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Introduction: Surgical resection is curative for some patients with early lung squamous cell carcinoma. Staging and clinical factors do not adequately predict recurrence risk. We sought to validate the discriminative performance of proposed prognostic gene expression signatures at a level of rigor sufficient to support clinical use., Methods: The two-stage validation used independent core laboratories, objective quality control standards, locked test parameters, and large multi-institutional specimen and data sets. The first stage validation confirmed a signature's ability to stratify patient survival. The second-stage validation determined which signature(s) optimally improved risk discrimination when added to baseline clinical predictors. Participants were prospectively enrolled in institutional (cohort I) or cooperative group (cohort II) biospecimen and data collection protocols. All cases underwent a central review of clinical, pathologic, and biospecimen parameters using objective criteria to determine final inclusion (cohort I: n = 249; cohort II: n = 234). Primary selection required that a signature significantly predict a 3-year survival after surgical resection in cohort I. Signatures meeting this criterion were further tested in cohort II, comparing risk prediction using baseline risk factors alone versus in combination with the signature., Results: Male sex, advanced age, and higher stage were associated with shorter survival in cohort I and established a baseline clinical model. Of the three signatures validated in cohort I, one signature was validated in cohort II and statistically significantly enhanced the prognosis relative to the baseline model (C-index difference 0.122; p < 0.05)., Conclusions: These results represent the first rigorous validation of a test appropriate to direct adjuvant treatment or clinical trials for patients with lung squamous cell carcinoma., (Copyright © 2020. Published by Elsevier Inc.)

Disorganized vessels in the tumor vasculature lead to impaired perfusion, resulting in reduced accessibility to immune cells and chemotherapeutic drugs. In the breast tumor-stroma interplay, paracrine factors such as interleukin-6 (IL-6) often facilitate disordered angiogenesis. We show here that epigenetic mechanisms regulate the crosstalk between IL-6 and vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways in myoepithelial (CD10 + ) and endothelial (CD31 + , CD105 + , CD146 + , and CD133 - ) cells isolated from malignant and nonmalignant tissues of clinically characterized human breast tumors. Tumor endothelial (Endo-T) cells in 3D cultures exhibited higher VEGFR2 expression levels, accelerated migration, invasion, and disorganized sprout formation in response to elevated IL-6 levels secreted by tumor myoepithelial (Epi-T) cells. Constitutively, compared with normal endothelial (Endo-N) cells, Endo-T cells differentially expressed DNA methyltransferase isoforms and had increased levels of IL-6 signaling intermediates such as IL-6R and signal transducer and activator of transcription 3 (STAT3). Upon IL-6 treatment, Endo-N and Endo-T cells displayed altered expression of the DNA methyltransferase 1 (DNMT1) isoform. Mechanistic studies revealed that IL-6 induced proteasomal degradation of DNMT1, but not of DNMT3A and DNMT3B and subsequently led to promoter hypomethylation and expression/activation of VEGFR2. IL-6-induced VEGFR2 up-regulation was inhibited by overexpression of DNMT1. Transfection of a dominant-negative STAT3 mutant, but not of STAT1, abrogated VEGFR2 expression. Our results indicate that in the breast tumor microenvironment, IL-6 secreted from myoepithelial cells influences DNMT1 stability, induces the expression of VEGFR2 in endothelial cells via a promoter methylation-dependent mechanism, and leads to disordered angiogenesis., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this manuscript., (© 2020 Hegde et al.)

The present study illustrates mitochondria-mediated impact of ionizing radiation which is paralleled by activation of several pro-adaptive responses in normal human dermal fibroblast cells. Irradiation of cells with X-rays (5 Gy) led to an increase in fragmentation and mitochondrial mass. Distinct temporal changes in cytosolic and mitochondrial reactive oxygen species (ROS) were noted in response to radiation, which was associated with depletion in mitochondrial membrane potential followed by decrease in ATP levels. Long Amplicon-Polymerase Chain Reaction (LA-PCR) analysis showed time-dependent increase in mitochondrial DNA damage that preceded mitochondrial ROS generation. Irradiation of cells led to an initial G 2 /M arrest at 8 h that persisted till 16 h, with subsequent block at G 0 /G 1 measured at 48 and 72 h time points. Interestingly, cells activated autophagy as a countermeasure against radiation-mediated cellular insults and aided in removal of damaged mitochondria. Blocking autophagy using 3-methyladenine led to cell death via activation of enhanced ROS, PARP-1 and caspase 3 cleavage. Upregulation of mitochondrial biogenesis factors Nrf1/PGC-1α, following irradiation was observed. Irradiated cells exhibited an increase in the phosphorylation of GCN2, PERK and eIF2α that might be responsible for the up-regulation of ATF4 and CHOP thereby regulating autophagy and components of integrated stress response. Apart from this, we measured accumulation of mitochondrial chaperones (HSP60/HSP10) and ATF5 which is a major molecule involved in mitochondrial stress. Taken together, mitochondria are one of the major cytoplasmic targets for ionizing radiation and possibly act as an early indicator of cellular insult. The findings also show that stressed mitochondria might influence endoplasmic reticulum (ER)-related signalling leading to the activation of adaptive mechanisms like cytoprotective autophagy, and molecules responsible for mitochondrial biogenesis and protein quality control in order to replenish mitochondrial pool and maintain cellular homeostasis., Competing Interests: Declaration of competing interest None declared., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bipolar disorder (BD) is multifactorial mood disorder characterized by alternating episodes of hyperactive mania and severe depression. Lithium is one of the most preferred drug used as mood stabilizer in treating BD. In this study, we examined the changes in plasma metabolome in BD subjects in the context of lithium responsiveness. Plasma samples from clinically defined, age and gender matched unrelated healthy controls and BD subjects (lithium responders and non-responders) were obtained and processed in positive and negative mode using untargeted liquid chromatography/mass spectrometry analysis. We identified significant alterations in plasma levels of dopamine along with its precursors (tyrosine and phenylalanine), branched chain amino acid such as valine and excitatory neurotransmitter glutamate between healthy control and BD subjects. Lipid molecules such as, eicosenoic acid and retinyl ester also showed distinguished patterns between control and BD individuals. Lithium responsiveness was markedly associated with significant differences in proline, L-gamma-glutamyl-isoleucine, dopamine, palmitic acid methyl ester, cholesterol sulfate, androsterone sulfate and 9S,12S,13S-triHOME levels. Altered metabolites enriched with key biochemical pathways associated with neuropsychiatry disorders. We hypothesize that BD pathogenesis and lithium responsiveness is associated with impaired homeostasis of amino acid and lipid metabolism., (Copyright © 2019. Published by Elsevier B.V.)

Background: The phosphoenolpyruvate carboxykinase (PEPCK) isoforms are considered as rate-limiting enzymes for gluconeogenesis and glyceroneogenesis pathways. PEPCK exhibits several interesting features such as a) organelle-specific isoforms (cytosolic and a mitochondrial) in vertebrate clade, b) tissue-specific expression of isoforms and c) organism-specific requirement of ATP or GTP as a cofactor. In higher organisms, PEPCK isoforms are intricately regulated and activated through several physiological and pathological stimuli such as corticoids, hormones, nutrient starvation and hypoxia. Isoform-specific transcriptional/translational regulation and their interplay in maintaining glucose homeostasis remain to be fully understood. Mounting evidence indicates the significant involvement of PEPCK isoforms in physiological processes (development and longevity) and in the progression of a variety of diseases (metabolic disorders, cancer, Smith-Magenis syndrome)., Objective: The present systematic review aimed to assimilate existing knowledge of transcriptional and translational regulation of PEPCK isoforms derived from cell, animal and clinical models., Conclusion: Based on current knowledge and extensive bioinformatics analysis, in this review we have provided a comparative (epi)genetic understanding of PCK1 and PCK2 genes encompassing regulatory elements, disease-associated polymorphisms, copy number variations, regulatory miRNAs and CpG densities. We have also discussed various exogenous and endogenous modulators of PEPCK isoforms and their signaling mechanisms. A comprehensive review of existing knowledge of PEPCK regulation and function may enable identification of the underlying gaps to design new pharmacological strategies and interventions for the diseases associated with gluconeogenesis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Metabolomics refers to the systematic identification and quantification of the small molecule metabolites (the metabolome) of a biological system in a given space (cell, tissue, organ, biological fluid, or organism) and time. Global metabolic profiling provides broad range of coverage for most of the analytes present in any tissue. Human retina is metabolically highly active, and retinal vascular diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), retinal vein occlusion (RVO), central retinal artery occlusion, and retinopathy of prematurity (ROP) are often associated with the disruptions in metabolic activities. A systematic study of total retinal metabolites from human diseased retina is a major challenge owing to the nonavailability of tissue specimens. Therefore, vitreous humor being very proximal to retina could be used as surrogate for retinal metabolomic analysis. As the extraction method adopted for such analysis determines the type of metabolites, two different types of solvent (methanol and chloroform)-based extraction methods could be used for retinal vascular patient samples (vitreous humor). Metabolites obtained from both the extraction methods are then subjected to LC-MS/MS for detection and identification.

Lead is an environmental hazard with great public health concern and has been known to inhibit delta-aminolevulinate dehydratase (ALAD) activity involved in the heme biosynthetic pathway. The study aimed to investigate the influence of ALAD polymorphism (G177C) on retention of Pb-B levels and ALAD activity on occupationally exposed lead workers. In the present study, we enrolled 561 lead exposed and 317 non-occupationally exposed subjects and performed a comprehensive analysis of Pb-B levels along with ALAD activity and genotyping. The frequency of ALAD variants observed in the total subjects (n = 878) was 70.04% for ALAD 1-1, 27.44% for heterozygous ALAD 1-2 and 2.5% for homozygous mutant ALAD 2-2. Our study revealed that ALAD 1-2 carriers presented higher Pb-B levels compared to wild type ALAD 1-1 carriers. Further, a significant difference was observed in the activity of ALAD between ALAD 1-2/ 2-2 and ALAD 1-1 carriers of non-occupationally exposed group indicating that the polymorphic nature of the enzyme may contribute to altered activity of ALAD irrespective of lead exposure. Hence, ALAD 2 allele might contribute to increased susceptibility to high Pb-B retention, and genotyping of ALAD in lead exposed subjects might be used as a prediction marker to impede tissue/organ damage due to lead toxicity., (Copyright © 2018. Published by Elsevier B.V.)

Individuals with type 2 diabetes (T2D) display vascular insulin resistance and decreased nitric oxide production leading to vasoconstriction and atherosclerosis. Soluble factors such as pro-inflammatory molecules, and various genetic and epigenetic mechanisms have been implicated to induce insulin resistance in vascular endothelial cells. Epigenetic mechanisms such as altered promoter DNA methylation have been demonstrated in development and progression of metabolic disorders and atherosclerosis. However, underlying precise epigenetic mechanisms regulating cross talk between insulin signaling genes and inflammation in vascular cells remains to be fully understood. Human endothelial cells when (a) treated with interleukin-6 (IL-6) and insulin together, (b) pretreated with IL-6, and (c) under hyperinsulinemic conditions led to a state of vascular insulin resistance resulting in decreased Akt/eNOS activation and subsequent stabilization of STAT3 phosphorylation. IL-6 abrogated insulin effects on angiogenesis in 3D spheroid and matrigel assays. IL-6-induced insulin resistance was associated with decreased activity of DNA methyltransferase isoforms and global DNA hypomethylation, which inversely correlated with S-phase of cell cycle. CpG microarray analysis in IL-6 treated endothelial cells revealed promoters associated hypo- and hypermethylation of 199 and 98 genes respectively. Promoter DNA methylation status of genes associated with insulin signaling and angiogenesis such as RPS6KA2, PIK3R2, FOXD3, EXOC7, MAP3K8, ITPKB, EPHA6, IGF1R, and FOXC2 were validated by bisulfite DNA sequencing. Concentration and time-dependent analysis revealed that IL-6 reduced DNMT1 and DNMT3B but not DNMT3A protein levels. Our data indicate a causal link between IL-6-induced changes in global and promoter-specific DNA methylation, due to reduced DNMT1 and DNMT3B protein levels leading to altered expression of critical genes involved in insulin signaling and angiogenesis.

In the present study, we have designed a laser-induced fluorescence (LIF) based instrumentation and developed a sensitive methodology for the effective separation, visualization, identification and analysis of proteins on a single platform. In this method, intrinsic fluorescence spectra of proteins were detected after separation on 1 or 2 dimensional Sodium Dodecyl Sulfate-Tris(2-carboxyethyl)phosphine (SDS-TCEP) polyacrylamide gel electrophoresis (PAGE) and the data were analyzed. The MATLAB assisted software was designed for the development of PAGE fingerprint for the visualization of protein after 1- and 2-dimensional protein separation. These provided objective parameters of intrinsic fluorescence intensity, emission peak, molecular weight and isoelectric point using a single platform. Further, the current architecture could differentiate the overlapping proteins in the PAGE gels which otherwise were not identifiable by conventional staining, imaging and tagging methods. Categorization of the proteins based on the presence or absence of tyrosine or tryptophan residues and assigning the corresponding emission peaks (309-356 nm) with pseudo colors allowed the detection of proportion of proteins within the given spectrum. The present methodology doesn't use stains or tags, hence amenable to couple with mass spectroscopic measurements. This technique may have relevance in the field of proteomics that is used for innumerable applications., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The present study is intended to analyze the occurrence of potent, low produce, naturally occurring stilbenes in protoplasts of wild species and hybrids of Dendrobium. The wild species selected for the study was Dendrobium ovatum, endemic to Western Ghats of India. Protoplasts were isolated from leaves and tepal tissues of all the species and were cultured purely to generate homofusants and cross-cultured to raise heterofusants. Phytochemical composition of protoplast culture with atypical and pure microcolonies was performed using mass spectrometry. Enzyme cocktail of 4% pectinase together with 2% cellulase displayed the highest competence for protoplast isolations. Maximum protoplast density of 30.11 × 10 4 /ml was obtained from D. ovatum leaves in 2 h. Subcellular features such as the presence of partially formed cell wall, the position of the nucleus, chloroplast density, colony existence, and integrity of the plasma membrane were analyzed. Among the pure and cross-cultured protoplasts, the number of heterofusants and homofusants formed were enumerated. The spectral feature extraction of the mass spectrometry indicated the presence of five phenolic marker compounds, viz., tristin, confusarin, gigantol, moscatilin, and resveratrol, some of them in pure and others in assorted protoplast cultures raised from Dendrobium leaves and tepals. The study demonstrated that protoplast fusion technique enabled phytochemical assemblage in vitro as stilbenes tend to get restricted either in a tissue or species specific manner. This is the first report showing the presence of resveratrol, moscatilin, tristin, gigantol, and confusarin in wild and hybrid species from cultured Dendrobium protoplasts in vitro.

Biobanking at Duke University has existed for decades and has grown over time in silos and based on specialized needs, as is true with most biomedical research centers. These silos developed informatics systems to support their own individual requirements, with no regard for semantic or syntactic interoperability. Duke undertook an initiative to implement an enterprise-wide biobanking information system to serve its many diverse biobanking entities. A significant part of this initiative was the development of a common terminology for use in the commercial software platform. Common terminology provides the foundation for interoperability across biobanks for data and information sharing. We engaged experts in research, informatics, and biobanking through a consensus-driven process to agree on 361 terms and their definitions that encompass the lifecycle of a biospecimen. Existing standards, common terms, and data elements from published articles provided a foundation on which to build the biobanking terminology; a broader set of stakeholders then provided additional input and feedback in a secondary vetting process. The resulting standardized biobanking terminology is now available for sharing with the biobanking community to serve as a foundation for other institutions who are considering a similar initiative.

Ethnopharmacological Relevance: Indian traditional medicinal system in Ayurveda suggests several preparations, known as medhya rasayanas, of diverse plant origin to enhance the health in general, reduce stress and improve brain function in particular during ageing. These effects in the context of contemporary knowledge and the underlying mechanisms are not clearly understood. Autophagy and DNA damage induced repair are inter-related quintessential pathways and are significantly altered during stress and ageing. Hence, medhya rasayana prepared from Clitoria ternatea (locally known as shankhpushpi) was used to test these effects in Wistar rat model of various age groups upon stereotaxic mediated kainic acid induced brain injury., Materials and Methods: The rodent experiments were carried out in one, twelve and eighteen months old male Wistar rats. The rats were orally fed with medhya rasayana prepared from Clitoria ternatea (3g per kg body weight/day) for 60 days. Stereotaxic mediated kainate stress to the hippocampus was performed on day 61. The rats were sacrificed on 66th day and the brain tissues were analyzed histologically and measured for autophagy, base excision repair and antioxidant enzyme activities. In addition, cognitive functions were analyzed by employing novel object recognition task and Morris water maze tests. The gene expression profile of hippocampus was assessed by microarray hybridization and two genes are validated., Results: Our study showed significant decrease of autophagy by medhya rasayana in both 12 and 18 months old rats. The hippocampal CA3 cellularity were increased in stereotaxic mediated stressed rats by medhya rasayana. There were no significant differences in constitutive base excision repair and antioxidant enzyme activities. Medhya rasayana treatment also significantly increased episodic memory in rats. Microarray experiments for pathway specific gene expression analysis showed altered expression of genes of long-term potentiation, axon guidance, neuroactive ligand-receptor interaction, regulation of autophagy, lysosome, homologous recombination and nucleotide excision repair pathways in adult rats by medhya rasayana., Conclusions: In the present study, we show that reduction in autophagy is crucial for medhya rasayana induced protection of rat hippocampal cells and that artificially enhanced autophagy protects the brain cell damage by maintaining the selective DNA damage repair pathway and removal of reactive oxygen species to inhibit apoptosis. These findings suggest autophagy directed pathways by medhya rasayana prepared from C. ternatea protects the brain cells from stress induced injury., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The discovery of rapid acting and powerful angiogenic proteins are of significant interest in the treatment of various human disorders associated with insufficient angiogenesis such as ischemia, menorrhagia and delayed wound healing. Snake venoms consist of a mixture of bioactive proteins and polypeptides and are rich sources of pharmacologically important molecules. Serine proteinases are one of the abundant proteins present in Bothrops snake venoms and possess multiple biological functions including the regulation of the blood coagulation cascade. In this study, serine proteinases from Bothrops atrox (B. atrox) and Bothrops brazili (B. brazili) that modulate angiogenesis were purified and characterized. Molecular size exclusion chromatography, affinity chromatography followed by ion exchange chromatography of the serine proteinases indicated molecular masses of around 32 kDa. Serine proteinases from both the species exhibited diverse catalytic activities such as the ability to induce amidolytic, fibrinogenolytic, gelatinolytic activities and also coagulated plasma with a minimal coagulation concentration of 2.4 μg/mL. Serine proteinases facilitated the sprouting of human umbilical vein endothelial cells (HUVEC) in three-dimensional culture systems and induced tubule formation in monolayer culture systems. Serine proteinase stimulated Akt ser473 and eNOS ser1177 phosphorylation in endothelial cells and addition of PI3K inhibitor LY294002 abrogated the effects of serine proteinases on sprout formation of endothelial cells in 3D collagen gels, suggesting that serine proteinase facilitated angiogenesis was mediated by PI3K/eNOS signaling axis. We also show in agarose plug assays using a mouse model, serine proteinases from Bothrops venoms significantly enhanced neovascularization. Our data suggests pro-angiogenic activity by the serine proteinases from B. atrox and B. brazili venom and further studies are warranted to explore the therapeutic applications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Constitutively active neutrophil extracellular traps (NETs) and elevated plasma homocysteine are independent risk factors for Type 2 Diabetes (T2D) associated vascular diseases. Here, we show robust NETosis due to elevated plasma homocysteine levels in T2D subjects and increased components of NETs such as neutrophil elastase and cell free DNA. Cooperative NETs formation was observed in neutrophils exposed to homocysteine, IL-6 and high glucose suggesting acute temporal changes tightly regulate constitutive NETosis. Homocysteine induced NETs by NADPH oxidase dependent and independent mechanisms. Constitutively higher levels of calcium and mitochondrial superoxides under hyperglycemic conditions were further elevated in response to homocysteine leading to accelerated NETosis. Homocysteine showed robust interaction between neutrophils and platelets by inducing platelet aggregation and NETosis in an interdependent manner. Our data demonstrates that homocysteine can alter innate immune function by promoting NETs formation and disturbs homeostasis between platelets and neutrophils which may lead to T2D associated vascular diseases.

Cytoplasmic and mitochondrial isoforms of phosphoenolpyruvate carboxykinase (PEPCK-C and PEPCK-M) regulate hepatic gluconeogenesis to control systemic glucose homeostasis. Transcriptional and post-transcriptional mechanisms may govern synthesis, maintenance and cooperative function of compartmentalized PEPCK enzymes. In a comparative analysis, we show that tumor cells consistently transcribe and translate higher levels of enzymatically active PEPCK-C than PEPCK-M and both the isoforms were present at lower levels in normal fibroblasts. Unlike in PEPCK-M, absence of glucose reduced the PEPCK-C mRNA and protein levels only in HepG2 cells. Interestingly, isoflavone genistein significantly increased PEPCK-C mRNA and protein levels in normal fibroblasts indicating cell type specific control mechanisms. Genistein also significantly affected RNA stability of PEPCK-C but not PEPCK-M in HepG2 cells. This was due to the conserved and functional mRNA destabilizing AU rich sequences at the 3'-UTR region of PEPCK-C gene and was confirmed by luciferase reporter assays suggesting that glucose deprivation and genistein targets these sequences for mRNA degradation in HepG2 cells but not in fibroblasts. Analysis of promoter methylation by luciferase reporter assays and bisulfite DNA sequencing suggested that PEPCK-C but not PEPCK-M promoter was activated by 5-aza-2-deoxycytidine by inducing cytosine demethylation at the specific CpG dinucleotides of 5'-UTR region. Taken together, our data suggests stable PEPCK-M activity and identifies intricate relationship between (1) mRNA stability and (2) promoter DNA methylation as two mechanisms of gene expression that distinguishes PEPCK-C and PEPCK-M enzyme activities in a context and cell type dependent manner during gluconeogenesis. J. Cell. Biochem. 117: 2506-2520, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Ethnopharmacological Relevance: Preparations from Phyllanthus emblica called Amalaki rasayana is used in the Indian traditional medicinal system of Ayurveda for healthy living in elderly. The biological effects and its mechanisms are not fully understood. Since the diminishing DNA repair is the hallmark of ageing, we tested the influence of Amalaki rasayana on recognized DNA repair activities in healthy aged individuals., Methods: Amalaki rasayana was prepared fresh and healthy aged randomized human volunteers were administrated with either rasayana or placebo for 45 days strictly as per the traditional text. The DNA repair was analyzed in peripheral blood mononuclear cells before and after rasayana administration and after 45 days post-rasayana treatment regimen. UVC-induced DNA strand break repair (DSBR) based on extent of DNA unwinding by fluorometric analysis, nucleotide excision repair (NER) by flow cytometry and constitutive base excision repair (BER) by gap filling method were analyzed., Results: Amalaki rasayana administration stably maintained/enhanced the DSBR in aged individuals. There were no adverse side effects. Further, subjects with different body mass index showed differential DNA strand break repair capacity. No change in unscheduled DNA synthesis during NER and BER was observed between the groups., Conclusion: Intake of Amalaki rasayana by aged individuals showed stable maintenance of DNA strand break repair without toxic effects. However, there was no change in nucleotide and base excision repair activities. Results warrant further studies on the effects of Amalaki rasayana on DSBR activities., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Leishmania are protozoan pathogens of humans that exist as extracellular promastigotes in the gut of their sand fly vectors and as obligate intracellular amastigotes within phagolysosomes of infected macrophages. Between infectious blood meal feeds, sand flies take plant juice meals that contain sucrose and store these sugars in their crop. Such sugars are regurgitated into the sand fly anterior midgut where they impact the developing promastigote parasite population. In this report we showed that promastigotes of all Leishmania species secreted an invertase/sucrase enzyme during their growth in vitro. In contrast, neither L. donovani nor L. mexicana amastigotes possessed any detectable invertase activity. Importantly, no released/secreted invertase activity was detected in culture supernatants from either Trypanosoma brucei or Trypanosoma cruzi. Using HPLC, the L. donovani secretory invertase was isolated and subjected to amino acid sequencing. Subsequently, we used a molecular approach to identify the LdINV and LmexINV genes encoding the ~72 kDa invertases produced by these organisms. Interestingly, we identified high fidelity LdINV-like homologs in the genomes of all Leishmania sp. but none were present in either T. brucei or T. cruzi. Northern blot and RT-PCR analyses showed that these genes were developmentally/differentially expressed in promastigotes but not amastigotes of these parasites. Homologous transfection studies demonstrated that these genes in fact encoded the functional secretory invertases produced by these parasites. Cumulatively, our results suggest that these secretory enzymes play critical roles in the survival/growth/development and transmission of all Leishmania parasites within their sand fly vector hosts.

Expression of GPI-anchored T-cadherin (T-cad) on vascular smooth muscle cells (VSMC) is elevated in vascular disorders such as atherosclerosis and restenosis which are associated with insulin resistance. Functions for T-cad and signal transduction pathway utilization by T-cad in VSMC are unknown. The present study examines the consequences of altered T-cad expression on VSMC for constitutive and insulin-induced Akt/mTOR axis signaling and contractile competence. Using viral vectors rat (WKY and SHR) and human aortic VSMCs were variously transduced with respect to T-cad-overexpression (Tcad+-VSMC) or T-cad-deficiency (shT-VSMC) and compared with their respective control transductants (E-VSMC or shC-VSMC). Tcad+-VSMC exhibited elevated constitutive levels of phosphorylated Akt(ser473), GSK3β(ser9), S6RP(ser235/236) and IRS-1(ser636/639). Total IRS-1 levels were reduced. Contractile machinery was constitutively altered in a manner indicative of reduced intrinsic contractile competence, namely decreased phosphorylation of MYPT1(thr696 or thr853) and MLC20(thr18/ser19), reduced RhoA activity and increased iNOS expression. Tcad+-VSMC-populated collagen lattices exhibited greater compaction which was due to increased collagen fibril packing/reorganization. T-cad+-VSMC exhibited a state of insulin insensitivity as evidenced by attenuation of the ability of insulin to stimulate Akt/mTOR axis signaling, phosphorylation of MLC20 and MYPT1, compaction of free-floating lattices and collagen fibril reorganization in unreleased lattices. The effects of T-cad-deficiency on constitutive characteristics and insulin responsiveness of VSMC were opposite to those of T-cad-overexpression. The study reveals novel cadherin-based modalities to modulate VSMC sensitivity to insulin through Akt/mTOR axis signaling as well as vascular function and tissue architecture through the effects on contractile competence and organization of extracellular matrix., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Neutrophils serve as an active constituent of innate immunity and are endowed with distinct ability for producing neutrophil extracellular traps (NETs) to eliminate pathogens. Earlier studies have demonstrated a dysfunction of the innate immune system in diabetic subjects leading to increased susceptibility to infections; however, the influence of hyperglycemic conditions on NETs is unknown. In the present study we demonstrate that (a) NETs are influenced by glucose homeostasis, (b) IL-6 is a potent inducer of energy dependent NET formation and (c) hyperglycemia mimics a state of constitutively active pro-inflammatory condition in neutrophils leading to reduced response to external stimuli making diabetic subjects susceptible to infections., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

T-cadherin (cadherin 13, H-cadherin, gene name CDH13) has been proposed to act as a tumor-suppressor gene as its expression is significantly diminished in several types of carcinomas, including melanomas. Allelic loss and promoter hypermethylation have been proposed as mechanisms for silencing of CDH13. However, they do not account for loss of T-cadherin expression in all carcinomas, and other genetic or epigenetic alterations can be presumed. The present study investigated transcriptional regulation of CDH13 in melanoma. Bioinformatical analysis pointed to the presence of known BRN2 (also known as POU3F2 and N-Oct-3)-binding motifs in the CDH13 promoter sequence. We found an inverse correlation between BRN2 and T-cadherin protein and transcript expression. Reporter gene analysis and electrophoretic mobility shift assays in melanoma cells demonstrated that CDH13 is a direct target of BRN2 and that BRN2 is a functional transcriptional repressor of CDH13 promoter activity. The regulatory binding element of BRN2 was located -219 bp of the CDH13 promoter proximal to the start codon and was identified as 5'-CATGCAAAA-3'. Ectopic expression of BRN2 in BRN2-negative/T-cadherin-positive melanoma cells resulted in suppression of CDH13 promoter activity, whereas BRN2 knockdown in BRN2-positive/T-cadherin-negative melanoma cells resulted in re-expression of T-cadherin transcripts and protein. Transcriptional repression of CDH13 by BRN2 may participate in malignant transformation of melanoma by increasing invasion and migration potentials of melanoma cells. The study has identified CDH13 as a novel direct BRN2 transcriptional target gene and has advanced knowledge of mechanisms underlying loss of T-cadherin expression in melanoma.

Background: Studies examining the impact of lower socioeconomic status (SES) on the outcomes of patients with nonsmall cell lung cancer (NSCLC) are inconsistent. The objective of this study was to clearly elucidate the association between SES, education, and clinical outcomes among patients with NSCLC., Methods: The study population was derived from a consecutive, retrospective cohort of patients with NSCLC who received treatment within the Duke Health System between 1995 and 2007. SES determinants were based on the individual's census tract and corresponding 2000 Census data. Determinants included the percentage of the population living below poverty, the median household income, and the percentages of residents with at least a high school diploma and at least a bachelor's degree. The SES and educational variables were divided into quartiles. Statistical comparisons were performed using the 25th and 75th percentiles., Results: Individuals who resided in areas with a low median household income or in which a high percentage of residents were living below the poverty line had a shorter cancer-specific 6-year survival than individuals who resided in converse areas (P = .0167 and P = .0067, respectively). Those living in areas in which a higher percentage of residents achieved a high school diploma had improved disease outcomes compared with those living in areas in which a lower percentage attained a high school diploma (P = .0033). A survival advantage also was observed for inhabitants of areas in which a higher percentage of residents attained a bachelor's degree (P = .0455)., Conclusions: Low SES was identified as an independent prognostic factor for poor survival in patients with both early and advanced stage NSCLC. Patients who lived in areas with high poverty levels, low median incomes, and low education levels had worse mortality., (Copyright © 2012 American Cancer Society.)

Genetic and epigenetic studies in different cancers, including cutaneous carcinomas, have implicated T-cadherin (T-cad) as a tumor suppressor. Immunohistochemical and in vitro studies have suggested that T-cad loss promotes incipient invasiveness in cutaneous squamous cell carcinoma (SCC). Molecular mechanisms are unknown. This study found that the main consequence of T-cad silencing in SCC is facilitation of ligand-dependent EGFR activation, whereas T-cad overexpression impedes EGFR activation. Gain- and loss-of-function studies in A431 SCC cells demonstrate T-cad-controlled responsiveness to EGF with respect to pharmacological inhibition of EGFR and to diverse signaling and functional events of the EGFR activation cascade (EGFR phosphorylation, internalization, nuclear translocation, cell retraction/de-adhesion, motility, invasion, integrin β1, and Rho small GTPases such as RhoA, Rac1, and Cdc42 activation). Further, T-cad modulates the EGFR pathway activity by influencing membrane compartmentalization of EGFR; T-cad upregulation promotes retention of EGFR in lipid rafts, whereas T-cad silencing releases EGFR from this compartment, rendering EGFR more accessible to ligand stimulation. This study reveals a mechanism for fine-tuning of EGFR activity in SCC, whereby T-cad represents an auxiliary "negative" regulator of the EGFR pathway, which impacts invasion-associated behavioral responses of SCC to EGF. This action of T-cad in SCC may serve as a paradigm explaining other malignancies displaying concomitant T-cad loss and enhanced EGFR activity.

Aims: T-cadherin (T-cad) is a glycosylphosphatidylinositol-anchored cadherin family member. Experimental, clinical, and genomic studies suggest a role for T-cad in vascular disorders such as atherosclerosis and hypertension, which are associated with endothelial dysfunction and insulin resistance (InsRes). In endothelial cells (EC), T-cad and insulin activate similar signalling pathways [e.g. PI3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR)] and processes (e.g. angiogenesis). We hypothesize that T-cad is a regulatory component of insulin signalling in EC and therefore a determinant of the development of endothelial InsRes., Methods and Results: We investigated T-cad-dependent effects on insulin sensitivity using human EC stably transduced with respect to T-cad overexpression or T-cad silencing. Responsiveness to insulin was examined at the level of effectors of the insulin signalling cascade, EC nitric oxide synthase (eNOS) activation, and angiogenic behaviour. Overexpression and ligation of T-cad on EC attenuates insulin-dependent activation of the PI3K/Akt/mTOR signalling axis, eNOS, EC migration, and angiogenesis. Conversely, T-cad silencing enhances these actions of insulin. Attenuation of EC responsiveness to insulin results from T-cad-mediated chronic activation of the Akt/mTOR-dependent negative feedback loop of the insulin cascade and enhanced degradation of the insulin receptor (IR) substrate. Co-immunoprecipitation experiments revealed an association between T-cad and IR. Filipin abrogated inhibitory effects of T-cad on insulin signalling, demonstrating localization of T-cad-insulin cross-talk to lipid raft plasma membrane domains. Hyperinsulinaemia up-regulates T-cad mRNA and protein levels in EC., Conclusion: T-cad expression modulates signalling and functional responses of EC to insulin. We have identified a novel signalling mechanism regulating insulin function in the endothelium and attribute a role for T-cad up-regulation in the pathogenesis of endothelial InsRes.

Previously, we characterized a gene encoding the unique nuclease (LdNuc(s)) from a Sudanese isolate of the human pathogen Leishmania donovani. This parasite secretory enzyme is involved in the salvage of host-derived purines and is constitutively expressed by both developmental forms of the parasite. Currently, we assessed whether an LdNuc(s)-like nuclease was conserved among other geographically disparate isolates of L. donovani and whether this enzyme was produced by intracellular amastigotes during human infections. Using RT-PCR and Southern blotting, we showed that LdNuc(s) gene homologs were present in each of the viscerotropic Leishmania tested (i.e., L. donovani isolates from the Sudan, Ethiopia and India as well as L. infantum). Further results of in situ enzyme activity gel analyses showed that each of these parasite isolates also expressed a released/secreted LdNuc(s)-like nuclease activity. In Western blots, our anti-LdNuc(s) (Sudan) peptide-specific antibody reacted with only a single ~35 kDa protein in each of the viscerotropic Leishmania isolates. Further, the ~35 kDa nuclease secreted by each of these isolates was specifically immunoprecipitated by the anti-LdNuc(s) antibody above. In situ gel analyses showed that each of these immunoprecipitates had LdNuc(s)-like nuclease activity. Moreover, sera from acute visceral leishmaniasis patients from India, Sudan and Brazil all immunoprecipitated an LdNuc(s)-HA expressed nuclease demonstrating, that these patients possessed antibodies against this parasite secretory enzyme. Cumulatively, these results showed that the LdNuc(s) homologs were functionally conserved among geographically disparate visceral Leishmania spp. and that amastigotes of these parasites must produce this nuclease enzyme during the course of human disease.

Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009-2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified., (Published by Elsevier Ltd.)

Aims: The presence of endothelial cell (EC)-derived surface molecules in the circulation is among hallmarks of endothelial activation and damage in vivo. Previous investigations suggest that upregulation of T-cadherin (T-cad) on the surface of ECs may be a characteristic marker of EC activation and stress. We investigated whether T-cad might also be shed from ECs and in amounts reflecting the extent of activation or damage., Methods and Results: Immunoblotting showed the presence of T-cad protein in the culture medium from normal proliferating ECs and higher levels in the medium from stressed/apoptotic ECs. Release of T-cad into the circulation occurs in vivo and in association with endothelial dysfunction. Sandwich ELISA revealed negligible T-cad protein in the plasma of healthy volunteers (0.90 ± 0.90 ng/mL, n = 30), and increased levels in the plasma from patients with non-significant atherosclerosis (9.23 ± 2.61 ng/mL, n = 63) and patients with chronic coronary artery disease (6.93 ± 1.31 ng/mL, n = 162). In both patient groups there was a significant (P = 0.043) dependency of T-cad and degree of endothelial dysfunction as measured by reactive hyperaemia peripheral tonometry. Flow cytometry analysis showed that the major fraction of T-cad was released into the EC culture medium and the plasma as a surface component of EC-derived annexin V- and CD144/CD31-positive microparticles (MPs). Gain-of-function and loss-of-function studies demonstrate that MP-bound T-cad induced Akt phosphorylation and activated angiogenic behaviour in target ECs via homophilic-based interactions., Conclusion: Our findings reveal a novel mechanism of T-cad-dependent signalling in the vascular endothelium. We identify T-cad as an endothelial MP antigen in vivo and demonstrate that its level in plasma is increased in early atherosclerosis and correlates with endothelial dysfunction.

Endoplasmic reticulum (ER) stress activated by perturbations in ER homeostasis induces the unfolded protein response (UPR) with chaperon Grp78 as the key activator of UPR signalling. The aim of UPR is to restore normal ER function; however prolonged or severe ER stress triggers apoptosis of damaged cells to ensure protection of the whole organism. Recent findings support an association of ER stress-induced apoptosis of vascular cells with cardiovascular pathologies. T-cadherin (T-cad), an atypical glycosylphosphatidylinositol-anchored member of the cadherin superfamily is upregulated in atherosclerotic lesions. Here we investigate the ability of T-cad to influence UPR signalling and endothelial cell (EC) survival during ER stress. EC were treated with a variety of ER stress-inducing compounds (thapsigargin, dithiothereitol, brefeldin A, tunicamycin, A23187 or homocysteine) and induction of ER stress validated by increases in levels of UPR signalling molecules Grp78 (glucose-regulated protein of 78kDa), phospho-eIF2alpha (phosphorylated eukaryotic initiation factor 2alpha) and CHOP (C/EBP homologous protein). All compounds also increased T-cad mRNA and protein levels. Overexpression or silencing of T-cad in EC respectively attenuated or amplified the ER stress-induced increase in phospho-eIF2alpha, Grp78, CHOP and active caspases. Effects of T-cad-overexpression or T-cad-silencing on ER stress responses in EC were not affected by inclusion of either N-acetylcysteine (reactive oxygen species scavenger), LY294002 (phosphatidylinositol-3-kinase inhibitor) or SP6000125 (Jun N-terminal kinase inhibitor). The data suggest that upregulation of T-cad on EC during ER stress attenuates the activation of the proapoptotic PERK (PKR (double-stranded RNA-activated protein kinase)-like ER kinase) branch of the UPR cascade and thereby protects EC from ER stress-induced apoptosis., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Lipases have been implicated to be of importance in the life cycle development, virulence, and transmission of a variety of parasitic organisms. Potential functions include the acquisition of host resources for energy metabolism and as simple building blocks for the synthesis of complex parasite lipids important for membrane remodeling and structural purposes. Using a molecular approach, we identified and characterized the structure of an LdLip3-lipase gene from the primitive trypanosomatid pathogen of humans, Leishmania donovani. The LdLip3 encodes a approximately 33 kDa protein, with a well-conserved substrate-binding and catalytic domains characteristic of members of the serine lipase-protein family. Further, we showed that LdLip3 mRNA is constitutively expressed by both the insect vector (i.e., promastigote) and mammalian (i.e., amastigote) life cycle developmental forms of this protozoan parasite. Moreover, a homologous episomal expression system was used to express an HA epitope-tagged LdLip3 chimeric construct (LdLip3::HA) in these parasites. Expression of the LdLip3 chimera was verified in these transfectants by Western blots and indirect immuno-fluorescence analyses. Results of coupled immuno-affinity purification and enzyme activity experiments demonstrated that the LdLip3::HA chimeric protein was secreted/released by transfected L. donovani parasites and that it possessed functional lipase enzyme activity. Taken together these observations suggest that this novel secretory lipase might play essential role(s) in the survival, growth, and development of this important group of human pathogens.

Neutrophil interaction with activated endothelial cells (EC) is required for transmigration. We examined consequences of this interaction on NETosis. Co-culture of activated EC with neutrophils induced neutrophil extracellular trap (NET) formation, which was partially dependent on production of IL-8 by activated EC. Extended neutophil/EC co-culture resulted in EC damage, which could be abrogated by inclusion of either diphenyleneiodonium to inhibit the NAPDH oxidase pathway required for NETosis, or DNAse to disrupt NETs. These findings offer new insight into mechanisms whereby NETs trigger damage to the endothelium in sepsis, small vessel vasculitis and possibly the villous trophoblast in preeclampsia., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

T-cadherin (T-cad) promotes survival, proliferation, and migration of endothelial cells and induces angiogenesis. We aimed to identify domains of T-cad functionally relevant to its effects on endothelial cell behavior. To specifically target the functional properties of the 5 cadherin repeat domains (EC1-EC5) of T-cad, endothelial cells were transduced with lentivectors containing specific T-cad-domain-deletion mutant constructs (DeltaI, DeltaII, DeltaIII, DeltaIV, DeltaV). Empty (E) lentivector-transduced cells served as control. Similarly to overexpression of native T-cad, cells expressing DeltaII, DeltaIII, or DeltaIV displayed elevated levels of p-Akt and p-GSK3beta and increased proliferation rates (for DeltaII, DeltaIII) vs. E. DeltaI- and DeltaV-transduced cells exhibited reduced levels of p-Akt and p-GSK3beta and retarded growth rates vs. E. Stimulatory effects of native T-cad overexpression on Akt and GSK3beta phosphorylation were dose dependently inhibited by coexpression of DeltaI or DeltaV. Subsequent functional analyses compared only DeltaI-, DeltaII-, and DeltaV-mutant constructs with E as a negative control. Unlike DeltaII cells, DeltaI and DeltaV cells failed to exhibit homophilic ligation and deadhesion responses on a substratum of T-cad protein. In the wound assay, migration was increased for DeltaII cells but impaired for DeltaI and DeltaV cells. In endothelial cell-spheroid assay, angiogenic sprouting was augmented for DeltaII cells but inhibited for DeltaI and DeltaV cells. We conclude that EC1 and EC5 domains of T-cad are essential for its proangiogenic effects. DeltaI and DeltaV constructs may serve as dominant-negative mutants and as potential tools targeting excessive angiogenesis.

Cadherins are a superfamily of transmembrane proteins that mediate calcium-dependent intercellular adhesion. T-cadherin (T-cad, H-cadherin or cadherin-13) is an atypical member, lacking transmembrane and cytosolic domains and possessing a glycosylphosphatidylinositol moiety that anchors T-cadherin to the plasma membrane. This article reviews current knowledge on the biomolecular characteristics of T-cadherin,its expression and function in different tissues in health and disease and its mechanisms of signal transduction. The structural characteristics of T-cadherin protein predict that it is unlikely to function as a"true" adhesion molecule in vivo. Studies from different fields suggest that it may act rather as a signalling receptor participating in recognition of the environment and regulation of cell motility, proliferation and phenotype. Cellular expression levels of T-cadherin in various tissues frequently correlate (be it negatively or positively) with the proliferative potential of the cells. Loss- and gain-of-function studies demonstrate the ability of T-cadherin to modulate cell motility and growth. Gathering evidence suggests that the "functional predestination" of T-cadherin is in control of tissue architecture through "guiding" navigation of moving structures, segregating functional tissue compartments and "guarding" integrity of functionally connected tissue layers.

T-cad (T-cadherin), a glycosylphosphatidylinositol-anchored cadherin superfamily member, is expressed widely in the brain and cardiovascular system, and absent, decreased, or even increased, in cancers. Mechanisms controlling T-cad expression are poorly understood. The present study investigated transcriptional regulation of T-cad in ECs (endothelial cells). Conditions of oxidative stress (serum-deprivation or presence of H(2)O(2)) elevate T-cad mRNA and protein levels in ECs. Reporter gene analysis, using serially deleted T-cad promoter stretches ranging from -99 to -2304 bp, located the minimal promoter region of T-cad within -285 bp from the translation start site. Reporter activity in ECs transfected with the -285 bp construct increased under conditions of oxidative stress, and this was normalized by antioxidant N-acetylcysteine. An electrophoretic-mobility-shift assay revealed a specific nucleoprotein complex unique to -156 to -203 bp, which increased when nuclear extracts from oxidatively stressed ECs were used, suggesting the presence of redox-sensitive binding element(s). MS analysis of the nucleoprotein complex unique to -156 to -203 bp after streptavidin-agarose pull-down detected the presence of the redox-active protein thioredoxin. The presence of thioredoxin-1 in a nuclear extract from oxidatively stressed ECs was demonstrated after immunoprecipitation and immunoblotting. Transfection of ECs with thioredoxin-1 small interfering RNA abrogated oxidative-stress-induced up-regulation of T-cad transcripts and protein. We conclude that thioredoxin-1 is an important determinant of redox-sensitive transcriptional up-regulation of T-cad in ECs.

Chitinases of trypanosomatid parasites have been proposed to fulfil various roles in their blood-feeding arthropod vectors but so far none have been directly tested using a molecular approach. We characterized the ability of Leishmania mexicana episomally transfected with LmexCht1 (the L. mexicana chitinase gene) to survive and grow within the permissive sand fly vector, Lutzomyia longipalpis. Compared with control plasmid transfectants, the overexpression of chitinase was found to increase the average number of parasites per sand fly and accelerate the escape of parasites from the peritrophic matrix-enclosed blood meal as revealed by earlier arrival at the stomodeal valve. Such flies also exhibited increased damage to the structure of the stomodeal valve, which may facilitate transmission by regurgitation. When exposed individually to BALB/c mice, those flies with chitinase-overexpressing parasites spent on average 2.4-2.5 times longer in contact with their host during feeding, compared with flies with control infections. Furthermore, the lesions that resulted from these single fly bite infections were both significantly larger and with higher final parasite burdens than controls. These data show that chitinase is a multifunctional virulence factor for L. mexicana which assists its survival in Lu. longipalpis. Specifically, this enzyme enables the parasites to colonize the anterior midgut of the sand fly more quickly, modify the sand fly stomodeal valve and affect its blood feeding, all of which combine to enhance transmission.

There is scant knowledge regarding how cell surface lipid-anchored T-cadherin (T-cad) transmits signals through the plasma membrane to its intracellular targets. This study aimed to identify membrane proteins colocalizing with atypical glycosylphosphatidylinositol (GPI)-anchored T-cad on the surface of endothelial cells and to evaluate their role as signaling adaptors for T-cad. Application of coimmunoprecipitation from endothelial cells expressing c-myc-tagged T-cad and high-performance liquid chromatography revealed putative association of T-cad with the following proteins: glucose-related protein GRP78, GABA-A receptor alpha1 subunit, integrin beta3, and two hypothetical proteins, LOC124245 and FLJ32070. Association of Grp78 and integrin beta3 with T-cad on the cell surface was confirmed by surface biotinylation and reciprocal immunoprecipitation and by confocal microscopy. Use of anti-Grp78 blocking antibodies, Grp78 small interfering RNA, and coexpression of constitutively active Akt demonstrated an essential role for surface Grp78 in T-cad-dependent survival signal transduction via Akt in endothelial cells. The findings herein are relevant in the context of both the identification of transmembrane signaling partners for GPI-anchored T-cad as well as the demonstration of a novel mechanism whereby Grp78 can influence endothelial cell survival as a cell surface signaling receptor rather than an intracellular chaperone.

Background: The domestic goat is one of the important livestock species of India. In the present study we assess genetic diversity of Indian goats using 17 microsatellite markers. Breeds were sampled from their natural habitat, covering different agroclimatic zones., Results: The mean number of alleles per locus (NA) ranged from 8.1 in Barbari to 9.7 in Jakhrana goats. The mean expected heterozygosity (He) ranged from 0.739 in Barbari to 0.783 in Jakhrana goats. Deviations from Hardy-Weinberg Equilibrium (HWE) were statistically significant (P < 0.05) for 5 loci breed combinations. The DA measure of genetic distance between pairs of breeds indicated that the lowest distance was between Marwari and Sirohi (0.135). The highest distance was between Pashmina and Black Bengal. An analysis of molecular variance indicated that 6.59% of variance exists among the Indian goat breeds. Both a phylogenetic tree and Principal Component Analysis showed the distribution of breeds in two major clusters with respect to their geographic distribution., Conclusion: Our study concludes that Indian goat populations can be classified into distinct genetic groups or breeds based on the microsatellites as well as mtDNA information.

The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients. In this study, we identified two unique 28-kDa, dithiothreitol-sensitive nucleases and showed that they are constitutively released/secreted by parasites during axenic culture. Using several different molecular approaches, we identified and characterized the structure of EhNucI and EhNucII, genes that encode ribonuclease T2 family proteins. Homologous episomal expression of epitope-tagged EhNucI and EhNucII chimeric constructs was used to define the functional and biochemical properties of these released/secreted enzymes. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that these "secretory" enzymes could hydrolyze a variety of synthetic polynucleotides, as well as the natural nucleic acid substrate RNA. Furthermore, our results demonstrated that sera from acutely infected amebiasis patients recognized and immunoprecipitated these parasite secretory enzymes. Based on these observations, we hypothesize that within its host, these secretory nucleases could function, at a distance away from the parasite, to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine and pyrimidine requirements of these organisms. Thus, these enzymes might play an important role in facilitating the survival, growth, and development of this important human pathogen.

Here, we show that Crithidia luciliae, a primitive trypanosomatid, purine auxotroph, up-expressed its unique, bi-functional, surface membrane 3'-nucleotidase/nuclease (Cl 3'NT/NU) activity by approximately 1000-fold in response to purine starvation. A second surface membrane phospho-monoesterase, i.e. a tartrate-resistant acid phosphatase (Cl MAcP) was also found to be up-expressed in such purine-starved cells. Here, we used homologous episomal-expression of an antisense construct of the Cl3'NT/NU to dissect the functional expression of these two surface membrane enzymes. In antisense transfected cells, a large excess of the antisense transcript was produced and no trace of any endogenous Cl3'NT/NU sense message was detected. Further, the purine-starvation hyper-induced levels of 3'NT/NU enzyme activity were completely abrogated in these transfected cells versus controls. Moreover, such antisense transcription completely abolished the ability of these transfectants to grow in poly(A)-containing medium demonstrating the essential nature of the 3'NT/NU for the growth/survival of this parasite. In contrast, antisense transcription had no apparent deleterious effects on either endogenous or purine-starvation-induced levels of MAcP enzyme activity, its steady-state mRNA levels, or the constitutive expression of house-keeping genes (e.g. Cl alpha-tubulin) in these transfectants. Cumulatively, results of our antisense experiments demonstrated that the functional nuclease activity of the surface membrane Cl 3'NT/NU was, in fact, critical/essential for the growth and development of these primitive parasites.

Glycosylphosphatidylinositol-anchored T-cadherin (T-cad) influences several parameters of angiogenesis including endothelial cell (EC) differentiation, migration, proliferation, and survival. This presupposes signal transduction networking via mediatory regulators and molecular adaptors since T-cad lacks transmembrane and cytosolic domains. Here, using pharmacological inhibition of PI3K, adenoviral-mediated T-cad-overexpression, siRNA-mediated T-cad-depletion, and agonistic antibody-mediated ligation, we demonstrate signaling by T-cad through PI3K-Akt-GSK3beta pathways in EC. T-cad-overexpressing EC exhibited increased levels and nuclear accumulation of active beta-catenin, which was transcriptionally active as shown by increased Lef/Tcf reporter activity and cyclin D1 levels. Cotransduction of EC with constitutively active GSK3beta (S9A-GSK3beta) abrogated the stimulatory effects of T-cad on active beta-catenin accumulation, proliferation, and survival. Integrin-linked kinase (ILK), a membrane proximal upstream regulator of Akt and GSK3beta, was considered a candidate signaling mediator for T-cad. T-cad was present in anti-ILK immunoprecipitates, and confocal microscopy revealed colocalization of T-cad and ILK within lamellipodia of migrating cells. ILK-siRNA abolished T-cad-dependent effects on (Ser-473)Akt/(Ser-9)GSK3beta phosphorylation, active beta-catenin accumulation, and survival. We conclude ILK is an essential mediator for T-cad signaling via Akt and GSK3beta in EC. This is the first demonstration that ILK can regulate inward signaling by GPI-anchored proteins. Furthermore, ILK-GSK3beta-dependent modulation of active beta-catenin levels by GPI-anchored T-cad represents a novel mechanism for controlling cellular beta-catenin activity.

This study addresses establishment of an "in vitro" melanoma angiogenesis model using multicellular tumor spheroids (MCTS) of differentiated (HBL) or undifferentiated (NA8) melanoma cell lines. DNA microarray assay and qRT-PCR indicated upregulation of pro-angiogenic factors IL-8, VEGF, Ephrin A1 and ANGPTL4 in NA8-MCTSs (vs. monolayers) whereas these were absent in MCTS and monolayer cultures of HBL. Upon co-culture with endothelial cell line HMEC-1 NA8-MCTS attract, whereas HBL-MCTS repulse, HMEC-1. Overexpression of T-cadherin in HMEC-1 leads to their increased invasion and network formation within NA8-MCTS. Given an appropriate angiogenic tumor microenvironment, T-cadherin upregulation on endothelial cells may potentiate intratumoral angiogenesis.

The primitive protozoan pathogen of humans, Leishmania donovani, resides and multiplies in highly restricted micro-environments within their hosts (i.e. as promastigotes in the gut lumen of their sandfly vectors and as amastigotes in the phagolysosomal compartments of infected mammalian macrophages). Like other trypanosomatid parasites, they are purine auxotrophs (i.e. lack the ability to synthesize purines de novo) and therefore are totally dependent upon salvaging these essential nutrients from their hosts. In that context, in this study we identified a unique 35-kDa, dithiothreitol-sensitive nuclease and showed that it was constitutively released/secreted by both promastigote and amastigote developmental forms of this parasite. By using several different molecular approaches, we identified and characterized the structure of LdNuc(s), a gene that encodes this new 35-kDa class I nuclease family member in these organisms. Homologous episomal expression of an epitope-tagged LdNuc(s) chimeric construct was used in conjunction with an anti-LdNuc(s) peptide antibody to delineate the functional and biochemical properties of this unique 35-kDa parasite released/secreted enzyme. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that this "secretory" enzyme could hydrolyze a variety of synthetic polynucleotides as well as several natural nucleic acid substrates, including RNA and single- and double-stranded DNA. Based on these cumulative observations, we hypothesize that within the micro-environments of its host, this leishmanial "secretory" nuclease could function at a distance away from the parasite to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine requirements of these organisms. Thus, this enzyme might play an important role(s) in facilitating the survival, growth, and development of this important human pathogen.

Background: Early stage lung cancer has a variable prognosis, and there are currently no markers that predict which patients will recur. This study examined the relation between tumor-regulatory T (Treg) cells and total tumor-infiltrating T-cell lymphocytes (TIL) to determine whether they correlated with recurrence., Methods: The authors reviewed all patients in our tissue databank from 1996 to 2001 and identified 64 consecutive pathologic stage I non-small cell lung cancer (NSCLC) patients who had surgical resection and at least a 2.5 years disease-free follow-up or documented recurrence within 2 years. Immunohistochemical analyses were performed on paraffin-embedded lung cancer tissue and the relation between Treg cells, TIL, and disease-specific survival was determined. A risk index was devised deductively for various possible combinations of Treg cells and TIL., Results: Treg cells and TIL were detected in 33 of 64 (51%) and 53 of 64 (83%) patients, respectively. When data were analyzed by using a Treg/TIL Combination Risk Index, patients with high-risk and intermediate-risk indices had hazard ratios of 8.2 (P = .007) and 3.3 (P = .109), respectively., Conclusions: Patients with stage I NSCLC who have a higher proportion of tumor Treg cells relative to TIL had a significantly higher risk of recurrence. These data may be useful, particularly if combined with a panel of tumor markers, to suggest at the time of diagnosis which patients with seemingly early-stage NSCLC will relapse., (Copyright 2006 American Cancer Society.)

We analyzed the complete mitochondrial genome of a 3-month-old female child with basal ganglionic lesions and other clinical features suggestive of Leigh syndrome, which is caused by variations in mitochondrial and nuclear genes. Our study revealed a novel, homoplasmic T11984C missense mutation in ND4 gene, which replaces a highly conserved amino acid tyrosine with histidine. Computational analysis showed that this mutation alters the secondary structure of ND4 subunit. As the mutation observed in this study was novel and homoplasmic, we speculate that there could be interplay of this mitochondrial mutation along with nuclear gene(s) in the pathogenesis.

Background: Patients with early stage nonsmall-cell lung cancer who have undergone complete resection have a recurrence rate of approximately 50%, predominately due to the development of systemic metastases. This study is a prospective analysis of the expression of seven serum protein markers of invasion and metastasis, collected preoperatively (baseline) and serially after resection, to determine the relationship between marker expression and recurrence., Methods: Serum was collected from 196 patients with clinical stage I nonsmall-cell lung cancer who underwent resection over a 5-year period (1996 to 2000). Samples were drawn before resection and 1, 4, 6, 12, 18, and 24 months postoperatively. All patients were followed for at least 24 months or until death. Serum protein levels of vascular endothelial growth factor, hepatocyte growth factor), E-selectin, CD44, basic fibroblast growth factor, urokinase plasminogen activator, and urokinase plasminogen activator receptor were determined using enzyme-linked immunosorbent assay., Results: To date, 73 patients (37%) have demonstrated recurrence. Baseline levels of only 1 marker (CD44) correlated with pathologic stage (p = 0.02). Analysis of the serial samples demonstrated that recurrence was predicted (before clinical or radiographic determination) by decreasing levels of E-selectin (p = 0.002), increasing levels of CD44 (p = 0.001), and increasing levels of urokinase plasminogen activator receptor (p = 0.03)., Conclusions: This study demonstrates the potential to predict recurrence after resection in patients with early-stage nonsmall-cell lung cancer using a panel of serum protein markers. Early identification of patients with recurrence may improve the efficacy of systemic therapy.

The development of an oncogenic state is a complex process involving the accumulation of multiple independent mutations that lead to deregulation of cell signalling pathways central to the control of cell growth and cell fate. The ability to define cancer subtypes, recurrence of disease and response to specific therapies using DNA microarray-based gene expression signatures has been demonstrated in multiple studies. Various studies have also demonstrated the potential for using gene expression profiles for the analysis of oncogenic pathways. Here we show that gene expression signatures can be identified that reflect the activation status of several oncogenic pathways. When evaluated in several large collections of human cancers, these gene expression signatures identify patterns of pathway deregulation in tumours and clinically relevant associations with disease outcomes. Combining signature-based predictions across several pathways identifies coordinated patterns of pathway deregulation that distinguish between specific cancers and tumour subtypes. Clustering tumours based on pathway signatures further defines prognosis in respective patient subsets, demonstrating that patterns of oncogenic pathway deregulation underlie the development of the oncogenic phenotype and reflect the biology and outcome of specific cancers. Predictions of pathway deregulation in cancer cell lines are also shown to predict the sensitivity to therapeutic agents that target components of the pathway. Linking pathway deregulation with sensitivity to therapeutics that target components of the pathway provides an opportunity to make use of these oncogenic pathway signatures to guide the use of targeted therapeutics.

We characterized a cDNA from Phlebotomus papatasi, PpChit1, which encodes a midgut specific chitinase and show the presence of a functional, blood-induced chitinolytic system in sand flies. PpChit1 is detected only in the midgut and is regulated by blood feeding. A recombinant protein (rPpChit1) produced in HEK 293-F cells exhibited a similar activity profile to that found in the native protein against several specific substrates, including an oligomeric glycol chitin and synthetic 4-methyl-umbelliferone labelled substrates. Western blotting showed that the native protein is recognized by mouse polyclonal antibodies against rPpChit1. Additionally, the rPpChit1 and the native chitinase displayed similar retention times in a HPLC size fractionation column. When added to rPpChit1 or to midgut lysates, PpChit1 sera reduced chitinolytic activity by 65-70%.

In vascular tissue, T-cadherin (T-cad) is up-regulated in vivo under disease conditions associated with oxidative stress and concomitant cell migration, proliferation and apoptosis/survival. Using cultures of human umbilical vein endothelial cells (HUVEC), we examined whether there is a functional relationship between oxidative stress, T-cad expression, and cell survival status. Culture of HUVEC under conditions of oxidative stress (e.g., serum deprivation, inclusion of H2O2) resulted in increased T-cad expression. Oxidative stress-induced increases in T-cad were inhibited by the free radical-scavenging antioxidant, N-acetylcysteine, and the flavin-containing oxidase inhibitor, diphenyleneiodonium. Thus reactive oxygen species (ROS) contribute to stress-induced elevation of T-cad in HUVEC. Compared with control cells, HUVEC overexpressing T-cad (T-cad+-HUVEC) had higher phosphorylation levels for phosphatidylinositol 3-kinase (PI3K) target Akt and mTOR target p70(S6K) (survival pathway regulators), but lower levels for p38MAPK (death pathway regulator). T-cad+-HUVEC exposed to stress (serum-deprivation, TNF-alpha, actinomycin D, staurosporine) exhibited reduced caspase activation together with increased cell survival. Protection against stress-induced apoptosis in T-cad+-HUVEC was abrogated by either PI3K-inhibitor wortmannin or mTOR-inhibitor rapamycin. We conclude that T-cad overexpression in HUVEC protects against stress-induced apoptosis through activation of the PI3K/Akt/mTOR survival signal pathway and concomitant suppression of the p38 MAPK proapoptotic pathway. ROS-induced changes in T-cad expression may play an important role in controlling tissue cellularity during vascular remodeling.

Purpose: To assess the relationship between molecular markers associated with chemotherapy resistance and survival in esophageal cancer patients treated with trimodality therapy., Experimental Design: The original pretreatment formalin-fixed, paraffin-embedded endoscopic esophageal tumor biopsy material was obtained from 99 patients treated with concurrent cisplatin plus 5-fluorouracil plus 45 Gy radiation followed by resection at Duke University Medical Center (Durham, NC) from 1986 to 1997. cDNA was derived from the biopsy and analyzed to determine mRNA expression relative to an internal reference gene (beta-actin) using fluorescence-based, real-time reverse transcription-PCR. Possible markers of platinum chemotherapy association [glutathione S-transferase pi (GSTP1) and excision cross-complementing gene 1 (ERCC1)] and 5-fluorouracil association [thymidylate synthase 1 (TS1)] were measured., Results: Cox proportional hazards model revealed a significant inverse, linear effect for TS1 with respect to survival (P = 0.007). An inverse relationship between TS1 expression and treatment response was also detected (P < or = 0.001). Univariate analysis identified an association with decreased survival for GSTP1 > or = 3.0 (P = 0.05). In multivariate analyses, TS1 >6.0, ERCC1 >3, and GSTP1 >3 were statistically significant predictors of decreased survival (P = 0.007). Additionally, the presence of ERCC1 >3.0 or TS1 >6.0 was associated with an approximately 2-fold increase in the risk of cancer recurrence (P = 0.086 and 0.003, respectively)., Conclusion: The measurement of relative gene expression of molecular markers associated with chemoresistance in endoscopic esophageal tumor biopsies may be a useful tool in assessing outcome in patients with trimodality-treated esophageal cancer. These data should be validated further in larger prospective studies.