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2.  Vapor of Volatile Oils from Litsea cubeba Seed Induces Apoptosis and Causes Cell Cycle Arrest in Lung Cancer Cells 
PLoS ONE  2012;7(10):e47014.
Non-small cell lung carcinoma (NSCLC) is a major killer in cancer related human death. Its therapeutic intervention requires superior efficient molecule(s) as it often becomes resistant to present chemotherapy options. Here we report that vapor of volatile oil compounds obtained from Litsea cubeba seeds killed human NSCLC cells, A549, through the induction of apoptosis and cell cycle arrest. Vapor generated from the combined oils (VCO) deactivated Akt, a key player in cancer cell survival and proliferation. Interestingly VCO dephosphorylated Akt at both Ser473 and Thr308; through the suppression of mTOR and pPDK1 respectively. As a consequence of this, diminished phosphorylation of Bad occurred along with the decreased Bcl-xL expression. This subsequently enhanced Bax levels permitting the release of mitochondrial cytochrome c into the cytosol which concomitantly activated caspase 9 and caspase 3 resulting apoptotic cell death. Impairment of Akt activation by VCO also deactivated Mdm2 that effected overexpression of p53 which in turn upregulated p21 expression. This causes enhanced p21 binding to cyclin D1 that halted G1 to S phase progression. Taken together, VCO produces two prong effects on lung cancer cells, it induces apoptosis and blocked cancer cell proliferation, both occurred due to the deactivation of Akt. In addition, it has another crucial advantage: VCO could be directly delivered to lung cancer tissue through inhalation.
PMCID: PMC3473030  PMID: 23091605
3.  Assessment of Corticotomy Facilitated Tooth Movement and Changes in Alveolar Bone Thickness - A CT Scan Study 
Introduction: Corticotomy is an effective method of accelerating the orthodontic treatment. The aim of this study was to compare the treatment time for the extraction space closure, between corticotomy assisted and conventional orthodontic tooth movement and to check the alveolar bone thickness before and after corticotomy procedure in the corticotomy group.
Settings and Design: Cross-sectional clinical study.
Materials and Methods: Twenty patients (age>15 y) requiring orthodontic treatment with upper anterior retraction in the extraction space of 1st premolar were selected and were randomised into control and corticotomy group each group consisted of 10 subjects. Pre retraction, corticotomy was performed in the maxillary anterior segment. The pre and post retraction CT scans were recorded and the thickness of the alveolar plates were measured at crestal level (S1), mid root level (S2) and apical level (S3) PreTreatment (T1). The same measurements were repeated after incisor retraction was completed PostTreatment (T2).
Statistical Analysis: Student’s t-test, Pearson correlation coefficient.
Results: There was a significant difference in retraction time (days) between control and corticotomy groups (p<0.001). Also, there were significant difference in total alveolar bone thickness at the crest region for all the four incisor teeth (p<0.05). A significant difference was observed in total alveolar bone thickness at the S2 and S3 level for 11, 21 and 11, 12 and 22 (p<0.05) respectively.
Conclusion: Alveolar corticotomies not only accelerates the orthodontic treatment but, also provides the advantage of increased alveolar width to support the teeth and overlying structures.
PMCID: PMC4253260  PMID: 25478442
Corticotomy facilitated orthodontics; Periodontally accelerated osteogenic orthodontics; Regional acceleratory phenomena
4.  A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica 
PLoS Pathogens  2014;10(10):e1004411.
Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics.
Author Summary
Entamoeba histolytica is one of the major causes of morbidity and mortality in developing countries. Phagocytosis plays an important role in both survival and virulence, and has been used as one of the virulence markers. E. histolytica displays a high rate of phagocytosis and offers a unique system to understand the mechanism of this important biological process seen in many eukaryotic cells. However, the molecular mechanism of the process is still largely unknown in E. histolytica, though this pathway has been characterized in many systems. We have been studying this pathway using red blood cells, and have identified a number of molecules that are involved during initiation. Here, we demonstrate that an alpha kinase like atypical kinase EhAK1 is an important component of the pathway that regulates erythrophagocytosis. We provide evidence that EhAK1 is recruited to the phagocytic cups through EhCaBP1. We also show that over expression of kinase defective mutant, or down regulation of the gene using antisense RNA, led to defects in phagocytosis. Actin appears to be one of the substrates of EhAK1 and phosphorylation of actin is required for phagocytosis. Our results suggest that E. histolytica has evolved a novel pathway to carry out phagocytosis.
PMCID: PMC4192601  PMID: 25299184
5.  Pentoxifylline Plus Prednisolone versus Pentoxifylline Only for Severe Alcoholic Hepatitis: A Randomized Controlled Clinical Trial 
Prednisolone and pentoxifylline (PTX) have been shown to be individually useful in severe alcoholic hepatitis with Maddrey discriminant function (MDF) score ≥32. Previous report suggests that PTX is probably superior to prednisolone alone. However the efficacy of PTX and prednisolone combination over PTX alone in the management of acute alcoholic hepatitis (MDF score ≥32) is yet unrevealed.
The present study was initiated to find out the efficacy of combined pentoxifylline and prednisolone versus PTX alone in acute alcoholic hepatitis in respect of short and intermediate term outcomes.
Subjects and Methods:
A total of 124 patients with severe alcoholic hepatitis (MDF score ≥ 32) initially were evaluated. 62 patients who fulfilled the inclusion and exclusion criteria were randomized and divided into 2 groups. Group 1 received PTX only, whereas Group 2 received PTX plus Prednisolone. The total duration of follow-up was 12 months. Student's t-test, Chi-square test, the Kaplan-Meier methods were used for statistical analysis.
A total of 60 patients, 30 in each group were available for final analysis. In Group-1, 6 patients expired at the end of 1 year (5 within 3 months and another after 3 months). In Group 2, 10 patients expired at the end of 1 year (9 within 3 months and another after 3 months). Though survival probability is higher among Group 1 patients but the difference is not statistically significant.
The combination of PTX plus Prednisolone yields no additional benefit in terms of mortality and morbidity from that of PTX monotherapy.
PMCID: PMC4199180  PMID: 25328799
Alcoholic hepatitis; Maddrey discriminant function score; Pentoxifylline; Prednisolone
6.  Impact of hepatitis B immunization among the Nicobarese tribe - antibody titres & seroprotection five years after vaccination 
Background & objectives:
A total of 237 Nicobarese subjects who had received hepatitis B vaccination as part of mass vaccination project during 2000-2001 were screened for anti-HBsAg titres by quantitative ELISA five years after vaccination.
Anti-HBsAg antibody was estimated using quantitative ELISA. Proportion of the subjects with protective levels of antibody and geometric mean antibody titres were calculated.
Among the 237 study subjects, 213 had received three doses of vaccine, 17 had received two doses and seven had received one dose. The geometric mean titres of anti-HBs antibodies were 201.7, 31.9 and 23.1 mIU/ml among those who received three, two and one dose of vaccine, respectively. Among those who received three doses of vaccination, 85.9 per cent had anti-HBs antibody levels of 10 mIU/ml or more, indicating seroprotection. The difference in the seroprotection rates among those who received three doses of vaccination (85.9%) and those who received less than three doses (58.3%) was significant. Seroprotection rates one month after the first, second and third dose of vaccination were 49.1, 86.9 and 96.7 per cent, respectively. It then declined to 89 per cent by the end of the second year and to 85.5 per cent by the end of the third year, but there was no decline thereafter.
Interpretation & conclusions:
Seroprotection rate reached at the maximum one month after the third dose of HBV vaccine. Although about 15 per cent of the vaccinated persons lost seroprotection by the end of the third year, no further loss in seroprotection was observed between the third year and the fifth year.
PMCID: PMC4069737  PMID: 24820837
Follow up; hepatitis B; Nicobarese; seroprotection; vaccination
7.  Erythrocytes Induce Proinflammatory Endothelial Activation in Hypoxia 
Although exposure to ambient hypoxia is known to cause proinflammatory vascular responses, the mechanisms initiating these responses are not understood. We tested the hypothesis that in systemic hypoxia, erythrocyte-derived H2O2 induces proinflammatory gene transcription in vascular endothelium. We exposed mice or isolated, perfused murine lungs to 4 hours of hypoxia (8% O2). Leukocyte counts increased in the bronchoalveolar lavage. The expression of leukocyte adhesion receptors, reactive oxygen species, and protein tyrosine phosphorylation increased in freshly recovered lung endothelial cells (FLECs). These effects were inhibited by extracellular catalase and by the removal of erythrocytes, indicating that the responses were attributable to erythrocyte-derived H2O2. Concomitant nuclear translocation of the p65 subunit of NF-κB and hypoxia-inducible factor–1α stabilization in FLECs occurred only in the presence of erythrocytes. Hemoglobin binding to the erythrocyte membrane protein, band 3, induced the release of H2O2 from erythrocytes and the p65 translocation in FLECs. These data indicate for the first time, to our knowledge, that erythrocytes are responsible for endothelial transcriptional responses in hypoxia.
PMCID: PMC3547079  PMID: 23043086
hypoxia; erythrocytes; endothelium; lung; inflammation
8.  Maternal obesity during pregnancy and premature mortality from cardiovascular event in adult offspring: follow-up of 1 323 275 person years 
Objectives To determine whether maternal obesity during pregnancy is associated with increased mortality from cardiovascular events in adult offspring.
Design Record linkage cohort analysis.
Setting Birth records from the Aberdeen Maternity and Neonatal databank linked to the General Register of Deaths, Scotland, and the Scottish Morbidity Record systems.
Population 37 709 people with birth records from 1950 to present day.
Main outcome measures Death and hospital admissions for cardiovascular events up to 1 January 2012 in offspring aged 34-61. Maternal body mass index (BMI) was calculated from height and weight measured at the first antenatal visit. The effect of maternal obesity on outcomes in offspring was tested with time to event analysis with Cox proportional hazard regression to compare outcomes in offspring of mothers in underweight, overweight, or obese categories of BMI compared with offspring of women with normal BMI.
Results All cause mortality was increased in offspring of obese mothers (BMI >30) compared with mothers with normal BMI after adjustment for maternal age at delivery, socioeconomic status, sex of offspring, current age, birth weight, gestation at delivery, and gestation at measurement of BMI (hazard ratio 1.35, 95% confidence interval 1.17 to 1.55). In adjusted models, offspring of obese mothers also had an increased risk of hospital admission for a cardiovascular event (1.29, 1.06 to 1.57) compared with offspring of mothers with normal BMI. The offspring of overweight mothers also had a higher risk of adverse outcomes.
Conclusions Maternal obesity is associated with an increased risk of premature death in adult offspring. As one in five women in the United Kingdom is obese at antenatal booking, strategies to optimise weight before pregnancy are urgently required.
PMCID: PMC3805484  PMID: 23943697
9.  Homologous Recombination Occurs in Entamoeba and Is Enhanced during Growth Stress and Stage Conversion 
PLoS ONE  2013;8(9):e74465.
Homologous recombination (HR) has not been demonstrated in the parasitic protists Entamoeba histolytica or Entamoeba invadens, as no convenient method is available to measure it. However, HR must exist to ensure genome integrity, and possible genetic exchange, especially during stage conversion from trophozoite to cyst. Here we show the up regulation of mitotic and meiotic HR genes in Entamoeba during serum starvation, and encystation. To directly demonstrate HR we use a simple PCR-based method involving inverted repeats, which gives a reliable read out, as the recombination junctions can be determined by sequencing the amplicons. Using this read out, we demonstrate enhanced HR under growth stress in E. histolytica, and during encystation in E. invadens. We also demonstrate recombination between chromosomal inverted repeats. This is the first experimental demonstration of HR in Entamoeba and will help future investigations into this process, and to explore the possibility of meiosis in Entamoeba.
PMCID: PMC3787063  PMID: 24098652
10.  Mitochondrial transfer from bone marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury 
Nature medicine  2012;18(5):759-765.
Bone marrow-derived stromal cells (BMSCs) protect against acute lung injury (ALI). To determine the role of BMSC mitochondria in the protection, we airway-instilled mice first with lipopolysaccharide (LPS), then with mouse BMSCs (mBMSCs). Live optical studies revealed that mBMSCs formed connexin 43 (Cx43)-containing gap junctional channels (GJCs) with the alveolar epithelium, releasing mitochondria-containing microvesicles that the epithelium engulfed. The presence of BMSC mitochondria in the epithelium was evident optically, as also by the presence of human mitochondrial DNA in mouse lungs in which we instilled human BMSCs (hBMSCs). The mitochondrial transfer increased alveolar ATP. LPS-induced ALI, indicated by alveolar leukocytosis and protein leak, inhibition of surfactant secretion and high mortality, was markedly abrogated by wild type mBMSCs, but not by mutant, GJC-incompetent mBMSCs, or by mBMSCs with dysfunctional mitochondria. This is the first evidence that BMSCs protect against ALI by restituting alveolar bioenergetics through Cx43-dependent alveolar attachment and mitochondrial transfer.
PMCID: PMC3727429  PMID: 22504485
11.  Genomic distribution of SINEs in Entamoeba histolytica strains: implication for genotyping 
BMC Genomics  2013;14:432.
The major clinical manifestations of Entamoeba histolytica infection include amebic colitis and liver abscess. However the majority of infections remain asymptomatic. Earlier reports have shown that some E. histolytica isolates are more virulent than others, suggesting that virulence may be linked to genotype. Here we have looked at the genomic distribution of the retrotransposable short interspersed nuclear elements EhSINE1 and EhSINE2. Due to their mobile nature, some EhSINE copies may occupy different genomic locations among isolates of E. histolytica possibly affecting adjacent gene expression; this variability in location can be exploited to differentiate strains.
We have looked for EhSINE1- and EhSINE2-occupied loci in the genome sequence of Entamoeba histolytica HM-1:IMSS and searched for homologous loci in other strains to determine the insertion status of these elements. A total of 393 EhSINE1 and 119 EhSINE2 loci were analyzed in the available sequenced strains (Rahman, DS4-868, HM1:CA, KU48, KU50, KU27 and MS96-3382. Seventeen loci (13 EhSINE1 and 4 EhSINE2) were identified where a EhSINE1/EhSINE2 sequence was missing from the corresponding locus of other strains. Most of these loci were unoccupied in more than one strain. Some of the loci were analyzed experimentally for SINE occupancy using DNA from strain Rahman. These data helped to correctly assemble the nucleotide sequence at three loci in Rahman. SINE occupancy was also checked at these three loci in 7 other axenically cultivated E. histolytica strains and 16 clinical isolates. Each locus gave a single, specific amplicon with the primer sets used, making this a suitable method for strain typing. Based on presence/absence of SINE and amplification with locus-specific primers, the 23 strains could be divided into eleven genotypes. The results obtained by our method correlated with the data from other typing methods. We also report a bioinformatic analysis of EhSINE2 copies.
Our results reveal several loci with extensive polymorphism of SINE occupancy among different strains of E. histolytica and prove the principle that the genomic distribution of SINEs is a valid method for typing of E. histolytica strains.
PMCID: PMC3716655  PMID: 23815468
Entamoeba histolytica; Genotype; EhSINE1; SINE occupancy; Polymorphism; Strain typing
12.  Dengue: A Growing Menace -- A Snapshot of Recent Facts, Figures & Remedies 
Dengue is specially owing to inadequate water supply and poor solid waste management , which are favorable for multiplication of the main vectors including the Aedes ageypti coupled with lack of proven anti viral therapy and no proven efficient vaccine .there are many cases of both dengue shock syndrome and dengue haemmorhagic fever making it a major public health burden sending ominous signal resulting both rising morbidity & mortality, deleterious effect on DALY [disability adjusted life year] & QALY [quality adjusted life year] & though it affect all section of society ,still it affect the poor & underprivileged section more, thereby growing menace in public health in general & in developing countries in particular.
PMCID: PMC3708269  PMID: 23847455
Dengue Virus; management; vaccine
13.  Seizure risk with AVM treatment or conservative management 
Neurology  2012;79(6):500-507.
To compare the risk of epileptic seizures in adults during conservative management or following invasive treatment for a brain arteriovenous malformation (AVM).
We used annual general practitioner follow-up, patient questionnaires, and medical records surveillance to quantify the 5-year risk of seizures and the chances of achieving 2-year seizure freedom for adults undergoing AVM treatment compared to adults managed conservatively in a prospective, population-based observational study of adults in Scotland, newly diagnosed with an AVM in 1999–2003.
We identified 229 adults with a new diagnosis of an AVM, of whom two-thirds received AVM treatment (154/229; 67%) during 1,862 person-years of follow-up (median completeness of follow-up 97%). There was no significant difference in the proportions with a first or recurrent seizure over 5 years following AVM treatment, compared to the first 5 years following clinical presentation in conservatively managed adults, in analyses stratified by mode of presentation (intracerebral hemorrhage, 35% vs 26%, p = 0.5; seizure, 67% vs 72%, p = 0.6; incidental, 21% vs 10%, p = 0.4). For patients with epilepsy, the chances of achieving 2-year seizure freedom during 5-year follow-up were similar following AVM treatment (n = 39; 52%, 95% confidence interval [CI] 36% to 68%) or conservative management (n = 21; 57%, 95% CI 35% to 79%; p = 0.7).
In this observational study, there was no difference in the 5-year risk of seizures with AVM treatment or conservative management, irrespective of whether the AVM had presented with hemorrhage or epileptic seizures.
PMCID: PMC3413766  PMID: 22764257
14.  Assessment of Potential In vitro Genotoxic and Cytotoxic Effects of Bupropion Hydrochloride (Wellbutrin) in Human Peripheral Lymphocytes and Human Cortical Neuron 
Toxicology International  2013;20(1):11-18.
Wellbutrin (bupropion hydrochloride; WB), an anti-depressant of the aminoketone class is new highly selective norepinephrine and dopamine reuptake inhibitor; it is effective in the treatment of patients with major depression.
Materials and Methods:
To investigate the in vitro effects of WB in human cultured peripheral blood lymphocytes and human cortical neural (HCN2) cell lines, micronucleus, sister chromatid exchange analysis, cellular viability, and comet assays were employed. The present study is to our knowledge, the first report on WB genotoxicity in cultured human peripheral blood lymphocytes and its cytotoxicity in the HCN2 cell line. We have also investigated the genotoxic potential of WB to induce chromosomal aberrations.
WB-induced cytotoxicity (measured as reduction of the nuclear division index) possibly prevented the division of damaged cells.
We conclude that although, WB exerts potential genotoxic effects in cultured lymphocytes, its cytogenetic effects are very unlikely to occur in blood cultures of WB-administered subjects.
PMCID: PMC3702119  PMID: 23833432
Comet assay; genotoxicity; micronucleus; wellbutrin
15.  The Calmodulin-like Calcium Binding Protein EhCaBP3 of Entamoeba histolytica Regulates Phagocytosis and Is Involved in Actin Dynamics 
PLoS Pathogens  2012;8(12):e1003055.
Phagocytosis is required for proliferation and pathogenesis of Entamoeba histolytica and erythrophagocytosis is considered to be a marker of invasive amoebiasis. Ca2+ has been found to play a central role in the process of phagocytosis. However, the molecular mechanisms and the signalling mediated by Ca2+ still remain largely unknown. Here we show that Calmodulin-like calcium binding protein EhCaBP3 of E. histolytica is directly involved in disease pathomechanism by its capacity to participate in cytoskeleton dynamics and scission machinery during erythrophagocytosis. Using imaging techniques EhCaBP3 was found in phagocytic cups and newly formed phagosomes along with actin and myosin IB. In vitro studies confirmed that EhCaBP3 directly binds actin, and affected both its polymerization and bundling activity. Moreover, it also binds myosin 1B in the presence of Ca2+. In cells where EhCaBP3 expression was down regulated by antisense RNA, the level of RBC uptake was reduced, myosin IB was found to be absent at the site of pseudopod cup closure and the time taken for phagocytosis increased, suggesting that EhCaBP3 along with myosin 1B mediate the closure of phagocytic cups. Experiments with EhCaBP3 mutant defective in Ca2+ -binding showed that Ca2+ binding is required for phagosome formation. Liposome binding assay revealed that EhCaBP3 recruitment and enrichment to membrane is independent of any cellular protein as it binds directly to phosphatidylserine. Taken together, our results suggest a novel pathway mediating phagocytosis in E. histolytica, and an unusual mechanism of modulation of cytoskeleton dynamics by two calcium binding proteins, EhCaBP1 and EhCaBP3 with mostly non-overlapping functions.
Author Summary
Entamoeba histolytica is one of the major causes of morbidity and mortality in developing countries. Phagocytosis plays an important role in both survival and virulence and has been used as a virulence marker. Inhibition of phagocytosis leads to a defect in cellular proliferation. Therefore, the molecules that participate in phagocytosis are good targets for developing new drugs. However, the molecular mechanism of the process is still largely unknown. Here, we demonstrate that Calmodulin-like calcium binding protein EhCaBP3 is involved in erythrophagocytosis. We show this by a number of different approaches including immunostaining of actin, myosin1B, EhCaBP1 and EhCaBP3 during uptake of RBC; over expression and down regulation of EhCaBP3, and over expression of calcium defective mutant of EhCaBP3. Our analysis suggests that EhCaBP3 can regulate actin dynamics. Along with actin and myosin 1B it can participate in both initiation and formation of phagosomes. The Ca2+-bound form of this protein is required only for progression from cups into early phagosomes but not for initiation. Our results demonstrate the complex role of Ca2+ binding proteins, EhCaBP1 and EhCaBP3 in regulation of phagocytosis in the protist parasite E. histolytica and the novel mechanisms of manipulating actin dynamics at multiple levels.
PMCID: PMC3531509  PMID: 23300437
16.  Turn over split fascial flap - a refinement for resurfacing shin defect 
Moderate size defects of the shin of tibia are frequently encountered following trauma and infection. They may be associated with or without a fracture. Such defects require resurfacing by a flap. Many different types of flaps have been described but most of them proved to be more bulky than desired. Although these procedures cover the defects successfully the results they produce are not aesthetically appropriate. The flap looks bulkier because the native subcutaneous tissue is thin over the shin and distal leg. Hence a search for a vascularized tissue of minimal bulk for suitable resurfacing was initiated. A turnover fascial flap fulfilled the requirement. Such a flap can be made thinner by splitting its distal part into two layers while maintaining a common vascular fascial pedicle with both the layers of the fascia. This allowed a larger surface area to be covered. Such refinement is based on the following parameters (a) fresh cadaveric dissection, (b) demonstration of live microcirculation individually in the superficial and deep layers of the deep fascia and (c) intraoperative flourescein study of the split fascial flap. The technique has been used in 5 cases over the upper and middle third of the shin of tibia. The split fascial flap was turned over and inset in the defect and covered with a split skin graft. The donor site was primarily closed. The functional and aesthetic results were highly satisfactory. The follow up of 18 months proved the durability and usefulness of the flap.
PMCID: PMC3462525  PMID: 23071906
Split fascial flap; shin defect; lower limb reconstruction
17.  CT angiographic evaluation of perforators in the lower limb and their reconstructive implication 
The perforator flaps evolved on the knowledge of the vascular tree from the main vascular trunk up to the subdermal plexus. Therefore, we thought that it's necessary to map the whole vascular arcade by CT angiography. The aim of this study is to evaluate the perforators and the whole vascular tree of the lower limb by peripheral CT angiography with 3D reconstruction and intraoperative evaluation. This study helps in designing flaps of different constituents based on the selected perforators.
Materials and Methods:
Twenty patients having lower limb defects were selected. CT angiography was done using a non-ionic iodinated contrast media injected through the antecubital vein. The lower limbs were imaged using volume rendering CT scan machine. Three dimensional reconstructions were made. The whole arterial tree, along with the perforators, were mapped. Findings of the audio-Doppler were correlated with the CT angiographic observations. Further these evaluations were confirmed by intraoperative findings.
The three dimensional CT angiographic reconstruction with bone and soft tissue provided advanced knowledge of this vascular network. It delineated the main vessel, the perforators, their caliber, distance from fixed bony landmarks and course up to the subdermal plexus. These findings were confirmed during dissection of the proposed flap. The perforators were mainly musculocutaneous in the proximal leg and septocutaneous distally.
The vascular details visualized by this technique made advancement over the existing methods namely color Doppler, audio Doppler, two dimensional angiography etc. It improved the understanding of perforator flaps and their successful clinical application.
PMCID: PMC3580348  PMID: 23450763
CT angiography; lower limb; perforator; reconstruction
18.  The Control of Stress Induced Type I Diabetes Mellitus in Humans through the Hepatic Synthesis of Insulin by the Stimulation of Nitric Oxide Production 
The role of stress induced development of Type-1 diabetes mellitus (T1DM) as opposed to autoimmunity remains obscure. It has been reported that a stress induced protein, identified to be dermcidin isoform 2 (dermcidin) inhibited insulin synthesis in both the pancreatic β cells and the hepatic cells. As dermcidin effect could be neutralized by the increased production of systemic nitric oxide (NO), investigations were carried out to determine the feasibility of controlling stress induced T1DM through the neutralization of dermcidin by systemic increase of NO. To determine the role of plasma dermcidin level in T1DM subjects (n=45), if any, when the plasma dermcidin level were determined, it was found that the protein level was increased in 65% of the participating volunteers. Efforts were made to normalize the plasma glucose level (median=175 mg/dL) in these T1DM subjects by systemic increase of NO by applying a cotton pad containing 0.28mmol sodium nitroprusside on the abdominal skin. It was found that the systemic increase of NO level decreased the blood glucose level of 275 mg/dL (median) to 115 mg/dL (median) in these volunteers within 24 h with concomitant increase of plasma insulin level from 7.5 μunits/dL to 101 μunits/dL at the same time. The increase of plasma insulin level was accompanied by the decrease of dermcidin level of 124.5 nM to 18 nM with increase of NO from 0.43 ± 0.19 nM to 4.1 ± 1.56 nM. The results suggested that the stress induced T1DM by dermcidin could be controlled by the systemic increase of NO which in consequence led to increased synthesis of insulin.
PMCID: PMC3615282  PMID: 23675270
dermcidin isoform 2; hepatic insulin synthesis; nitric oxide; sodium nitroprusside; type IB diabetes mellitus
19.  Computational prediction and validation of C/D, H/ACA and Eh_U3 snoRNAs of Entamoeba histolytica 
BMC Genomics  2012;13:390.
Small nucleolar RNAs are a highly conserved group of small RNAs found in eukaryotic cells. Genes encoding these RNAs are diversely located throughout the genome. They are functionally conserved, performing post transcriptional modification (methylation and pseudouridylation) of rRNA and other nuclear RNAs. They belong to two major categories: the C/D box and H/ACA box containing snoRNAs. U3 snoRNA is an exceptional member of C/D box snoRNAs and is involved in early processing of pre-rRNA. An antisense sequence is present in each snoRNA which guides the modification or processing of target RNA. However, some snoRNAs lack this sequence and often they are called orphan snoRNAs.
We have searched snoRNAs of Entamoeba histolytica from the genome sequence using computational programmes (snoscan and snoSeeker) and we obtained 99 snoRNAs (C/D and H/ACA box snoRNAs) along with 5 copies of Eh_U3 snoRNAs. These are located diversely in the genome, mostly in intergenic regions, while some are found in ORFs of protein coding genes, intron and UTRs. The computationally predicted snoRNAs were validated by RT-PCR and northern blotting. The expected sizes were in agreement with the observed sizes for all C/D box snoRNAs tested, while for some of the H/ACA box there was indication of processing to generate shorter products.
Our results showed the presence of snoRNAs in E. histolytica, an early branching eukaryote, and the structural features of E. histolytica snoRNAs were well conserved when compared with yeast and human snoRNAs. This study will help in understanding the evolution of these conserved RNAs in diverse phylogenetic groups.
PMCID: PMC3542256  PMID: 22892049
U3 snoRNA; Guide/ orphan snoRNAs; Entamoeba histolytica
20.  Reproductive Outcomes Following Ectopic Pregnancy: Register-Based Retrospective Cohort Study 
PLoS Medicine  2012;9(6):e1001243.
Using Scottish national registry data, Sohinee Bhattacharya and colleagues investigate pregnancy outcomes following ectopic pregnancy in comparison to livebirth, miscarriage, or termination in a first pregnancy.
We aimed to compare reproductive outcomes following ectopic pregnancy (EP) versus livebirth, miscarriage, or termination in a first pregnancy.
Methods And Findings
A retrospective cohort study design was used. Scottish national data on all women whose first pregnancy occurred between 1981 and 2000 were linked to records of a subsequent pregnancy. The exposed cohort comprised women with an EP in their first pregnancy. There were three unexposed cohorts: women with livebirth, miscarriage, and termination of their first pregnancies. Any differences in rates of second pregnancy, livebirth, EP, miscarriage, or terminations and complications of a second ongoing pregnancy and delivery were assessed among the different exposure groups. A total of 2,969 women had an initial EP; 667,299 had a livebirth, 39,705 women miscarried, and 78,697 terminated their first pregnancies. Women with an initial EP had an increased chance of another pregnancy within 2 years (adjusted hazard ratio (AHR) 2.76 [95% CI 2.58–2.95]) or after 6 years (AHR 1.57 [95% CI 1.29–1.91]) compared to women with a livebirth. In comparison with women with an initial miscarriage, women who had an EP had a lower chance of a second pregnancy (AHR 0.53 [95% CI 0.50–0.56]). Compared to women with an initial termination, women with an EP had an increased chance of a second pregnancy (AHR 2.38 [95% CI 2.23–2.55]) within 2 years. Women with an initial EP suffered an increased risk of another EP compared to women with a livebirth (AHR 13.0 [95% CI 11.63–16.86]), miscarriage (AHR 6.07 [95% CI 4.83–7.62]), or termination (AHR 12.84 [95% CI 10.07–16.37]). Perinatal complications in a pregnancy following EP were not significantly higher than those in primigravidae or in women with a previous miscarriage or termination.
Women with an initial EP have a lower chance of conception than those who miscarry but an increased risk of a repeat EP in comparison with all three comparison groups. A major limitation of this study was the inability to separate women using contraception from those who were intending to conceive.
Please see later in the article for the Editors' Summary
Editors' Summary
An ectopic pregnancy occurs when the embryo (fertilized egg) implants outside the uterine cavity, usually in the fallopian tubes but sometimes in the cervix, ovaries, or abdomen. The prevalence for this condition is between 1%–2% of all pregnancies, and risk factors are thought to include pelvic infection, smoking, previous pelvic surgery, and use of certain types of intrauterine contraceptive devices. Ectopic pregnancies are potentially life threatening because as the fetus grows, it can lead to tubal rupture and abdominal bleeding—for example, in the UK, ectopic pregnancies are responsible for almost three-quarters of early pregnancy-related deaths. However, due to improvements in early diagnosis, in high income countries, deaths from ectopic pregnancies have become increasingly rare.
Why Was This Study Done?
Having an ectopic pregnancy can have serious implications for future fertility and subsequent pregnancies but to date, there is little information on reproductive outcomes in women who have had an ectopic pregnancy. So in this study, the researchers used a population-based cohort of women in Scotland to examine future reproductive outcomes in women who had an initial ectopic pregnancy and then compare these outcomes to those in women following a successful (live birth) or unsuccessful (miscarriage or termination) intrauterine pregnancy.
What Did The Researchers Do And Find?
The researchers used a national database (The Scottish Morbidity Record) and hospital discharge information to identify women who had ectopic pregnancies, miscarriages, terminations, or on-going pregnancies between 1981–2000. Then, using unique linking identifiers, they were able to examine the outcomes of subsequent pregnancies and conducted a statistical analysis to investigate whether the first pregnancy outcome had any effect on second pregnancy outcomes.
 The researchers found that during the time period studied, in their first pregnancy, 2,969 women had an ectopic pregnancy, 39,705 women miscarried, 78,697 women underwent termination, and the majority, 667,299, gave birth to a live infant. The researchers then found that compared to women with an initial live birth, women with an ectopic pregnancy were 2.76 times more likely to conceive a second pregnancy within two years. However, compared to women whose first pregnancies ended in miscarriage, women with an initial ectopic pregnancy were significantly less likely to conceive a second time but had an increased chance of a second pregnancy within two years compared to women who terminated their first pregnancy. Importantly, the researchers found that women with an initial ectopic pregnancy had a higher risk of a further ectopic pregnancy compared to all the other groups of women. Furthermore, these women had a significantly higher risk of preeclampsia, preterm delivery, and emergency cesarean delivery in their next continuing pregnancy compared to women who had a previous live birth. However, these risks were not significantly higher than women who had an early loss in a first pregnancy.
What Do These Findings Mean?
These findings suggest that women with an initial ectopic pregnancy have a lower chance of conception than those who miscarry and also have an increased risk of a repeat ectopic pregnancy compared to women who experience miscarriage, termination, or a live birth in their first pregnancy. However, as the researchers did not have any information on contraception use, a major limitation of this study is the inability to separate women using contraception from those who were intending to conceive—women who experienced an ectopic pregnancy may not want to conceive again after a traumatic experience rather than being unable to conceive because of tubal damage. However, the results of this study may help doctors to counsel women with an ectopic pregnancy at the time of initial diagnosis and treatment, and in those willing to conceive again, offer follow-up to discuss future fertility and possible risks of subsequent pregnancy. Further research will help to investigate whether the site of ectopic pregnancy affects future reproductive outcomes.
Additional Information
Please access these Web sites via the online version of this summary at
The American Pregnancy Association and the UK National Health Service (NHS) Choices give information on ectopic pregnancy
The UK nonprofit organization Ectopic Pregnancy Trust provides support for individuals affected by ectopic pregnancy
PMCID: PMC3378618  PMID: 22723747
21.  A comparison between volume-controlled ventilation and pressure-controlled ventilation in providing better oxygenation in obese patients undergoing laparoscopic cholecystectomy 
Indian Journal of Anaesthesia  2012;56(3):276-282.
The maintenance of oxygenation is a commonly encountered problem in obese patients undergoing laparoscopic cholecystectomy. There is no specific guideline on the ventilation modes for this group of patients. Although several studies have been performed to determine the optimal ventilatory settings in these patients, the answer is yet to be found. The aim of this study was to evaluate the efficacy of pressure-controlled ventilation (PCV) in comparison with volume-controlled ventilation (VCV) for maintaining oxygenation during laparoscopic cholecystectomy in obese patients.
One hundred and two adult patients of ASA physical status I and II, Body Mass Index of 30–40 kg/m2, scheduled for laparoscopic cholecystectomy were included in this prospective randomized open-label parallel group study. To start with, all patients received VCV. Fifteen minutes after creation of pneumoperitoneum, they were randomized to receive either VCV (Group V) or PCV (Group P). The ventilatory parameters were adjusted accordingly to maintain the end-tidal CO2 between 35 and 40 mmHg. Respiratory rate, tidal volume, minute ventilation and peak airway pressure were noted. Arterial blood gas analyses were done 15 min after creation of pneumoperitoneum and at 20-min intervals thereafter till the end of the surgery. All data were analysed statistically.
Patients in Group P showed a statistically significant (P < 0.05) higher level of PaO2 and lower value of PAO2–PaO2 than those in Group V.
PCV is a more effective mode of ventilation in comparison with VCV regarding oxygenation in obese patients undergoing laparoscopic cholecystectomy.
PMCID: PMC3425289  PMID: 22923828
Laparoscopic cholecystectomy; obesity; pressure-controlled ventilation; volume-controlled ventilation
22.  Lipids induce expression of serum-responsive transmembrane kinase EhTMKB1-9 in an early branching eukaryote Entamoeba histolytica 
Scientific Reports  2012;2:333.
Mechanisms underlying the initiation of proliferative response are known only for a few organisms, and are not understood for the medically important organisms including Entamoeba histolytica. The trans membrane kinase EhTMKB1-9 of E. histolytica is one of the early indicators of proliferation and its' expression is regulated by serum, one of the components necessary for cellular proliferation in vitro. In this study we show that bovine serum albumin (BSA) can induce EhTMKB1-9 expression in place of serum, and that both follow the same mechanism. Both serum and BSA use the same promoter element and the activation process is initiated through a PI3 kinase-mediated pathway. We further show that BSA activates EhTMKB1-9 due to the lipids associated with it and that unsaturated fatty acids are responsible for activation. These results suggest that lipid molecules are ligand(s) for initiation of a signaling system that stimulates EhTMKB1-9 expression.
PMCID: PMC3312203  PMID: 22451862
23.  Self-circularizing 5′-ETS RNAs accumulate along with unprocessed pre ribosomal RNAs in growth-stressed Entamoeba histolytica 
Scientific Reports  2012;2:303.
The primary transcript of rRNA genes is a large pre rRNA which is precisely processed to release the mature rRNAs. The 5′-external transcribed spacer (ETS) of rRNA genes contains important sites for pre rRNA processing. Once the processing is accomplished the ETS is rapidly degraded. We show that in growth-stressed cells of the human parasitic protist Entamoeba histolytica the A'-A0 sub-fragment of the 5′-ETS accumulates to high levels as a family of RNA molecules of size 666 to 912 nt. These etsRNAs are circular in vivo and can spontaneously self-circularize in vitro. The accumulation of etsRNAs is accompanied by accumulation of unprocessed pre rRNA, indicating a possible role of etsRNAs in inhibition of processing during growth stress. Our data shows for the first time that processed etsRNA is not a mere by-product destined for degradation but is stabilized by circularization and could play a regulatory role as noncoding RNA.
PMCID: PMC3294279  PMID: 22396851
24.  Arabinosylated Lipoarabinomannan Skews Th2 Phenotype towards Th1 during Leishmania Infection by Chromatin Modification: Involvement of MAPK Signaling 
PLoS ONE  2011;6(9):e24141.
The parasitic protozoan Leishmania donovani is the causative organism for visceral leishmaniasis (VL) which persists in the host macrophages by deactivating its signaling machinery resulting in a critical shift from proinflammatory (Th1) to an anti-inflammatory (Th2) response. The severity of this disease is mainly determined by the production of IL-12 and IL-10 which could be reversed by use of effective immunoprophylactics. In this study we have evaluated the potential of Arabinosylated Lipoarabinomannan (Ara-LAM), a cell wall glycolipid isolated from non pathogenic Mycobacterium smegmatis, in regulating the host effector response via effective regulation of mitogen-activated protein kinases (MAPK) signaling cascades in Leishmania donovani infected macrophages isolated from BALB/C mice. Ara-LAM, a Toll-like receptor 2 (TLR2) specific ligand, was found to activate p38 MAPK signaling along with subsequent abrogation of extracellular signal–regulated kinase (ERKs) signaling. The use of pharmacological inhibitors of p38MAPK and ERK signaling showed the importance of these signaling pathways in the regulation of IL-10 and IL-12 in Ara-LAM pretreated parasitized macrophages. Molecular characterization of this regulation of IL-10 and IL-12 was revealed by chromatin immunoprecipitation assay (CHIP) which showed that in Ara-LAM pretreated parasitized murine macrophages there was a significant induction of IL-12 by selective phosphorylation and acetylation of histone H3 residues at its promoter region. While, IL-10 production was attenuated by Ara-LAM pretreatment via abrogation of histone H3 phosphorylation and acetylation at its promoter region. This Ara-LAM mediated antagonistic regulations in the induction of IL-10 and IL-12 genes were further correlated to changes in the transcriptional regulators Signal transducer and activator of transcription 3 (STAT3) and Suppressor of cytokine signaling 3 (SOCS3). These results demonstrate the crucial role played by Ara-LAM in regulating the MAPK signaling pathway along with subsequent changes in host effector response during VL which might provide crucial clues in understanding the Ara-LAM mediated protection during Leishmania induced pathogenesis.
PMCID: PMC3173371  PMID: 21935379
25.  Differential distribution of a SINE element in the Entamoeba histolytica and Entamoeba dispar genomes: Role of the LINE-encoded endonuclease 
BMC Genomics  2011;12:267.
Entamoeba histolytica and Entamoeba dispar are closely related protistan parasites but while E. histolytica can be invasive, E. dispar is completely non pathogenic. Transposable elements constitute a significant portion of the genome in these species; there being three families of LINEs and SINEs. These elements can profoundly influence the expression of neighboring genes. Thus their genomic location can have important phenotypic consequences. A genome-wide comparison of the location of these elements in the E. histolytica and E. dispar genomes has not been carried out. It is also not known whether the retrotransposition machinery works similarly in both species. The present study was undertaken to address these issues.
Here we extracted all genomic occurrences of full-length copies of EhSINE1 in the E. histolytica genome and matched them with the homologous regions in E. dispar, and vice versa, wherever it was possible to establish synteny. We found that only about 20% of syntenic sites were occupied by SINE1 in both species. We checked whether the different genomic location in the two species was due to differences in the activity of the LINE-encoded endonuclease which is required for nicking the target site. We found that the endonucleases of both species were essentially very similar, both in their kinetic properties and in their substrate sequence specificity. Hence the differential distribution of SINEs in these species is not likely to be influenced by the endonuclease. Further we found that the physical properties of the DNA sequences adjoining the insertion sites were similar in both species.
Our data shows that the basic retrotransposition machinery is conserved in these sibling species. SINEs may indeed have occupied all of the insertion sites in the genome of the common ancestor of E. histolytica and E. dispar but these may have been subsequently lost from some locations. Alternatively, SINE expansion took place after the divergence of the two species. The absence of SINE1 in 80% of syntenic loci could affect the phenotype of the two species, including their pathogenic properties, which needs to be explored.
PMCID: PMC3118788  PMID: 21612594

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