4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway

4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G₂/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested.     

A case of Kartagener’s syndrome: Importance of early diagnosis and treatment

Kartagener’s syndrome is a very rare congenital malformation comprising of a classic triad of sinusitis, situs inversus and bronchiectasis. Primary ciliary dyskinesia is a genetic disorder with manifestations present from early life and this distinguishes it from acquired mucociliary disorders. Approximately one half of patients with primary ciliary dyskinesia have situs inversus and, thus are having Kartagener syndrome. We present a case of 12 year old boy with sinusitis, situs inversus and bronchiectasis. The correct diagnosis of this rare congenital autosomal recessive disorder in early life is important in the overall prognosis of the syndrome, as many of the complications can be prevented if timely management is instituted, as was done in this in this case.     

Overexpression of WsSGTL1 Gene of Withania somnifera Enhances Salt Tolerance,Heat Tolerance and Cold Acclimation Ability in Transgenic Arabidopsis Plants

Background

Sterol glycosyltrnasferases (SGT) are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant’s adaptation to abiotic stress.

Methodology

The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses - salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA) and the 3D structures were predicted by using Discovery Studio Ver. 2.5.

Results

The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana.

Conclusions

Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found to have stress responsive elements. The 3D structure showed functional similarity with sterol glycosyltransferases.     

Detailed Analysis of Gene Polymorphisms Associated with Ischemic Stroke in South Asians

The burden of stroke is disproportionately high in the South Asian subcontinent with South Asian ethnicity conferring a greater risk of ischemic stroke than European ancestry regardless of country inhabited. While genes associated with stroke in European populations have been investigated, they remain largely unknown in South Asians. We conducted a comprehensive meta-analysis of known genetic polymorphisms associated with South Asian ischemic stroke, and compared effect size of the MTHFR C677T-stroke association with effect sizes predicted from homocysteine-stroke association. Electronic databases were searched up to August 2012 for published case control studies investigating genetic polymorphisms associated with ischemic stroke in South Asians. Pooled odds ratios (OR) for each gene-disease association were calculated using a random-effects model. We identified 26 studies (approximately 2529 stroke cases and 2881 controls) interrogating 33 independent genetic polymorphisms in 22 genes. Ten studies described MTHFR C677T (108 with TT genotype and 2018 with CC genotype) -homocysteine relationship and six studies (735 stroke cases and 713 controls) described homocysteine-ischemic stroke relationship. Risk association ORs were calculated for ACE I/D (OR 5.00; 95% CI, 1.17–21.37; p = 0.03), PDE4D SNP 83 (OR 2.20; 95% CI 1.21–3.99; p = 0.01), PDE4D SNP 32 (OR 1.57; 95% CI 1.01–2.45, p = 0.045) and IL10 G1082A (OR 1.44; 95% CI, 1.09–1.91, p = 0.01). Significant association was observed between elevated plasma homocysteine levels and MTHFR/677 TT genotypes in healthy South Asians (Mean difference (ΔX) 5.18 µmol/L; 95% CI 2.03–8.34: p = 0.001). Our results demonstrate that the genetic etiology of ischemic stroke in South Asians is broadly similar to the risk conferred in Europeans, although the dataset is considerably smaller and warrants the same clinical considerations for risk profiling.     

Ex Vivo Neurogenesis within Enteric Ganglia Occurs in a PTEN Dependent Manner

A population of multipotent stem cells capable of differentiating into neurons and glia has been isolated from adult intestine in humans and rodents. While these cells may provide a pool of stem cells for neurogenesis in the enteric nervous system (ENS), such a function has been difficult to demonstrate in vivo. An extensive study by Joseph et al. involving 108 rats and 51 mice submitted to various insults demonstrated neuronal uptake of thymidine analog BrdU in only 1 rat. Here we introduce a novel approach to study neurogenesis in the ENS using an ex vivo organotypic tissue culturing system. Culturing longitudinal muscle and myenteric plexus tissue, we show that the enteric nervous system has tremendous replicative capacity with the majority of neural crest cells demonstrating EdU uptake by 48 hours. EdU⁺ cells express both neuronal and glial markers. Proliferation appears dependent on the PTEN/PI3K/Akt pathway with decreased PTEN mRNA expression and increased PTEN phosphorylation (inactivation) corresponding to increased Akt activity and proliferation. Inhibition of PTEN with bpV(phen) augments proliferation while {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, blocks it. These data suggest that the ENS is capable of neurogenesis in a PTEN dependent manner.     

Cost Effectiveness of Implementing Integrated Management of Neonatal and Childhood Illnesses Program in District Faridabad,India

Introduction

Despite the evidence for preventing childhood morbidity and mortality, financial resources are cited as a constraint for Governments to scale up the key health interventions in some countries. We evaluate the cost effectiveness of implementing IMNCI program in India from a health system and societal perspective.

Methods

We parameterized a decision analytic model to assess incremental cost effectiveness of IMNCI program as against routine child health services for infant population at district level in India. Using a 15-years time horizon from 2007 to 2022, we populated the model using data on costs and effects as found from a cluster-randomized trial to assess effectiveness of IMNCI program in Haryana state. Effectiveness was estimated as reduction in infant illness episodes, deaths and disability adjusted life years (DALY). Incremental cost per DALY averted was used to estimate cost effectiveness of IMNCI. Future costs and effects were discounted at a rate of 3%. Probabilistic sensitivity analysis was undertaken to estimate the probability of IMNCI to be cost effective at varying willingness to pay thresholds.

Results

Implementation of IMNCI results in a cumulative reduction of 57384 illness episodes, 2369 deaths and 76158 DALYs among infants at district level from 2007 to 2022. Overall, from a health system perspective, IMNCI program incurs an incremental cost of USD 34.5 (INR 1554) per DALY averted, USD 34.5 (INR 1554) per life year gained, USD 1110 (INR 49963) per infant death averted. There is 90% probability for ICER to be cost effective at INR 2300 willingness to pay, which is 5.5% of India’s GDP per capita. From a societal perspective, IMNCI program incurs an additional cost of USD 24.1 (INR 1082) per DALY averted, USD 773 (INR 34799) per infant death averted and USD 26.3 (INR 1183) per illness averted in during infancy.

Conclusion

IMNCI program in Indian context is very cost effective and should be scaled-up as a major child survival strategy.     

Fuzzy logic feedback control for fed-batch enzymatic hydrolysis of lignocellulosic biomass

A fuzzy logic feedback control system was developed for process monitoring and feeding control in fed-batch enzymatic hydrolysis of a lignocellulosic biomass, dilute acid-pretreated corn stover. Digested glucose from hydrolysis reaction was assigned as input while doser feeding time and speed of pretreated biomass were responses from fuzzy logic control system. Membership functions for these three variables and rule-base were created based on batch hydrolysis data. The system response was first tested in LabVIEW environment then the performance was evaluated through real-time hydrolysis reaction. The feeding operations were determined timely by fuzzy logic control system and efficient responses were shown to plateau phases during hydrolysis. Feeding of proper amount of cellulose and maintaining solids content was well balanced. Fuzzy logic proved to be a robust and effective online feeding control tool for fed-batch enzymatic hydrolysis.     

Airway Microbiota in Severe Asthma and Relationship to Asthma Severity and Phenotypes

BackgroundThe lower airways harbor a community of bacterial species which is altered in asthma.ObjectivesWe examined whether the lower airway microbiota were related to measures of asthma severity.MethodsWe prospectively recruited 26 severe asthma, 18 non-severe asthma and 12 healthy subjects. DNA was extracted from induced sputum and PCR amplification of the V3-V5 region of bacterial 16S rRNA gene was performed.ResultsWe obtained 138,218 high quality sequences which were rarefied at 133 sequences/sample. Twenty OTUs had sequences ≥1% of total. There were marked differences in the distribution of Phyla between groups (P = 2.8x10^-118). Bacteroidetes and Fusobacteria were reduced in non-severe and severe asthmatic groups. Proteobacteria were more common in non-severe asthmatics compared to controls (OR = 2.26; 95% CI = 1.94–2.64) and Firmicutes were increased in severe asthmatics compared to controls (OR = 2.15; 95%CI = 1.89–2.45). Streptococcal OTUs amongst the Firmicutes were associated with recent onset asthma, rhinosinusitis and sputum eosinophilia.ConclusionsSputum microbiota in severe asthma differs from healthy controls and non-severe asthmatics, and is characterized by the presence of Streptococcus spp with eosinophilia. Whether these organisms are causative for the pathophysiology of asthma remains to be determined.     

Ion channel expression and function in normal and osteoarthritic human synovial fluid progenitor cells

Osteoarthritis (OA) is a chronic disease affecting the cartilage of over 15% of Canadians. Synovial fluid mesenchymal progenitor cells (sfMPCs) are present in joints and are thought to contribute to healing. OA sfMPCs have a greater proliferative ability but decreased chondrogenic potential. However, little is known about the factors influencing/regulating the differences between normal and OA sfMPCs. Recently, our lab has shown that sfMPC chondrogenic differentiation in vitro is favorably biased toward a similar osmotic environment as they experience in vivo. The current study now examines the expression and functionality of a variety of ion channels in sfMPCs derived from normal individuals and early OA patients. Results indicated that there is differential ion channel regulation at the functional level and expression level in early OA sfMPCs. All ion channels were upregulated in early OA compared to normal sfMPCs with the exception of KCNMA1 at the mRNA level. At the protein level, TRPV4 was over expressed in early OA sfMPCs, while KCNJ12 and KCNMA1 were unchanged between normal and early OA sfMPCs. At the functional level, the inward rectifying potassium channel was under expressed in early OA sfMPCs, however the membrane potential was unchanged between normal and early OA sfMPCs. In the synovial environment itself, a number of differences in ion concentration between normal and early OA synovial fluid were observed. These findings suggest that normal and OA progenitor cells demonstrate functional differences in how they interact with the synovial ion environment.     

Direct Observation of Treatment Provided by a Family Member as Compared to Non-Family Member among Children with New Tuberculosis: A Pragmatic,Non-Inferiority,Cluster-Randomized Trial in Gujarat,India

Background

The World Health Organization recommends direct observation of treatment (DOT) to support patients with tuberculosis (TB) and to ensure treatment completion. As per national programme guidelines in India, a DOT provider can be anyone who is acceptable and accessible to the patient and accountable to the health system, except a family member. This poses challenges among children with TB who may be more comfortable receiving medicines from their parents or family members than from unfamiliar DOT providers. We conducted a non-inferiority trial to assess the effect of family DOT on treatment success rates among children with newly diagnosed TB registered for treatment during June–September 2012.

Methods

We randomly assigned all districts (n = 30) in Gujarat to the intervention (n = 15) or usual-practice group (n = 15). Adult family members in the intervention districts were given the choice to become their child’s DOT provider. DOT was provided by a non-family member in the usual-practice districts. Using routinely collected clinic-based TB treatment cards, we compared treatment success rates (cured and treatment completed) between the two groups and the non-inferiority limit was kept at 5%.

Results

Of 624 children with newly diagnosed TB, 359 (58%) were from intervention districts and 265 (42%) were from usual-practice districts. The two groups were similar with respect to baseline characteristics including age, sex, type of TB, and initial body weight. The treatment success rates were 344 (95.8%) and 247 (93.2%) (p = 0.11) among the intervention and usual-practice groups respectively.

Conclusion

DOT provided by a family member is not inferior to DOT provided by a non-family member among new TB cases in children and can attain international targets for treatment success.

Trial Registration

Clinical Trials Registry–India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2015/09/006229