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IntroductionNoninvasive High-Frequency Oscillatory Ventilation (NHFOV) is increasingly being adopted to reduce the need for invasive ventilation after extubation.ObjectivesTo evaluate the benefits and harms of NHFOV as post-extubation respiratory support in newborns compared to other non-invasive respiratory support modes.Material & methodsWe included randomized controlled trials comparing NHFOV with other non-invasive modes post-extubation in newborns. Data sources were MEDLINE (via Pubmed), Cochrane Central Register of Controlled Trials, China National Knowledge Infrastructure, WHO international clinical trials registry platform and Clinical Trial Registry, forward and backward citation search. Methodological quality of studies was assessed by Cochrane's Risk of Bias tool 1.0.ResultsThis systematic review included 21 studies and 3294 participants, the majority of whom were preterm. NHFOV compared to nasal continuous positive airway pressure (NCPAP) reduced reintubation within seven days (RR 0.34, 95% CI 0.22 to 0.53) after extubation. It also reduced extubation failure (RR 0.39, 95% CI 0.30 to 0.51) and reintubation within 72 hrs (RR 0.40, 95% CI 0.31 to 0.53), bronchopulmonary dysplasia (RR 0.59, 95% CI 0.37 to 0.94) and pulmonary air leak (RR 0.46, 95% CI 0.27 to 0.79) compared to NCPAP. The rate of reintubation within seven days (RR 0.62, 95% CI 0.18 to 2.14) was similar whereas extubation failure (RR 0.65, 95% CI 0.50 to 0.83) and reintubation (RR 0.68, 95% CI 0.52 to 0.89) within 72 hrs were lower in NHFOV group compared to nasal intermittent positive pressure ventilation. There was no effect on other outcomes. Overall quality of the evidence was low to very low in both comparisons.ConclusionsNHFOV may reduce the rate of reintubation and extubation failure post-extubation without increasing complications. Majority of the trials were exclusively done in preterm neonates. Further research with high methodological quality is warranted.

Human paraoxonase 1 (h-PON1) is a serum enzyme that can hydrolyze a variety of substrates. The enzyme exhibits anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial and organophosphate-hydrolyzing activities. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against a variety conditions in human. However, the crystal structure of h-PON1 is not solved and the molecular details of how the enzyme hydrolyzes different substrates are not clear yet. Understanding the catalytic mechanism(s) of h-PON1 is important in developing the enzyme for therapeutic use. Literature suggests that R/Q polymorphism at position 192 in h-PON1 dramatically modulates the substrate specificity of the enzyme. In order to understand the role of the amino acid residue at position 192 of h-PON1 in its various hydrolytic activities, site-specific mutagenesis at position 192 was done in this study. The mutant enzymes were produced using Escherichia coli expression system and their hydrolytic activities were compared against a panel of substrates. Molecular dynamics simulation studies were employed on selected recombinant h-PON1 (rh-PON1) mutants to understand the effect of amino acid substitutions at position 192 on the structural features of the active site of the enzyme. Our results suggest that, depending on the type of substrate, presence of a particular amino acid residue at position 192 differentially alters the micro-environment of the active site of the enzyme resulting in the engagement of different subsets of amino acid residues in the binding and the processing of substrates. The result advances our understanding of the catalytic mechanism of h-PON1.

Mitochondrial genome sequences have been widely used for evolutionary and phylogenetic studies. Among vertebrates, fish are an important, diverse group, and their mitogenome sequences are growing rapidly in public repositories. To facilitate mitochondrial genome analysis and to explore the valuable genetic information, we developed the Fish Mitogenome Resource (FMiR) database to provide a workbench for mitogenome annotation, species identification and microsatellite marker mining. The microsatellites are also known as simple sequence repeats (SSRs) and used as molecular markers in studies on population genetics, gene duplication and marker assisted selection. Here, easy-to-use tools have been implemented for mining SSRs and for designing primers to identify species/habitat specific markers. In addition, FMiR can analyze complete or partial mitochondrial genome sequence to identify species and to deduce relational distances among sequences across species. The database presently contains curated mitochondrial genomes from 1302 fish species belonging to 297 families and 47 orders reported from saltwater and freshwater ecosystems. In addition, the database covers information on fish species such as conservation status, ecosystem, family, distribution and occurrence downloaded from the FishBase and IUCN Red List databases. Those fish information have been used to browse mitogenome information for the species belonging to a particular category. The database is scalable in terms of content and inclusion of other analytical modules. The FMiR is running under Linux operating platform on high performance server accessible at URL http://mail.nbfgr.res.in/fmir.

Treatment options for triple negative breast cancer (TNBC) are generally limited to cytotoxic chemotherapy. Recently, anti-epidermal growth factor receptor (EGFR) therapy has been introduced for TNBC patients. We engineered a novel nanobioconjugate based on a poly(β-L-malic acid) (PMLA) nanoplatform for TNBC treatment. The nanobioconjugate carries anti-tumor nucleosome-specific monoclonal antibody (mAb) 2C5 to target breast cancer cells, anti-mouse transferrin receptor (TfR) antibody for drug delivery through the host endothelial system, and Morpholino antisense oligonucleotide (AON) to inhibit EGFR synthesis. The nanobioconjugates variants were: (1) P (BioPolymer) with AON, 2C5 and anti-TfR for tumor endothelial and cancer cell targeting, and EGFR suppression (P/AON/2C5/TfR), and (2) P with AON and 2C5 (P/AON/2C5). Controls included (3) P with 2C5 but without AON (P/2C5), (4) PBS, and (5) P with PEG and leucine ester (LOEt) for endosomal escape (P/mPEG/LOEt). Drugs were injected intravenously to MDA-MB-468 TNBC bearing mice. Tissue accumulation of injected nanobioconjugates labeled with Alexa Fluor 680 was examined by Xenogen IVIS 200 (live imaging) and confocal microscopy of tissue sections. Levels of EGFR, phosphorylated and total Akt in tumor samples were detected by western blotting. In vitro western blot showed that the leading nanobioconjugate P/AON/2C5/TfR inhibited EGFR synthesis significantly better than naked AON. In vivo imaging revealed that 2C5 increased drug-tumor accumulation. Significant tumor growth inhibition was observed in mice treated with the lead nanobioconjugate (1) [P = 0.03 vs. controls; P

Chemotherapeutic drugs and newly developed therapeutic monoclonal antibodies are adequately delivered to most solid and systemic tumors. However, drug delivery into primary brain tumors and metastases is impeded by the blood-brain tumor barrier (BTB), significantly limiting drug use in brain cancer treatment.We examined the effect of phosphodiesterase 5 (PDE5) inhibitors in nude mice on drug delivery to intracranially implanted human lung and breast tumors as the most common primary tumors forming brain metastases, and studied underlying mechanisms of drug transport. In vitro assays demonstrated that PDE5 inhibitors enhanced the uptake of [(14)C]dextran and trastuzumab (Herceptin, a humanized monoclonal antibody against HER2/neu) by cultured mouse brain endothelial cells (MBEC). The mechanism of drug delivery was examined using inhibitors for caveolae-mediated endocytosis, macropinocytosis and coated pit/clathrin endocytosis. Inhibitor analysis strongly implicated caveolae and macropinocytosis endocytic pathways involvement in the PDE5 inhibitor-enhanced Herceptin uptake by MBEC. Oral administration of PDE5 inhibitor, vardenafil, to mice with HER2-positive intracranial lung tumors led to an increased tumor permeability to high molecular weight [(14)C]dextran (2.6-fold increase) and to Herceptin (2-fold increase). Survival time of intracranial lung cancer-bearing mice treated with Herceptin in combination with vardenafil was significantly increased as compared to the untreated, vardenafil- or Herceptin-treated mice (p0.05).These findings suggest that PDE5 inhibitors may effectively modulate BTB permeability, and enhance delivery and therapeutic efficacy of monoclonal antibodies in hard-to-treat brain metastases from different primary tumors that had metastasized to the brain.

Introduction: The most crucial abilities for a Paediatric Dentist involve evaluating and managing children based on their behaviour. Children are often motivated to choose coloured restorative materials. They tend to hold their new fillings in high regard and are usually fascinated by the new materials in their oral cavity. Consequently, they are more likely to maintain the restoration, which greatly enhances their overall oral hygiene and fosters a favourable attitude towards therapy. Aim: To evaluate the behaviour of children and their acceptance of multicoloured and conventional composite materials: A randomised controlled trial. Materials and Methods: A split-mouth, single-blinded, randomised clinical study was conducted on 25 patients with Class I occlusal caries at the Department of Paediatric and Preventive Dentistry, Government Dental College and Hospital (GDCH), Nagpur, Maharashtra, India, from July 2023 to January 2024. Healthy children aged between 5 and 10 years, who were visiting the dentist for the first time, were selected from the Department of Paediatric and Preventive Dentistry. The chidren were divided in two groups-group 1, 13 (52%) children with blue coloured composite restorations and group 2, 12 (48%) chidren who received conventional composite restorations. Behaviour was assessed using the modified behaviour rating scale before and after treatment. Data analysis was performed using Statistical Package for Social Sciences (SPSS) software version 16.0 with the Wilcoxon’s Signed-rank Test. Results: Among total of 25 children, 14 (56%) were males and 11 (44%) were females with a mean age of 8.47±1.59 years. There was a significant improvement in the behaviour of children after treatment with blue coloured compomer (p-value

Neurorehabilitation is the major pathway for motor recovery, but its effect on biomarkers of stroke has rarely been considered. Only brain-derived neurotrophic factor and matrix metalloproteinase are related to the rehabilitation process. Radiological and inflammatory biomarkers need to be correlated with the neurorehabilitation process. The clinical and physiological effects of neurorehabilitation on biomarkers and motor recovery should be established, to maximise the effect of neurorehabilitation. In this systematic review with narrative synthesis we provide the current state and understanding of neurorehabilitation and its effect on biomarkers for motor recovery in acute ischaemic stroke patients.

Abstract Background Breast cancer cells (BCCs) can remain undetected for decades in dormancy. These quiescent cells are similar to cancer stem cells (CSCs); hence their ability to initiate tertiary metastasis. Dormancy can be regulated by components of the tissue microenvironment such as bone marrow mesenchymal stem cells (MSCs) that release exosomes to dedifferentiate BCCs into CSCs. The exosomes cargo includes histone 3, lysine 4 (H3K4) methyltransferases - KMT2B and KMT2D. A less studied mechanism of CSC maintenance is the process of cell-autonomous regulation, leading us to examine the roles for KMT2B and KMT2D in sustaining CSCs, and their potential as drug targets. Methods Use of pharmacological inhibitor of H3K4 (WDR5–0103), knockdown (KD) of KMT2B or KMT2D in BCCs, real time PCR, western blot, response to chemotherapy, RNA-seq, and flow cytometry for circulating markers of CSCs and DNA hydroxylases in BC patients. In vivo studies using a dormancy model studied the effects of KMT2B/D to chemotherapy. Results H3K4 methyltransferases sustain cell autonomous regulation of CSCs, impart chemoresistance, maintain cycling quiescence, and reduce migration and proliferation of BCCs. In vivo studies validated KMT2’s role in dormancy and identified these genes as potential drug targets. DNA methylase (DNMT), predicted within a network with KMT2 to regulate CSCs, was determined to sustain circulating CSC-like in the blood of patients. Conclusion H3K4 methyltransferases and DNA methylation mediate cell autonomous regulation to sustain CSC. The findings provide crucial insights into epigenetic regulatory mechanisms underlying BC dormancy with KMT2B and KMT2D as potential therapeutic targets, along with standard care. Stem cell and epigenetic markers in circulating BCCs could monitor treatment response and this could be significant for long BC remission to partly address health disparity.

The histone deacetylase 2 (HDAC2), an enzyme involved in gene regulation, is a potent drug target for the treatment of colon cancer. Phytocompounds having anticancer properties show the ability to interact with HDAC2 enzyme. Among the compounds, docking scores of caffeic acid (CA) and p-coumaric acid (pCA) with HDAC2 showed good binding efficacy of -5.46 kcal/mol and -5.16 kcal/mol, respectively, with small inhibition constants. The higher binding efficacy of CA compared to pCA can be credited to the presence of an extra oxygen atom in the CA molecule, which forms an additional hydrogen bond with Tyr297. The HDAC2 in complex with these molecules was found to be stable by analyzing RMSD, RMSF, Rg, and SASA values obtained through MD simulations. Furthermore, CA and pCA exhibited low MM/GBSA free energies of -16.32 ± 2.62 kcal/mol and -17.01 ± 2.87 kcal/mol, respectively. The HOMO and LUMO energy gaps, dipole moments, global reactivity descriptor values, and MEP surfaces showed the reactivity of the molecules. The favourable physicochemical and pharmacokinetic properties, along with absence of toxicity of the molecules determined using ADMET analysis, suggested both the acids to be regarded as effective drugs in the treatment of colon cancer.

BackgroundLow- and middle-income countries are facing the emerging burden of chronic noncommunicable diseases (NCDs). Apart from loss of human lives and premature deaths, NCDs result in huge costs for treatment to individuals and the health system. Although NCDs develop in later life, the risk factors begin at an early age. The key to the control of the global epidemic of NCDs is primary prevention based on comprehensive community-based programs. ObjectiveThis study aims to develop, implement, and evaluate the effect of a participatory health promotion initiative utilizing the existing mechanisms of Village Health Nutrition and Sanitation Committees (VHNSCs), women’s self-help groups (SHGs), and schools on modifiable risk factors for NCDs among young people aged 10-30 years. MethodsThe proposed type II hybrid effectiveness implementation cluster randomized field trial will be conducted in the catchment area of 4 primary health centers (PHCs) in Wardha district, India, comprising 100 villages with a population of 144,000. Each PHC will be randomly allocated to one of the 3 intervention arms or the control arm. The 3-intervention arm PHCs will utilize a unique strategy with either VHNSC or SHG members or school students as change agents for health action against common modifiable NCD risk factors. This study will be implemented in 3 phases from January 2022 to December 2024. First, the preparatory phase for baseline assessments includes anthropometry, behavioral and biochemical risk factors for NCDs, and participatory development of the health promotion intervention modules. Second, the implementation phase will focus on capacity building of the change agents and implementation of the participatory health promotion initiative. The implementation will include organization of community-based events, 6-monthly participatory assessment of change, and preparation of a sustainability and exit plan toward the end of this phase. Third, the evaluation phase will consist of studying the effectiveness of each intervention strategy in the reduction of risk factor prevalence at the population level. ResultsWe will assess 12,000 (3000 in each arm) randomly selected individuals for behavioral risk factors and 1600 (400 in each arm) individuals for biochemical risk factors during baseline as well as endline assessments. Difference in differences, ANOVA or multivariate analysis of covariance, and regression analysis will be performed to assess the effectiveness of the interventions. Qualitative methods such as focus group discussions and stories of change will be documented and analyzed using thematic framework analysis. The implementation outcomes will be reported using the PRISM (Practical Robust Implementation and Sustainability Model) RE-AIM (Reach, Effectiveness, Adoption, Implementation, Maintenance) framework. The results are expected to be published by mid-2025. ConclusionsThis study will show the magnitude of risk factors for NCDs, its determinants, feasibility, effectiveness of community-based interventions, and health promotion models for NCD prevention. Trial RegistrationClinical Trials Registration India CTRI/2020/10/028700; https://ctri.nic.in/Clinicaltrials/showallp.php?mid1=47597&EncHid=&userName=V-CaN International Registered Report Identifier (IRRID)DERR1-10.2196/42450

Worsening heart failure (WHF) is a distinct under-diagnosed and under-treated condition, independent of location of care. Heart failure (HF) progression is punctuated by repeated WHF events, each resulting in reduced cardiac function. One-third of the patients with HF with reduced ejection fraction experience a decompensation event. These decompensation events often result in the emergency department visits and HF hospitalization. Despite its inclusion in recent guidelines, there is no precise definition of WHF or its various forms. It is worth noting that WHF signals a need for treatment optimization as per guideline-directed medical therapy and the addition of novel drugs like a stimulator of soluble guanylate cyclase that benefit this high-risk patient population. This practical document is based on the expert opinion of cardiologists, cardiothoracic surgeons, and physicians that discussed the definition, assessment, pharmacological management, and monitoring of WHF patients in a hospitalized setting. In addition, there is also a need for an expert opinion for the management of WHF in an outpatient setting.

Thoracic aortic diseases involving the thoracic aorta, from the ascending aorta, the aortic arch to the descending aorta, can present in the form of aneurysms, dissection, tear and coarctation which usually lead to various complications, requiring surgical intervention. The same can be addressed surgically by ascending aorta replacement or reduction aortoplasty with/without Aortic Valve Replacement (AVR). The anaesthetic implications might vary depending on the pathology of the thoracic aortic disease which can be acute or chronic and, silent or symptomatic. Anaesthetic management of four patients (1 female and 3 males) with varied thoracic aortic diseases have been described in the series, including bicuspid aortic valve with severe aortic stenosis, Ischaemic Heart Disease (IHD) with severe Aortic Regurgitation (AR) with ascending aortic aneurysm, ascending aortic dilatation in a known Takayasu arteritis patient and coarctation of aorta with atrial septal defect. These patients underwent aortoplasty with or without aortic root replacement. Full cardiopulmonary bypass with Deep Hypothermic Circulatory Arrest (DHCA) at 16-20°C was the technique used for these procedures as it prevents stroke and ensures cognitive function. This technique had no additional cannulas, less chances of intimal injury or embolisation and clear surgical fields. During the process of rewarming, Inj. nitroglycerine was started which reduced preload, conserving the myocardium against ischaemic injuries. These patients were successfully managed perioperatively and were discharged with good outcomes postoperatively.

Abstract Inducible and tunable expression systems are essential for the microbial production of biochemicals. Five different carbon source- and substrate-inducible promoter systems were developed and further evaluated in Pseudomonas putida KT2440 by analyzing the expression of green fluorescent protein (GFP) as a reporter protein. These systems can be induced by low-cost compounds such as glucose, 3-hydroxypropionic acid (3HP), levulinic acid (LA), and xylose. 3HP-inducible HpdR/P hpdH was also efficiently induced by LA. LvaR/P lvaA and XutR/P xutA systems were induced even at low concentrations of LA (0.1 mM) and xylose (0.5 mM), respectively. Glucose-inducible HexR/P zwf1 showed weak GFP expression. These inducer agents can be used as potent starting materials for both cell growth and the production of a wide range of biochemicals. The efficiency of the reported systems was comparable to that of conventional chemical-inducible systems. Hence, the newly investigated promoter systems are highly useful for the expression of target genes in the widely used synthetic biology chassis P. putida KT2440 for industrial and medical applications.

Abstract In plants, during growth and development, photoreceptors monitor fluctuations in their environment and adjust their metabolism as a strategy of surveillance. Phytochromes (Phys) play an essential role in plant growth and development, from germination to fruit development. FR-light (FR) insensitive mutant (fri) carries a recessive mutation in Phytochrome A and is characterized by the failure to de-etiolate in continuous FR. Here we used iTRAQ-based quantitative proteomics along with metabolomics to unravel the role of Phytochrome A in regulating central metabolism in tomato seedlings grown under FR. Our results indicate that Phytochrome A has a predominant role in FR-mediated establishment of the mature seedling proteome. Further, we observed temporal regulation in the expression of several of the late response proteins associated with central metabolism. The proteomics investigations identified a decreased abundance of enzymes involved in photosynthesis and carbon fixation in the mutant. Profound accumulation of storage proteins in the mutant ascertained the possible conversion of sugars into storage material instead of being used or the retention of an earlier profile associated with the mature embryo. The enhanced accumulation of organic sugars in the seedlings indicates the absence of photomorphogenesis in the mutant.

Despite increasing immunisation rates, developing countries continue to report subacute sclerosing pan encephalitis (SSPE). The defective measles virus causng SSPE persists in brain cells because of hypermutated M protein and deranged host's immune responses. Patients usually present with cognitive decline and myoclonus. However, atypical presentations such as seizures and visual loss are also quite common, causing wrong or delayed diagnosis in a significant number of cases. Diagnosis is based on suggestive clinical features, electroencephalographic findings and elevated cerebrospinal fluid (CSF) antimeasles antibody titre. Newer methods of reporting antibody levels such as CSF/serum quotient (CSQrel) result in increased specificity, but individual values of serum and CSF antimeasles antibody titres should also be checked if CSQrel is negative or equivocal. In highly suspicious cases with negative CSF antimeasles antibody profile, repeat testing should be done. Combination therapy with interferon-alpha and isoprinosine is the most common starting regimen. Intraventricular administration of interferon-alpha is theoretically the most effective route but requires meticulous hygiene and complications are frequent. Hence, the proper route and frequency of interferon-alpha treatment should be chosen depending on efficacy, affordability, disease stage and parent's expectations. Though treatment has largely remained unsatisfactory, reported rates of improvement or stabilisation (34%–35%) are much better than that for spontaneous remission (5%–10%). Fusion inhibitors and adenovirus-delivered small interfering RNA are being studied as new therapies. However, increasing immunisation rates can be the only long-term answer to tackle the menace of measles and its complications.

Introduction: Drug-Induced Gingival Overgrowth (DIGO) is caused due to prolonged use of anti convulsants, immunosuppressant, and calcium channel blockers given for non dental purpose. It affects the maintenance of oral hygiene and may cause speech, mastication, tooth eruption and aesthetic problems. General physicians can play a key role as they can inform the patient about gingival overgrowth as an adverse effect of these drugs. Aim: To evaluate the awareness regarding drug-induced gingival overgrowth and to know the impact of educational qualification on their awareness among physicians. Materials and Methods: This cross-sectional questionnaire survey was conducted from January 2019 to June 2019 in Latur district of Maharashtra, India. A total of 196 practicing physicians were approached with self-structured questionnaire and answers were collected in the presence of the investigator. Survey responses were divided into two groups based on educational qualification as group A: physicians educationally qualified to practice Allopathy, group B: physicians educationally qualified to practice alternative medicine (Ayurveda, Homeopathy etc.,). Comparison of responses for qualitative variables was carried among groups using Chi-square test with p-value set as p

With the intent to better management human wildlife conflict (HWC) and wildlife conservation in mid-hills outside protected areas of Gandaki province, Nepal, we analyzed the patterns and drivers of HWC. Using data collected from literature, government records and questionnaire survey, we investigated temporal, seasonal and spatial distribution of human casualties caused by wildlife attacks. We also appraised the perception of local people towards wildlife conservation. We have recorded 77 cases (69 human injuries and 8 mortalities) during the period of nine year between 2011 and 2019. The number of wildlife attacks increased over this period. Wildlife attacks were more frequent in winter with 50% (42) of attacks occurred between September and December. Common leopard (Panthera pardus) and Himalayan black bear (Ursus thibetanus laniger) were the major species involved in these conflicts. Common leopard was the most feared species that causes highest number of human mortalities (87%, n = 67); the most severe type of HWC outcome. Forty-eight percent (n = 37) attacks were reported at human settlement areas followed by 27% attacks in agriculture land (n = 21) and 24% (n = 19) in forest. Generalized linear model analysis on spatial variables showed that the probability of human attacks increases with decreasing elevation (β = -0.0021, Z = -1.762, p = 0.078) and distance from the forest (β = -0.608, Z = -0.789, p = 0.429). We recommend to decrease habitat degradation / fragmentation, carry out habitat management program within forest to increase prey availability to decrease the wildlife invasion into human settlement area, and decrease dependency of people on forest resources by providing alternative livelihood opportunities. Simplified relief fund distribution mechanism at local level also helps alleviate the impact of HWC. The knowledge obtained by this study and management measures are important for better human-wildlife co-existence.

Isatin (1H indole 2, 3-dione) is a heterocyclic, endogenous lead molecule recognized in humans and different plants. The isatin nucleus and its derivatives are owed the attention of researchers due to their diverse pharmacological activities such as anticancer, anti-TB, antifungal, antimicrobial, antioxidant, anti-inflammatory, anticonvulsant, anti-HIV, and so on. Many research chemists take advantage of the gentle structure of isatins, such as NH at position 1 and carbonyl functions at positions 2 and 3, for designing biologically active analogues via different approaches. Literature surveys based on reported preclinical, clinical, and patented details confirm the multitarget profile of isatin analogues and thus their importance in the field of medicinal chemistry as a potent chemotherapeutic agent. This review represents the recent development of isatin analogues possessing potential pharmacological action in the years 2016–2020. The structure–activity relationship is also discussed to provide a pharmacophoric pattern that may contribute in the future to the design and synthesis of potent and less toxic therapeutics.

Abstract Warburg effect is an emerging hallmark of cancer cells with pyruvate kinase M2 (PKM2) as its key regulator. Curcumin is an extensively-studied anti-cancer compound, however, its role in affecting cancer metabolism remains poorly understood. Herein, we show that curcumin inhibits glucose uptake and lactate production (Warburg effect) in a variety of cancer cell lines by down-regulating PKM2 expression, via inhibition of mTOR-HIF1α axis. Stable PKM2 silencing revealed that PKM2 is required for Warburg effect and proliferation of cancer cells. PKM2 over-expression abrogated the effects of curcumin, demonstrating that inhibition of Warburg effect by curcumin is PKM2-mediated. High PKM2 expression correlated strongly with poor overall survival in cancer, suggesting the requirement of PKM2 in cancer progression. The study unravels novel PKM2-mediated inhibitory effect of curcumin on metabolic capacities of cancer cells. To the best of our knowledge, this is the first study linking curcumin with PKM2-driven cancer glycolysis, thus, providing new perspectives into the mechanism of its anticancer activity.

Myelodysplastic Syndromes (MDSs) affect the elderly and can progress to Acute Myeloid Leukemia (AML). Epigenetic alterations including DNA methylation and chromatin modification may contribute to the initiation and progression of these malignancies. DNA hypomethylating agents such as decitabine and azacitidine are used as therapeutic treatments and have shown to promote expression of genes involved in tumor suppression, apoptosis, and immune response. Another anti-cancer drug, the proteasome inhibitor bortezomib, is used as a chemotherapeutic treatment for multiple myeloma (MM). Phase III clinical trials of decitabine and azacitidine used alone and in combination with other chemotherapeutics demonstrated their capacity to treat hematological malignancies and prolong the survival of MDS and AML patients. Although phase III clinical trials examining bortezomib’s role in MDS and AML patients are limited, its underlying mechanisms in MM highlight its potential as a chemotherapeutic for such malignancies. Further research is needed to better understand how the epigenetic mechanisms mediated by these chemotherapeutic agents and their targeted gene networks are associated with the development and progression of MDS into AML. This review discusses the mechanisms by which decitabine, azacitidine, and bortezomib alter epigenetic programs and their results from phase III clinical trials.

The topological characteristics of biological networks enable us to identify the key nodes in terms of modularity. However, due to a large size of the biological networks with many hubs and functional modules across intertwined layers within the network, it often becomes difficult to accomplish the task of identifying potential key regulators. We use for the first time a generalized formalism of Hamiltonian Energy (HE) with a recursive approach. The concept, when applied to the Apoptosis Regulatory Gene Network (ARGN), helped us identify 11 Motif hubs (MHs), which influenced the network up to motif levels. The approach adopted allowed to classify MHs into 5 significant motif hubs (S-MHs) and 6 non-significant motif hubs (NS-MHs). The significant motif hubs had a higher HE value and were considered as high-active key regulators; while the non-significant motif hubs had a relatively lower HE value and were considered as low-active key regulators, in network control mechanism. Further, we compared the results of the HE analyses with the topological characterization, after subjecting to the three conditions independently: (i) removing all MHs, (ii) removing only S-MHs, and (iii) removing only NS-MHs from the ARGN. This procedure allowed us to cross-validate the role of 5 S-MHs, NFk-B1, BRCA1, CEBPB, AR, and POU2F1 as the potential key regulators. The changes in HE calculations further showed that the removal of 5 S-MHs could cause perturbation at all levels of the network, a feature not discernible by topological analysis alone.

Breast cancer is the most prevalent cancer among women, with the basal-like triple negative (TNBC) being the most agressive one, displaying the poorest prognosis within the ductal carcinoma subtype. Due to the lack of adequate molecular targets, the diagnosis and treatment of patients with the TNBC phenotype has been a great challenge. In a previous work, we identified CD90/Thy-1 as being highly expressed in the aggressive high malignancy grade Hs578T basal-like breast tumor cell line, pointing to this molecule as a promising breast tumor marker, which should be further investigated. Here, CD90 expression was analyzed in human breast cancer samples and its functional role was investigated to better assess the oncogenic nature of CD90 in mammary cells. Quantification of CD90 expression in human breast cancer samples, by tissue microarray, showed that high CD90 positivity correlates with metastasis and poor patient survival in the basal-like subtype. The functional genetic approach, by overexpression in the CD90 cDNA in a basal-like normal mammary cell line (MCF10A) and knockdown in a highly malignant cell line (Hs578T), allowed us to demonstrate that CD90 is involved with several cellular processes that lead to malignant transformation, such as: morphological change, increased cell proliferation, invasiveness, metastasis and activation of the EGFR pathway. Therefore, our results reveal that CD90 is involved with malignant transformation in breast cancer cell lines and is correlated with metastasis and poor patient survival in the basal-like subtype, being considered as a promising new breast cancer target.

An isolated and unilateral foot drop due to intracranial lesion is quite rare. Presenting herein a case of a 14-year-old female who complained of inability to wear and hold slipper in her left foot. Detailed neurological examination revealed left foot dorsiflexion which had 1/5 muscle power along with brisk left ankle reflex. Magnetic resonance imaging of the brain revealed multiple conglomerate inflammatory granulomas in cerebrum and cerebellum, larger one in right parasagittal region with perifocal oedema. Magnetic resonance spectrum was suggestive of tuberculoma. Her chest X-ray chest revealed milliary shadowing. She was put on antitubercular drugs, steroid and a prophylactic anti-epileptic drug. The dorsiflexion improved to grade 4/5 after three weeks of treatment. The motor homunculus for foot is located in parasagittal area. Therefore, in patients with foot drop, we must keep high index of suspicion for parasagittal lesions, so that prompt diagnosis and early management can be done to prevent complications and improve the quality of life of patient.

Plants can regenerate from a variety of tissues on culturing in appropriate media. However, the metabolic shifts involved in callus formation and shoot regeneration are largely unknown. The metabolic profiles of callus generated from tomato (Solanum lycopersicum) cotyledons and that of shoot regenerated from callus were compared with the pct1-2 mutant that exhibits enhanced polar auxin transport and the shr mutant that exhibits elevated nitric oxide levels. The transformation from cotyledon to callus involved a major shift in metabolite profiles with denser metabolic networks in the callus. In contrast, the transformation from callus to shoot involved minor changes in the networks. The metabolic networks in pct1-2 and shr mutants were distinct from wild type and were rewired with shifts in endogenous hormones and metabolite interactions. The callus formation was accompanied by a reduction in the levels of metabolites involved in cell wall lignification and cellular immunity. On the contrary, the levels of monoamines were upregulated in the callus and regenerated shoot. The callus formation and shoot regeneration were accompanied by an increase in salicylic acid in wild type and mutants. The transformation to the callus and also to the shoot downregulated LST8 and upregulated TOR transcript levels indicating a putative linkage between metabolic shift and TOR signalling pathway. The network analysis indicates that shift in metabolite profiles during callus formation and shoot regeneration is governed by a complex interaction between metabolites and endogenous hormones.

Introduction: COPD is a common preventable and treatable disease and a major cause of morbidity and mortality globally. ECG is a very simple, widely available and convenient bedside investigation that can be used to detect various cardiac abnormalities. Electrical activities of the heart are often influenced by COPD. ECG changes like P-wave abnormalities have to be carefully assessed before coming to an inference. Methodology: The present study was an institutional based prospective study, conducted from July 2014 to June 2015. The study was designed to assess the various ECG abnormalities in stable COPD patients and to determine echocardiography findings in relation to ECG changes. Results: Among the P-wave abnormalities P-wave axis verticalisation (PWAV) was observed in 59(76.6%) patients. Twenty (26%) patients showed negative P-wave in V1, significant-Ptf(P-terminal force) was observed in 3(3.9%) patients. P-mitrale was seen in 18(31.2%), and P-pulmonale in 16 (20.8%) patients. Conclusion: Though not specific, ECG may reveal various functional and structural abnormalities of the heart in relation to COPD like PWAV. PWAV may be the most commonly seen P-wave abnormalities in COPD patients. Echocardiography findings suggest that presence of P-mitrale is not conclusive of left atrial overload in patients of COPD. Echocardiography should be done routinely in all COPD patients to confirm ECG findings and to diagnose pulmonary hypertension, cor-pulmonale and other subclinical cardiovascular co-morbidities like left ventricular diastolic dysfunction.

Objective: To evaluate the utility of fiberoptic bronchoscopy in sputum smear negative PTB patients. Material and Methods: A total of 66 adult patients with sputum smear negative for Acid Fast Bacilli (AFB) and chest X-ray suggestive of pulmonary tuberculosis underwent fiberopticbronchoscopy (FOB). A thorough examination of bronchial tree was carried out and bronchoalveolarlavage (BAL) was taken and was sent for Ziehl-Neelsen staining, MGIT960 TBculture, pyogenicand fungal culture. Bronchial brushing, endo-bronchial and transbronchial lung biopsy(TBLB) wherever indicated were performed and Ziehl-Neelsen staining was performed and post bronchoscopy sputum(PBS) was also sent for Ziehl-Neelsen stain.Results are summarized in tables and percentages. Quantitative data is summarized using means& standard deviation. Cross tabulation with outcome variable of interest was done using statistical software Epi-info version 7 (7.1.1.0). A p-value of less than 0.05 was considered statistically significant. Results: Males constituted majority of our study population. The most common age group involved in the study was 18-28 years (36.3%). cough was the most common symptom reported by 62 patients (93.93%). The past history of PTB was present 6 patients (9.09%).Majority of study population, 39 patients (59.09%) had unilateral lesion on CXR. Out of 66 clinically suspected SSN-PTB patients, 52 patients (78.7%) were finally diagnosed as having active PTB. The diagnosis other than PTB was established in 6 (9.09%) cases, which included 3 cases of fungal (Candida) pneumonia and 3 cases of bacterial (pseudomonas species, citrobacter species, serratiamarsecens) pneumonia. Conclusions: FOB and various bronchoscopy guided procedures can provide a rapid and definitive diagnosis of PTB in sputum negative patients.

The ancestor of tomato underwent whole genome triplication ca. 71 Myr ago followed by widespread gene loss. However, few of the triplicated genes are retained in modern day tomato including lycopene beta cyclase that mediates conversion of lycopene to β-carotene. The fruit specific β-carotene formation is mediated by a chromoplast-specific paralog of lycopene beta cyclase (CYC-B) gene. Presently limited information is available about how the variations in CYC-B gene contributed to its neofunctionalization. CYC-B gene in tomato clade contained several SNPs and In-Dels in the coding sequence (33 haplotypes) and promoter region (44 haplotypes). The CYC-B gene coding sequence in tomato appeared to undergo purifying selection. The transit peptide sequence of CYC-B protein was predicted to have a stronger plastid targeting signal than its chloroplast specific paralog indicating a possible neofunctionalization. In promoter of two Bog (Beta old gold) mutants, a NUPT (nuclear plastid) DNA fragment of 256 bp, likely derived from a S. chilense accession, was present. In transient expression assay, this promoter was more efficient than the "Beta type" promoter. CARGATCONSENSUS box sequences are required for the binding of the MADS-box regulatory protein RIPENING INHIBITOR (RIN). The loss of CARGATCONSENSUS box sequence from CYC-B promoter in tomato may be related to attenuation of its efficiency to promote higher accumulation of β-carotene than lycopene during fruit ripening.

Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS), carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1) involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima'D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future.

Objective: To know the frequency and predictors of peripheral neuropathy and the abnormal visual evoked potentials in stable patients with chronic obstructive pulmonary disease. Material & Methods: All consecutive patients of stable COPD of age ≥ 40 years were screened for peripheral neuropathy. Total 57 patients of stable COPD of age ≥ 40 Years were selected. Spirometric examination was done in all the patients using an electronic portable based spirometer with printer. Neuro-physiological evaluation (neurological disability score-NDS), Mini Mental State Examination-questionnaire (MMSE), Visual analogue scale (VAS) were done on all subjects. The following nerves were evaluated for latency, amplitude, and conduction velocity. For motor nerve conduction: - median, ulnar, common peroneal and tibial nerves were evaluated. For sensory nerve conduction: - median, ulnar and sural nerves were evaluated. VEP was evaluated in all the 57 subjects. The continuous variables were described using means and analysis was done using independent t test. Results: Sub-clinical neuropathy was seen in 28 (49%) out of 57 stable COPD patients. Sensory neuropathy was seen in majority of patients. Sural nerve was the most commonly involved nerve. Neuropathy was predominantly axonal in nature. Abnormal VEP was found in 18 (32%) patients in whom P100 latency was more than 107. Conclusions: Electro-physiological testing showed the presence of peripheral neuropathy in 49% patients and abnormal VEP in 32% patients with stable COPD, who had no neurological symptoms. Maximum number of stable COPD patients with severe obstruction, heavy smoking index and bio-mass exposure had peripheral neuropathy, but statistically this was not found significant.

Despite early detection through the use of mammograms and aggressive intervention, breast cancer (BC) remains a clinical dilemma. BC can resurge after >10 years of remission. Studies indicate that BC cells (BCCs) with self-renewal and chemoresistance could be involved in dormancy. The majority of studies use in vitro, two-dimensional (2-D) monolayer cultures, which do not recapitulate the in vivo microenvironment. Thus, to determine the effect of three-dimensional (3-D) microenvironment on BCCs, this study fabricated tissue engineering scaffolds made of poly (ε-caprolactone) (PCL) having aligned or random fibers. Random and aligned fibers mimic, respectively, the random and highly organized collagen fibers found in the tumor extracellular matrix. Chemoresistant BCCs were obtained by treating with carboplatin. Western blot analysis of carboplatin resistant (treated) MDA-MB-231 (highly invasive, basal-like) and T47D (low-invasive, luminal) BCCs showed an increase in Bcl-2, Oct-4 and Sox-2, suggesting protection from apoptosis and increase in stem-like markers. Further studies with MDA-MB-231 BCCs seeded on the scaffolds showed little to no change in cell number over time for non-treated BCCs whereas on tissue culture polystyrene (TCP), non-treated BCCs displayed a significant increase in cell number at days 4 and 7 as compared to day 1 (p

Role of, 29-non-synonymous, 15-intronic, 3-close to UTR, single nucleotide polymorphisms (SNPs) and 2 mutations of Human Pyruvate Kinase (PK) M2 were investigated by in-silico and in-vitro functional studies. Prediction of deleterious substitutions based on sequence homology and structure based servers, SIFT, PANTHER, SNPs&GO, PhD-SNP, SNAP and PolyPhen, depicted that 19% emerged common between all the mentioned programs. SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y) in-silico as deleterious and functionally important. In-vitro activity assays showed C31F and S437Y variants of PKM2 with reduced activity, while Q310P variant was catalytically inactive. The allosteric activation due to binding of fructose 1-6 bisphosphate (FBP) was compromised in case of S437Y nsSNP variant protein. This was corroborated through molecular dynamics (MD) simulation study, which was also carried out in other two variant proteins. The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431) could generate an alternative transcript by influencing splicing factor binding to PKM2. We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

Cysticercosis is a rare disease caused by the ingestion of the parasite Cysticercus cellulosae, a larval stage of Taenia solium. The definitive host is human who harbors the adult worm and may accidentally or incidentally become the host. The larval form of cyst is commonly seen in the brain, meninges and eyes. Cases in the maxillofacial region including oral cavity and cheek muscles are rarely reported. Cysticercosis is not commonly considered in the diagnosis of swellings of the head and neck and a diagnostic and therapeutic dilemma for clinicians. Hence, they are of utmost interest to the practitioner and have to be studied. We present an unusual case of cysticercosis presenting as a solitary cystic nodule in the upper left vestibule of the oral cavity in an 18 year male and the diagnosis was made on histopathological examination.

Non-destructive monitoring of engineered tissues is needed for translation of these products from the lab to the clinic. In this study, non-invasive, high resolution spectral ultrasound imaging (SUSI) was used to monitor the differentiation of MC3T3 pre-osteoblasts seeded within collagen hydrogels. SUSI was used to measure the diameter, concentration and acoustic attenuation of scatterers within such constructs cultured in either control or osteogenic medium over 21 days. Conventional biochemical assays were used on parallel samples to determine DNA content and calcium deposition. Construct volume and morphology were accurately imaged using ultrasound. Cell diameter was estimated to be approximately 12.5-15.5 µm using SUSI, which corresponded well to measurements of fluorescently stained cells. The total number of cells per construct assessed by quantitation of DNA content decreased from 5.6±2.4×10(4) at day 1 to 0.9±0.2×10(4) at day 21. SUSI estimation of the equivalent number of acoustic scatters showed a similar decreasing trend, except at day 21 in the osteogenic samples, which showed a marked increase in both scatterer number and acoustic impedance, suggestive of mineral deposition by the differentiating MC3T3 cells. Estimation of calcium content by SUSI was 41.7±11.4 µg/ml, which agreed well with the biochemical measurement of 38.7±16.7 µg/ml. Color coded maps of parameter values were overlaid on B-mode images to show spatiotemporal changes in cell diameter and calcium deposition. This study demonstrates the use of non-destructive ultrasound imaging to provide quantitative information on the number and differentiated state of cells embedded within 3D engineered constructs, and therefore presents a valuable tool for longitudinal monitoring of engineered tissue development.

Gemcitabine, an effective agent in treatment of cancer of pancreas, has undergone failures in many instances after multiple cycles of therapy due to emergence of drug resistance. Combination of dietary compounds with clinically validated drugs has emerged as an effective therapeutic approach to treat pancreatic tumors, refractory to gemcitabine therapy. In order to optimize a possible synergistic combination of Gemcitabine (GCB) with dietary molecules, Betuilnic acid (BA) and Thymoquinone (TQ), stand-alone IC50 dose of GCB, BA and TQ was calculated for pancreatic cancer cell lines. Fixed IC50 dose ratio of the dietary molecules in combination with reduced IC50 dose of GCB was tested on GCB resistant PANC-1 and sensitive MIA PaCa-2 cells for synergism, additive response and antagonism, using calcusyn. Combination index (CI) revealed that pre-treatment of BA and TQ along with GCB synergistically inhibited the cancer cell proliferation in in-vitro experiments. Pyruvate kinase (PK) M2 isoform, a promising target involved in cancer cell metabolism, showed down-regulation in presence of TQ or BA in combination with GCB. GCB with BA acted preferentially on tumor mitochondria and triggered mitochondrial permeability transition. Pre-exposure of the cell lines, MIA PaCa-2 and PANC-1, to TQ in combination with GCB induced apoptosis. Thus, the effectiveness of BA or TQ in combination with GCB to inhibit cell proliferation, induce apoptosis and down-regulate the expression of PKM2, reflects promise in pancreatic cancer treatment.

Type 2 diabetes (T2D) is a syndrome of multiple metabolic disorders and is genetically heterogeneous. India comprises one of the largest global populations with highest number of reported type 2 diabetes cases. However, limited information about T2D associated loci is available for Indian populations. It is, therefore, pertinent to evaluate the previously associated candidates as well as identify novel genetic variations in Indian populations to understand the extent of genetic heterogeneity. We chose to do a cost effective high-throughput mass-array genotyping and studied the candidate gene variations associated with T2D in literature. In this case-control candidate genes association study, 91 SNPs from 55 candidate genes have been analyzed in three geographically independent population groups from India. We report the genetic variants in five candidate genes: TCF7L2, HHEX, ENPP1, IDE and FTO, are significantly associated (after Bonferroni correction, p

The glycoprotein YKL-40 (CHI3L1) is a secreted chitinase family protein that induces angiogenesis, cell survival, and cell proliferation, and plays roles in tissue remodeling and immune regulation. It is expressed primarily in cells of mesenchymal origin, is overexpressed in numerous aggressive carcinomas and sarcomas, but is rarely expressed in normal ectodermal tissues. Bone marrow-derived mesenchymal stem cells (MSCs) can be induced to differentiate into various mesenchymal tissues and trans-differentiate into some non-mesenchymal cell types. Since YKL-40 has been used as a mesenchymal marker, we followed YKL-40 expression as undifferentiated MSCs were induced to differentiate into bone, cartilage, and neural phenotypes. Undifferentiated MSCs contain significant levels of YKL-40 mRNA but do not synthesize detectable levels of YKL-40 protein. MSCs induced to differentiate into chondrocytes and osteocytes soon began to express and secrete YKL-40 protein, as do ex vivo cultured chondrocytes and primary osteocytes. In contrast, MSCs induced to trans-differentiate into neurons did not synthesize YKL-40 protein, consistent with the general absence of YKL-40 protein in normal CNS parenchyma. However, these trans-differentiated neurons retained significant levels of YKL-40 mRNA, suggesting the mechanisms which prevented YKL-40 translation in undifferentiated MSCs remained in place, and that these trans-differentiated neurons differ in at least this way from neurons derived from neuronal stem cells. Utilization of a differentiation protocol containing β-mercaptoethanol resulted in cells that expressed significant amounts of intracellular YKL-40 protein that was not secreted, which is not seen in normal cells. Thus the synthesis of YKL-40 protein is a marker for MSC differentiation into mature mesenchymal phenotypes, and the presence of untranslated YKL-40 mRNA in non-mesenchymal cells derived from MSCs reflects differences between differentiated and trans-differentiated phenotypes.

Our previous studies have implicated genes mainly involved in the activity of pancreatic β cells in type 2 diabetes (T2D) susceptibility in the North Indian population. Recent literature on the role of SIRT1 as a potential master switch modulating insulin secretion and regulating gene expression in pancreatic β cells has warranted an evaluation of SIRT1 promoter region polymorphisms in the North Indian population, which is the main focus of the present study. 1542 samples (692 T2D patients and 850 controls) were sequenced for the 1.46 kb region upstream the translation start site of the SIRT1 gene. We performed a functional characterization of the SIRT1 promoter region polymorphisms using luciferase assay and observed a single-nucleotide polymorphism (SNP), rs12778366, in association with SIRT1 expression. We propose that TT, the high-expressing genotype of SNP rs12778366 in the SIRT1 promoter region and present in >80% of the North Indian population, was favored under conditions of feast-famine cycles in evolution, which has turned out to be a cause of concern in the present sedentary lifestyle under ad libitum conditions. Case-control association analysis did not implicate rs12778366 in T2DM per se in the studied population. However, our earlier reported risk genotype combinations of mt-ND3, PGC1α, and UCP2-866, when compared with the protective genotype combinations, in the background of the high-expressing TT genotype of SIRT1 SNP rs12778366, showed a very high additive risk [corrected odd ratio (OR) = 8.91; p = 6.5×10(-11)]. The risk level was considerably low in the genotype backgrounds of TX (OR = 6.68; p = 2.71×10(-12)) and CX (OR = 3.74; p = 4.0×10(-3)). In addition, we screened other reported T2D-associated polymorphisms: PIK3R1 rs3730089, IRS1 rs1801278, and PPP1R3 rs1799999, which did not show any significant association in North Indian population. The present paper emphasizes the importance of gene interactions in the biological pathways of T2D, a complex lifestyle disease.

Metabolism of cancer cells with pyruvate kinase M2 (PKM2) at its centre stage has assumed a prime significance in cancer research in recent times. Cancer cell metabolism, characterized by enhanced glucose uptake, production of lactate and anabolism is considered an ideal target for therapeutic interventions. Expression of PKM2 switches metabolism in favor of cancer cells, therefore, the present study was designed to investigate the hitherto unknown effect of resveratrol, a phytoalexin, on PKM2 expression and resultant implications on cancer metabolism. We observed that resveratrol down-regulated PKM2 expression by inhibiting mTOR signaling and suppressed cancer metabolism, adjudged by decreased glucose uptake, lactate production (aerobic glycolysis) and reduced anabolism (macromolecule synthesis) in various cancer cell lines. A contingent decrease in intracellular levels of ribose-5-phosphate (R5P), a critical intermediate of pentose phosphate pathway, accounted for a reduced anabolism. Consequently, the state of suppressed cancer metabolism resulted in decreased cellular proliferation. Interestingly, shRNA-mediated silencing of PKM2 inhibited glucose uptake and lactate production, providing evidence for the critical role of PKM2 and its mediation in the observed effects of resveratrol on cancer metabolism. Further, an over-expression of PKM2 abolished the observed effects of resveratrol, signifying the role of PKM2 downregulation as a critical function of resveratrol. The study reports a novel PKM2-mediated effect of resveratrol on cancer metabolism and provides a new dimension to its therapeutic potential.

BACKGROUND: An understanding of BC cell (BCC) entry into bone marrow (BM) at low tumor burden is limited when compared to highly metastatic events during heavy tumor burden. BCCs can achieve quiescence, without interfering with hematopoiesis. This occurs partly through the generation of gap junctions with BM stroma, located close to the endosteum. These events are partly mediated by the evolutionary conserved gene, Tac1. METHODOLOGY/PRINCIPAL FINDINGS: This study focuses on the role of mesenchymal stem cells (MSCs), Tac1, SDF-1 and CXCR4 in BCC entry into BM. The model is established in studies with low numbers of tumor cells, and focuses on cancer cells with low metastatic and invasion potential. This allowed us to recapitulate early event, and to study cancer cells with low invasive potential, even when they are part of larger numbers of highly metastatic cells. A novel migration assay showed a facilitating role of MSCs in BCC migration across BM endothelial cells. siRNA and ectopic expression studies showed a central role for Tac1 and secondary roles for SDF-1alpha and CXCR4. We also observed differences in the mechanisms between low invasive and highly metastatic cells. The in vitro studies were verified in xenogeneic mouse models that showed a preference for low invasive BCCs to BM, but comparable movement to lung and BM by highly metastatic BCCs. The expressions of Tac1 and production of SDF-1alpha were verified in primary BCCs from paired samples of BM aspirates and peripheral blood. CONCLUSIONS/SIGNIFICANCE: MSC facilitate BCC entry into BM, partly through Tac1-mediated regulation of SDF-1alpha and CXCR4. We propose a particular population of BCC with preference for BM could be isolated for characterization. This population might be the subset that enter BM at an early time period, and could be responsible for cancer resurgence and resistance to current therapies.

Abstract Background Targeting Induced Local Lesions in Genomes (TILLING) is a high throughput reverse genetics tool which detects mismatches (single point mutations or small indels) in large number of individuals of mutagenized populations. Currently, TILLING is intensively used for genomics assisted molecular breeding of several crop plants for desired traits. Most commonly used platform for mutation detection is Li-COR DNA Analyzer, where PCR amplified products treated with single strand mismatch specific nuclease are resolved on denaturing gels. The molecular size of any cut product can be easily estimated by comparing with IR dye labeled markers of known sizes. Similar fluorescent dye labeled size markers are also used for several genotyping experiments. Currently, commercially available size standards are expensive and are restricted up to only 700 bp which renders estimation of products of sizes greater than 700 bases inaccurate. Findings A simple protocol was developed for labeling 5' end of multiple DNA size markers with fluorescent dyes. This method involves cloning a pool of different size markers of DNA in a plasmid vector. PCR amplification of plasmid using IR dye labeled universal primers generates 5' fluorescent labeled products of various sizes. The size of products constituting the ladder can be customized as per the need. The generated size markers can be used without any further purification and were found to be stable up to one year at -20°C. Conclusions A simple method was developed for generating fluorescent dye labeled size standards. This method can be customized to generate different size standards as per experimental needs. The protocol described can also be adapted for developing labeled size standards for detection on platforms other than Li-COR i.e. other than infra red range of the spectrum.