-0·78]). This translates into a relative reduction of tuberculosis incidence of 39% (all forms) to 48% (microbiologically confirmed pulmonary tuberculosis) in the intervention group. An estimated 30 households (111 household contacts) would need to be provided nutritional supplementation to prevent one incident tuberculosis., Interpretation: To our knowledge, this is the first randomised trial looking at the effect of nutritional support on tuberculosis incidence in household contacts, whereby the nutritional intervention was associated with substantial (39-48%) reduction in tuberculosis incidence in the household during 2 years of follow-up. This biosocial intervention can accelerate reduction in tuberculosis incidence in countries or communities with a tuberculosis and undernutrition syndemic., Funding: Indian Council of Medical Research-India TB Research Consortium., Competing Interests: Declaration of interests MP serves on the Scientific Advisory Committee of Foundation of Innovative New Diagnostics, which is a non-profit global alliance for diagnostics, and is also an advisor to the Bill & Melinda Gates Foundation. MP has no financial or industry conflicts. All other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Gaucher disease (GD) is an autosomal recessive inherited lysosomal storage disease that often presents in early childhood and is associated with damage to multiple organ systems. Many challenges associated with GD diagnosis and management arise from the considerable heterogeneity of disease presentations and natural history. Phenotypic classification has traditionally been based on the absence (in type 1 GD) or presence (in types 2 and 3 GD) of neurological involvement of varying severity. However, patient management and prediction of prognosis may be best served by a dynamic, evolving definition of individual phenotype rather than by a rigid system of classification. Patients may experience considerable delays in diagnosis, which can potentially be reduced by effective screening programs; however, program implementation can involve ethical and practical challenges. Variation in the clinical course of GD and an uncertain prognosis also complicate decisions concerning treatment initiation, with differing stakeholder perspectives around efficacy and acceptable cost/benefit ratio. We review the challenges faced by physicians in the diagnosis and management of GD in pediatric patients. We also consider future directions and goals, including acceleration of accurate diagnosis, improvements in the understanding of disease heterogeneity (natural history, response to treatment, and prognosis), the need for new treatments to address unmet needs for all forms of GD, and refinement of the tools for monitoring disease progression and treatment efficacy, such as specific biomarkers., Competing Interests: Declaration of Competing Interest NJW has consulted for Pfizer, Sanofi Genzyme and Takeda, has participated in speaking engagements for Sanofi Genzyme, and is a member of the data safety monitoring board for AVROBIO. OG-A has served on advisory boards and received consulting fees from 4DMT, Amicus Therapeutic, Sangamo Therapeutics, Sanofi Genzyme and Takeda, has research contracts with 4DMT, Amicus Therapeutics, AVROBIO, Freeline Therapeutics, Genentech, Protalix BioTherapeutics, Sangamo Therapeutics, Sanofi Genzyme and Takeda, and has participated in a speaker bureau for Sanofi Genzyme and Takeda. PSK has received research/grant support from Pfizer, Sanofi Genzyme and Takeda, has received consulting fees and honoraria from Sanofi Genzyme and Takeda, and is a member of the Gaucher Disease Registry Advisory Board for Sanofi Genzyme and the Advisory Board for Takeda. NL has participated in advisory boards and clinical trials for Amicus Therapeutics, Pfizer, Protalix BioTherapeutics, Sanofi Genzyme and Shire (a Takeda company). TAB is a member of the American Gaucher Disease Registry Board. He has participated in advisory board meetings for and received research funding, travel reimbursement, and honoraria for speaking from Sanofi Genzyme and Takeda. JAB receives research support from AVROBIO, BioMarin Pharmaceutical, Idorsia Pharmaceuticals, Pfizer, Protalix BioTherapeutics, Sangamo Therapeutics, Sanofi Genzyme and Takeda and has served on advisory boards for Sanofi Genzyme and Takeda. PG has participated in advisory boards and clinical trials for BioMarin Pharmaceutical, Pfizer, Sanofi Genzyme and Shire (a Takeda company) and is on the speaker bureau for Sanofi Genzyme. NH has nothing to disclose. HP has participated in advisory boards and/or speaking engagements and/or clinical trials for Alexion Pharmaceuticals, Amicus Therapeutics, AVROBIO, Sanofi Genzyme and Takeda. CP has consulted for Takeda and has participated in a speaker bureau for Sanofi Genzyme. DV has nothing to disclose. RRP is a full-time employee of Takeda and a stockholder of Takeda Pharmaceuticals Company Limited. EW is a full-time employee of Takeda and a stockholder of Takeda Pharmaceuticals Company Limited. CF has served as an advisor or consultant for Horizon Therapeutics, Orphan Technologies, Recordati, Sanofi Genzyme, Shire (a Takeda company) and Swedish Orphan Biovitrum. He has received grants for clinical research from Homology Medicines, Orphan Technologies, Passage Bio, REGENXBIO, Sanofi Genzyme, Takeda and Vtesse., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: Gaucher disease (GD) is a rare autosomal recessive condition. Type 1 GD (GD1) is the most prevalent form of GD in Western countries; enzyme replacement therapy (ERT) is a treatment option for patients with GD1. To understand the economic value of the GD1 ERT velaglucerase alfa, a budget impact model (BIM) was developed from a United States (US) payer perspective., Methods: We estimated the budget impact of velaglucerase alfa for a 10-million-member US health plan by comparing the annual total costs of therapy between a scenario using current velaglucerase alfa uptake to a projected scenario with increased velaglucerase alfa uptake. Total drug costs for both scenarios were estimated as the sum of the product of the number of eligible patients on each treatment and the annual per-patient cost of each medication. Average per-patient costs for ERTs were calculated by adding the yearly drug acquisition, drug administration, and site-of-care markup costs. The budget impact was measured over years 1-3., Results: An estimated 65 patients would receive velaglucerase alfa treatment in year 1, increasing to 90 patients by year 3. Across analyses, cost savings were realized with velaglucerase alfa compared with imiglucerase ($115,909) and taliglucerase alfa ($80,401). An annual total budget savings of $8.67 million could be realized for a hypothetical 10-million-member US health plan with increased velaglucerase alfa uptake. The per-member per-month costs decreased by $0.0241 across years 1-3., Conclusions: BIM results show that increased velaglucerase alfa uptake for GD1 treatment is cost-saving for US health plans.

Introduction: India has the largest burden of cases and deaths related to tuberculosis (TB). Undernutrition is the leading risk factor accounting for TB incidence, while severe undernutrition is a common risk factor for mortality in patients with TB in India. The impact of nutritional supplementation on TB incidence is unknown, while few underpowered studies have assessed its impact on TB mortality. We designed an open-label, field-based cluster randomised trial to assess the impact of nutritional supplementation (with food rations) on TB incidence in a group at higher risk of TB infection and disease, viz household contacts (HHC) of patients with microbiologically confirmed pulmonary TB (PTB) in Jharkhand, a state with a high prevalence of undernutrition., Methods and Analysis: We shall enrol 2800 adult patients with PTB of the national TB programme, across 28 treatment units in 4 districts, and their approximately 11 200 eligible contacts. The sample size has 80% power to detect the primary outcome of 50% reduction in incidence of active TB in HHC over 2 years of follow-up. Patients and HHC in both the arms will undergo nutritional assessment and counselling. Patients will receive monthly food rations (supplying 1200 kcal and 52 g proteins/day) and multivitamins along with antitubercular treatment. The HHC in the intervention arm will receive food rations (supplying 750 kcal and 23 g proteins/day) and multivitamins while HHC in control arm will be on usual diet. The secondary outcomes in HHC will include effects on nutritional status, non-TB infections. Secondary outcomes in patients are effects on TB mortality, adherence, adverse effects, nutritional and performance status. Substudies will examine micronutrient status and effects on dietary intake, body composition, muscle strength and immune function., Ethics and Dissemination: The institutional ethics committee of ICMR-NIRT, Chennai, approved the study (289/NIRT-IEC/2018). The results will be disseminated in publications and presentations., Trial Registration Number: Clinical Trial Registry of India: CTRI/2019/08/020490., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives/hypothesis: Characterization of the localized adaptive immune response in the airway scar of patients with idiopathic subglottic stenosis (iSGS)., Study Design: Basic Science., Methods: Utilizing 36 patients with subglottic stenosis (25 idiopathic subglottic stenosis [iSGS], 10 iatrogenic post-intubation stenosis [iLTS], and one granulomatosis with polyangiitis [GPA]) we applied immunohistochemical and immunologic techniques coupled with RNA sequencing., Results: iSGS, iLTS, and GPA demonstrate a significant immune infiltrate in the subglottic scar consisting of adaptive cell subsets (T cells along with dendritic cells). Interrogation of T cell subtypes showed significantly more CD69 + CD103 + CD8 + tissue resident memory T cells (T RM ) in the iSGS airway scar than iLTS specimens (iSGS vs. iLTS; 50% vs. 28%, P = .0065). Additionally, subglottic CD8 + clones possessed T-cell receptor (TCR) sequences with known antigen specificity for viral and intracellular pathogens., Conclusions: The human subglottis is significantly enriched for CD8 + tissue resident memory T cells in iSGS, which possess TCR sequences proven to recognize viral and intracellular pathogens. These results inform our understanding of iSGS, provide a direction for future discovery, and demonstrate immunologic function in the human proximal airway. Laryngoscope, 131:610-617, 2021., (© 2020 The American Laryngological, Rhinological and Otological Society, Inc.)

G-proteins are implicated in plant productivity, but their genome-wide roles in regulating agronomically important traits remain uncharacterized. Transcriptomic analyses of rice G-protein alpha subunit mutant (rga1) revealed 2270 differentially expressed genes (DEGs) including those involved in C/N and lipid metabolism, cell wall, hormones and stress. Many DEGs were associated with root, leaf, culm, inflorescence, panicle, grain yield and heading date. The mutant performed better in total weight of filled grains, ratio of filled to unfilled grains and tillers per plant. Protein-protein interaction (PPI) network analysis using experimentally validated interactors revealed many RGA1-responsive genes involved in tiller development. qPCR validated the differential expression of genes involved in strigolactone-mediated tiller formation and grain development. Further, the mutant growth and biomass were unaffected by submergence indicating its role in submergence response. Transcription factor network analysis revealed the importance of RGA1 in nitrogen signaling with DEGs such as Nin-like, WRKY, NAC, bHLH families, nitrite reductase, glutamine synthetase, OsCIPK23 and urea transporter. Sub-clustering of DEGs-associated PPI network revealed that RGA1 regulates metabolism, stress and gene regulation among others. Predicted rice G-protein networks mapped DEGs and revealed potential effectors. Thus, this study expands the roles of RGA1 to agronomically important traits and reveals their underlying processes.

Nitrate (N) response is modulated by light, but not understood from a genome-wide perspective. Comparative transcriptomic analyses of nitrate response in light-grown and etiolated rice leaves revealed 303 and 249 differentially expressed genes (DEGs) respectively. A majority of them were exclusive to light (270) or dark (216) condition, whereas 33 DEGs were common. The latter may constitute response to N signaling regardless of light. Functional annotation and pathway enrichment analyses of the DEGs showed that nitrate primarily modulates conserved N signaling and metabolism in light, whereas oxidation-reduction processes, pentose-phosphate shunt, starch-, sucrose- and glycerolipid-metabolisms in the dark. Differential N-regulation of these pathways by light could be attributed to the involvement of distinctive sets of transporters, transcription factors, enriched cis-acting motifs in the promoters of DEGs as well as differential modulation of N-responsive transcriptional regulatory networks in light and dark. Sub-clustering of DEGs-associated protein-protein interaction network constructed using experimentally validated interactors revealed that nitrate regulates a molecular complex consisting of nitrite reductase, ferredoxin-NADP reductase and ferredoxin. This complex is associated with flowering time, revealing a meeting point for N-regulation of N-response and N-use efficiency. Together, our results provide novel insights into distinct pathways of N-signaling in light and dark conditions.

Autologous cell transplantation holds enormous promise to restore organ and tissue functions in the treatment of various pathologies including endocrine, cardiovascular, and neurological diseases among others. Even though immune rejection is circumvented with autologous transplantation, clinical adoption remains limited due to poor cell retention and survival. Cell transplant success requires homing to vascularized environment, cell engraftment and importantly, maintenance of inherent cell function. To address this need, we developed a three dimensional (3D) printed cell encapsulation device created with polylactic acid (PLA), termed neovascularized implantable cell homing and encapsulation (NICHE). In this paper, we present the development and systematic evaluation of the NICHE in vitro, and the in vivo validation with encapsulated testosterone-secreting Leydig cells in Rag1-/- castrated mice. Enhanced subcutaneous vascularization of NICHE via platelet-rich plasma (PRP) hydrogel coating and filling was demonstrated in vivo via a chorioallantoic membrane (CAM) assay as well as in mice. After establishment of a pre-vascularized bed within the NICHE, transcutaneously transplanted Leydig cells, maintained viability and robust testosterone secretion for the duration of the study. Immunohistochemical analysis revealed extensive Leydig cell colonization in the NICHE. Furthermore, transplanted cells achieved physiologic testosterone levels in castrated mice. The promising results provide a proof of concept for the NICHE as a viable platform technology for autologous cell transplantation for the treatment of a variety of diseases., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Patient-derived xenografts (PDX) are an increasingly valuable tool in oncology, providing biologically faithful models of many different cancer types, and potential platforms for the development of precision oncology approaches. However, PDX have primarily been established in immunodeficient rodent models, with accompanying cost and efficiency constraints that pose barriers to more widespread adoption. The chicken egg chorioallantoic membrane (CAM) is an alternative in vivo PDX model. We provide here a comprehensive review of studies that grafted primary human tissue, as opposed to cell lines, onto the CAM. Twenty publications met our criteria of having inoculated patient-derived tumor tissue onto the CAM. Successful engraftment has been reported for over a dozen tumor subtypes, supporting the appropriateness of the CAM as a PDX platform. Resemblance of xenografts to the original patient tumor, increased vascularity of the CAM following engraftment, and micrometastasis into the chick mesenchyme were frequently reported. Application of standard or experimental cancer therapies to xenografts has also been undertaken, with the discovery of both synergistic drug effects and positive associations between the assay and clinical outcome. The CAM provides opportunities for RNA and DNA based sequencing of patient tumors, and the ability to efficiently (in 5-10 days) test multiple targeted therapies on fragments derived from the same tumor. While routine use of the CAM-based PDX model would benefit from a more-complete understanding of the stromal environment of CAM xenografts and interaction with the developing avian immune system, current literature supports the model's potential as an efficient, scalable precision medicine platform., Competing Interests: MTL is a limited partner in StemMed LTD, a manager in StemMed Holdings LLC, and holds an equity stake in Tvardi Therapeutics Inc. AGS holds patents BAYM.P0196US.P1 and BAYM.PO172US.P1, based on CAM-related technologies.

Tumor immune microenvironment and tumor metabolism are major determinants of chemoradiotherapy response. The interdependency and prognostic significance of specific immune and metabolic phenotypes in head and neck squamous cell carcinoma (HNSCC) were assessed and changes in reactive oxygen species were evaluated as a mechanism of treatment response in tumor spheroid/immunocyte co-cultures. Pretreatment tumor biopsies were immunohistochemically characterized in 73 HNSCC patients treated by definitive chemoradiotherapy and correlated with survival. The prognostic significance of CD8A, GLUT1, and COX5B gene expression was analyzed within The Cancer Genome Atlas database. HNSCC spheroids were co-cultured in vitro with peripheral blood mononuclear cells (PBMCs) in the presence of the glycolysis inhibitor 2-deoxyglucose and radiation treatment followed by PBMC chemotaxis determination via fluorescence microscopy. In the chemoradiotherapy-treated HNSCC cohort, mitochondrial-rich (COX5B) metabolism correlated with increased and glucose-dependent (GLUT1) metabolism with decreased intratumoral CD8/CD4 ratios. High CD8/CD4, together with mitochondrial-rich or glucose-independent metabolism, was associated with improved short-term survival. The Cancer Genome Atlas analysis confirmed that patients with a favorable immune and metabolic gene signature (high CD8A, high COX5B, low GLUT1) had improved short- and long-term survival. In vitro, 2-deoxyglucose and radiation synergistically up-regulated reactive oxygen species-dependent PBMC chemotaxis to HNSCC spheroids. These results suggest that glucose-independent tumor metabolism is associated with CD8-dominant antitumor immune infiltrate, and together, these contribute to improved chemoradiotherapy response in HNSCC., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Therapeutic protein kinase inhibitors are designed on the basis of kinase structures. Here, we define intrinsically disordered regions (IDRs) in structurally hybrid kinases. We reveal that 65% of kinases have an IDR adjacent to their kinase domain (KD). These IDRs are evolutionarily more conserved than IDRs distant to KDs. Strikingly, 36 kinases have adjacent IDRs extending into their KDs, defining a unique structural and functional subset of the kinome. Functional network analysis of this subset of the kinome uncovered FAK1 as topologically the most connected hub kinase. We identify that KD-flanking IDR of FAK1 is more conserved and undergoes more post-translational modifications than other IDRs. It preferentially interacts with proteins regulating scaffolding and kinase activity, which contribute to cytoskeletal remodeling. In summary, spatially and evolutionarily conserved IDRs in kinases may influence their functions, which can be exploited for targeted therapies in diseases including those that involve aberrant cytoskeletal remodeling., Competing Interests: The authors declare no competing interests., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We present data on the evolution of intrinsically disordered regions (IDRs) taking into account the entire human protein kinome. The evolutionary data of the IDRs with respect to the kinase domains (KDs) and kinases as a whole protein (WP) are reported. Further, we have reported its post translational modifications of FAK1 IDRs and their contribution to the cytoskeletal remodeling. We also report the data to build a protein-protein interaction (PPI) network of primary and secondary FAK1-interacting hybrid proteins. Detailed analysis of the data and its effect on FAK1-related functions have been described in "Structural pliability adjacent to the kinase domain highlights contribution of FAK1 IDRs to cytoskeletal remodeling" (Kathiriya et. al., 2016) [1].

Introduction: Seven district-level Nutritional Rehabilitation Centres (NRCs) in Bihar, India provide clinical and nutritional care for children with severe acute malnutrition (SAM)., Aim: To assess whether intensified case finding (ICF) strategies at NRCs can lead to pediatric case detection among SAM children and link them to TB treatment under the Revised National Tuberculosis Control Programme (RNTCP)., Materials and Methods: A retrospective cohort study was conducted that included medical record reviews of SAM children registered for TB screening and RNTCP care during July-December 2012., Results: Among 440 SAM children screened, 39 (8.8%) were diagnosed with TB. Among these, 34 (87%) initiated TB treatment and 18 (53%) were registered with the RNTCP. Of 16 children not registered under the RNTCP, nine (56%) weighed below six kilograms-the current weight requirement for receiving drugs under RNTCP., Conclusion: ICF approaches are feasible at NRCs; however, screening for TB entails diagnostic challenges, especially among SAM children. However, only half of the children diagnosed with TB were treated by the RNTCP. More effort is needed to link this vulnerable population to TB services in addition to introducing child-friendly drug formulations for covering children weighing less than six kilograms.

The genome-wide role of heterotrimeric G-proteins in abiotic stress response in rice has not been examined from a functional genomics perspective, despite the availability of mutants and evidences involving individual genes/processes/stresses. Our rice whole transcriptome microarray analysis (GSE 20925 at NCBI GEO) using the G-alpha subunit (RGA1) null mutant (Daikoku 1 or d1) and its corresponding wild type (Oryza sativa Japonica Nipponbare) identified 2270 unique differentially expressed genes (DEGs). Out of them, we mined for all the potentially abiotic stress-responsive genes using Gene Ontology terms, STIFDB2.0 and Rice DB. The first two approaches produced smaller subsets of the 1886 genes found at Rice DB. The GO approach revealed similar regulation of several families of stress-responsive genes in RGA1 mutant. The Genevestigator analysis of the stress-responsive subset of the RGA1-regulated genes from STIFDB revealed cold and drought-responsive clusters. Meta data analysis at Rice DB revealed large stress-response categories such as cold (878 up/810 down), drought (882 up/837 down), heat (913 up/777 down), and salt stress (889 up/841 down). One thousand four hundred ninety-eight of them are common to all the four abiotic stresses, followed by fewer genes common to smaller groups of stresses. The RGA1-regulated genes that uniquely respond to individual stresses include 111 in heat stress, eight each in cold only and drought only stresses, and two genes in salt stress only. The common DEGs (1498) belong to pathways such as the synthesis of polyamine, glycine-betaine, proline, and trehalose. Some of the common DEGs belong to abiotic stress signaling pathways such as calcium-dependent pathway, ABA independent and dependent pathway, and MAP kinase pathway in the RGA1 mutant. Gene ontology of the common stress responsive DEGs revealed 62 unique molecular functions such as transporters, enzyme regulators, transferases, hydrolases, carbon and protein metabolism, binding to nucleotides, carbohydrates, receptors and lipids, morphogenesis, flower development, and cell homeostasis. We also mined 63 miRNAs that bind to the stress responsive transcripts identified in this study, indicating their post-transcriptional regulation. Overall, these results indicate the potentially extensive role of RGA1 in the regulation of multiple abiotic stresses in rice for further validation.

Heterotrimeric G-proteins are implicated in several plant processes, but the mechanisms of signal-response coupling and the roles of G-protein coupled receptors in general and GCR1 in particular, remain poorly understood. We isolated a knock-out mutant of the Arabidopsis G-protein α subunit (gpa1-5) and analysed its transcriptome to understand the genomewide role of GPA1 and compared it with that of our similar analysis of a GCR1 mutant (Chakraborty et al. 2015, PLoS ONE 10(2):e0117819). We found 394 GPA1-regulated genes spanning 79 biological processes, including biotic and abiotic stresses, development, flavonoid biosynthesis, transcription factors, transporters and nitrate/phosphate responses. Many of them are either unknown or unclaimed explicitly in other published gpa1 mutant transcriptome analyses. A comparison of all known GPA1-regulated genes (including the above 394) with 350 GCR1-regulated genes revealed 114 common genes. This can be best explained by GCR1-GPA1 coupling, or by convergence of their independent signaling pathways. Though the common genes in our GPA1 and GCR1 mutant datasets constitute only 26% of the GPA1-regulated and 30% of the GCR1-responsive genes, they belong to nearly half of all the processes affected in both the mutants. Thus, GCR1 and GPA1 regulate not only some common genes, but also different genes belonging to the same processes to achieve similar outcomes. Overall, we validate some known and report many hitherto unknown roles of GPA1 in plants, including agronomically important ones such as biotic stress and nutrient response, and also provide compelling genetic evidence to revisit the role of GCR1 in G-protein signalling.

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

Objectives: The study aims to understand the process and factors influencing the implementation of structured oral examination (SOE) for undergraduate medical students; in comparison with conventional oral examination (COE) in pharmacology., Methods: In a randomized, parallel group study, 123 students of pharmacology were divided into two groups, SOE (n = 63) and COE (n = 60). Students of each group were subdivided into two, and four examiners took viva voce individually. Three sets of questionnaires from autonomic nervous system were prepared, each having 15 items with increasing difficulty levels and were validated by subject experts and pretested. Ten minutes were allotted for each student for each viva. Feedback of students and faculty about the novel method was obtained., Results: SOE yielded significantly lower marks as compared to COE. There were significant inter-examiner variations in marks awarded in SOE and COE. Other factors influencing implementation were difficulty in structuring viva, rigid time limits, lack of flexibility in knowledge content, monotony, and fatigue. The students perceived this format not different from COE but felt that it required in-depth preparation of topic. Faculty opined that SOE led to less drift from main topic and provided uniform coverage of topics in given time., Conclusion: Conducting SOE is a resource-intensive exercise. Despite structuring, inter-examiner variability was not completely eliminated. The students' performance was depended on factors related to examiners such as teaching experience, vernacular language used, and lack of training. Orientation and training of examiners in assessment strategies is necessary. Standardization of questionnaire is necessary before the implementation of SOE for summative assessment.

The controversy over the existence or the need for G-protein coupled receptors (GPCRs) in plant G-protein signalling has overshadowed a more fundamental quest for the role of AtGCR1, the most studied and often considered the best candidate for GPCR in plants. Our whole transcriptome microarray analysis of the GCR1-knock-out mutant (gcr1-5) in Arabidopsis thaliana revealed 350 differentially expressed genes spanning all chromosomes. Many of them were hitherto unknown in the context of GCR1 or G-protein signalling, such as in phosphate starvation, storage compound and fatty acid biosynthesis, cell fate, etc. We also found some GCR1-responsive genes/processes that are reported to be regulated by heterotrimeric G-proteins, such as biotic and abiotic stress, hormone response and secondary metabolism. Thus, GCR1 could have G-protein-mediated as well as independent roles and regardless of whether it works as a GPCR, further analysis of the organism-wide role of GCR1 has a significance of its own.

Since aberrant cell signaling pathways underlie majority of pathophysiological morbidities, kinase inhibitors are routinely used for pharmacotherapy. However, most kinase inhibitors suffer from adverse off-target effects. Inhibition of one kinase in a pathogenic signaling pathway elicits multiple compensatory feedback signaling loops, reinforcing the pathway rather than inhibiting it, leading to chemoresistance. Thus, development of novel computational strategies providing predictive evidence to inhibit a specific set of kinases to mitigate an aberrant signaling pathway with minimum side-effects is imperative. First, our analyses reveal that many kinases contain intrinsically disordered regions, which may participate in facilitating protein-protein interactions at the kinome level. Second, we employ a kinome-wide approach to identify intrinsic disorder and streamline a methodology that adds to the knowledge of therapeutically targeting kinase cascades to treat diseases. Furthermore, we find that within the kinome network, some kinases with intrinsically disordered regions have a high topological score, likely acting as kinome modulators. Third, using network analysis, we demonstrate that 5 kinases emerge as topologically most significant, forming kinome sub-networks, comprising of other kinases and transcription factors that are known to serve as drivers of disease pathogenesis. To support these findings, we have biologically validated the interplay between kinome modulators SRC and AKT kinases and uncovered their novel function in regulating transcription factors of the SMAD family. Taken together, we identify novel kinome modulators driven by intrinsic disorder, and biologically validate the thesis that therapeutic disruption of the function of kinome modulators engaged in regulatory cross-talk between disparate pathways can lead to reduced oncogenic potential in cancer cells.

Assimilation and integration of "omics" technologies, including genomics, epigenomics, proteomics, and metabolomics has readily altered the landscape of medical research in the last decade. The vast and complex nature of omics data can only be interpreted by linking molecular information at the organismic level, forming the foundation of systems biology. Research in pulmonary biology/medicine has necessitated integration of omics, network, systems and computational biology data to differentially diagnose, interpret, and prognosticate pulmonary diseases, facilitating improvement in therapy and treatment modalities. This review describes how to leverage this emerging technology in understanding pulmonary diseases at the systems level -called a "systomic" approach. Considering the operational wholeness of cellular and organ systems, diseased genome, proteome, and the metabolome needs to be conceptualized at the systems level to understand disease pathogenesis and progression. Currently available omics technology and resources require a certain degree of training and proficiency in addition to dedicated hardware and applications, making them relatively less user friendly for the pulmonary biologist and clinicians. Herein, we discuss the various strategies, computational tools and approaches required to study pulmonary diseases at the systems level for biomedical scientists and clinical researchers., (© 2014 S. Karger AG, Basel.)

Elevated levels of systemic and pulmonary leptin are associated with diseases related to lung injury and lung cancer. However, the role of leptin in lung biology and pathology, including the mechanism of leptin gene expression in the pathogenesis of lung diseases, including lung cancer, remains elusive. Here, using conditional deletion of tumor suppressor gene Pten in the lung epithelium in vivo in transgenic mice and human PTEN-null lung epithelial cells, we identify the leptin-driven feed-forward signaling loop in the lung epithelial cells. Leptin-mediated leptin/leptin-receptor gene expression likely amplifies leptin signaling that may contribute to the pathogenesis and severity of lung diseases, resulting in poor clinical outcomes. Loss of Pten in the lung epithelial cells in vivo activated adipokine signaling and induced leptin synthesis as ascertained by genome-wide mRNA profiling and pathway analysis. Leptin gene transcription was mediated by binding of transcription factors NRF-1 and CCAAT/enhancer-binding protein δ (C/EBP) to the proximal promoter regions and STAT3 to the distal promoter regions as revealed by leptin promoter-mutation, chromatin immunoprecipitation, and gain- and loss-of-function studies in lung epithelial cells. Leptin treatment induced expression of the leptin/leptin receptor in the lung epithelial cells via activation of MEK/ERK, PI3K/AKT/mammalian target of rapamycin (mTOR), and JAK2/STAT3 signaling pathways. Expression of constitutively active MEK-1, AKT, and STAT3 proteins increased expression, and treatment with MEK, PI3K, AKT, and mTOR inhibitors decreased LEP expression, indicating that leptin via MAPK/ERK1/2, PI3K/AKT/mTOR, and JAK2/STAT3 pathways, in turn, further induces its own gene expression. Thus, targeted inhibition of the leptin-mediated feed-forward loop provides a novel rationale for pharmacotherapy of disease associated with lung injury and remodeling, including lung cancer.

IDPs, while structurally poor, are functionally rich by virtue of their flexibility and modularity. However, how mutations in IDPs elicit diseases, remain elusive. Herein, we have identified tumor suppressor PTEN as an intrinsically disordered protein (IDP) and elucidated the molecular principles by which its intrinsically disordered region (IDR) at the carboxyl-terminus (C-tail) executes its functions. Post-translational modifications, conserved eukaryotic linear motifs and molecular recognition features present in the C-tail IDR enhance PTEN's protein-protein interactions that are required for its myriad cellular functions. PTEN primary and secondary interactomes are also enriched in IDPs, most being cancer related, revealing that PTEN functions emanate from and are nucleated by the C-tail IDR, which form pliable network-hubs. Together, PTEN higher order functional networks operate via multiple IDP-IDP interactions facilitated by its C-tail IDR. Targeting PTEN IDR and its interaction hubs emerges as a new paradigm for treatment of PTEN related pathologies.

The biochemical landscape of lysine acetylation has expanded from a small number of proteins in the nucleus to a multitude of proteins in the cytoplasm. Since the first report confirming acetylation of the tumor suppressor protein p53 by a lysine acetyltransferase (KAT), there has been a surge in the identification of new, non-histone targets of KATs. Added to the known substrates of KATs are metabolic enzymes, cytoskeletal proteins, molecular chaperones, ribosomal proteins and nuclear import factors. Emerging studies demonstrate that no fewer than 2000 proteins in any particular cell type may undergo lysine acetylation. As described in this review, our analyses of cellular acetylated proteins using DAVID 6.7 bioinformatics resources have facilitated organization of acetylated proteins into functional clusters integral to cell signaling, the stress response, proteolysis, apoptosis, metabolism, and neuronal development. In addition, these clusters also depict association of acetylated proteins with human diseases. These findings not only support lysine acetylation as a widespread cellular phenomenon, but also impel questions to clarify the underlying molecular and cellular mechanisms governing target selectivity by KATs. Present challenges are to understand the molecular basis for the overlapping roles of KAT-containing co-activators, to differentiate between global versus dynamic acetylation marks, and to elucidate the physiological roles of acetylated proteins in biochemical pathways. In addition to discussing the cellular 'acetylome', a focus of this work is to present the widespread and dynamic nature of lysine acetylation and highlight the nexus that exists between epigenetic-directed transcriptional regulation and metabolism.

Arthrospira (Spirulina) platensis is widely used as a food supplement and has been an economically important species for centuries. However, the genetic aspect of studies of this particular organism has always been neglected, mainly because of the nonavailability of suitable methods for isolation of nucleic acids and the difficulties faced during further manipulations. Although total RNA has been isolated using commercially available kits, we present a method optimized to obtain DNA-free total RNA of higher yields and higher purity in less time than is required by other methods (<2 h). It involves hot phenol - chloroform - IAA extraction using an aqueous to organic phase ratio of 1:2 followed by lithium chloride precipitation and 70% ethanol wash. This method, optimized for the cyanobacterium Arthrospira (Spirulina) platensis, eliminates the need for DNase treatment and produces high-quality RNA, as validated by bioanalyzer, RT-PCR, and cloning. With the recent release of the Arthrospira genome, the current method will be of great value for carrying out high-throughput studies like microarray and real-time PCR.

Nitrate response at the plant level is mediated by the transcriptional regulation of several hundreds of genes, but no common cis-acting nitrate-responsive elements (NREs) have been identified so far. Earlier, we bioinformatically ruled out the possibility that the previously published [(a/t)7Ag/cTCA] motif could act as NRE on its own (Das et al., 2007, Mol. Genet. Genomics, 278: 519-525). In the present study, we examined other motifs such as Dof and GATA binding elements in homologous as well as heterologous pairwise combinations in the Arabidopsis genome in silico. None of the above three motifs revealed any unique association with nitrate responsive genes or their subsets in any combination, either within their ORFs or 1 kb flanking sequences on either side. Additionally, twelve new, top-scoring candidate motifs that were generated using different online motif samplers were analyzed in silico using a subset of 21 'early' nitrate responsive genes, but did not reveal any specificity of occurence. These results underscore the need to continue the search for novel candidate NREs, as possible sites of intervention to understand/improve nitrate-responsive gene expression and nitrate use efficiency.

Nitrate response element (NRE) was originally reported to be comprised of an Ag/cTCA core sequence motif preceded by a 7-bp AT rich region, based on promoter deletion analyses in nitrate and nitrite reductases from Arabidopsis thaliana and birch. In view of hundreds of new nitrate responsive genes discovered recently, we sought to computationally verify whether the above motif indeed qualifies to be the cis-acting NRE for all the responsive genes. We searched for the specific occurrence of at least two copies of the above motif in and around the nitrate responsive genes and elsewhere in the Arabidopsis and rice (Oryza sativa) genomes, with respect to their positional, orientational and strand-specific bias. This is the first comprehensive analysis of NREs for 625 nitrate responsive genes of Arabidopsis and their rice homologs, representing dicots and monocots, respectively. We report that the above motifs are present almost randomly throughout these genomes and do not reveal any specificity or bias towards nitrate responsive genes. This also seems to be true for smaller subsets of nitrate responsive genes in Arabidopsis, such as the 21 early responsive genes, 261 and 90 genes for root-specific and shoot-specific response, respectively, and 25 housekeeping genes. This necessitates a fresh search for candidate sequences that qualify to be NREs in these and other plants.