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1.  Comparative study on production of a-Amylase from Bacillus licheniformis strains 
Brazilian Journal of Microbiology  2011;42(4):1397-1404.
Alpha amylase (α-1, 4-glucan-glucanhydrolase, EC, an extracellular enzyme, degrades α, 1–4 glucosidic linkages of starch and related substrates in an endo-fashion producing oligosaccharides including maltose, glucose and alpha limit dextrin (7). The present study deals with the production and comparative study of production of α-amylase from two strains of Bacillus licheniformis, MTCC 2617 and 2618, by using four different substrates, starch, rice, wheat and ragi powder as carbon source by submerged fermentation. The effect of varying pH and incubation temperature, activator, inhibitor, and substrate concentration was investigated on the activity of α-amylase produced by MTCC strain 2618. The results shows that the production of the α-amylase by the B.licheniformis strain MTCC 2618, using four different substrates were found to be maximum (Starch 3.64 IU/ml/minutes, Rice powder 2.93 IU/ml/minutes, Wheat powder 2.67 IU/ml/minutes, Ragi powder 2.36 IU/ml/minutes) on comparing the enzyme production of two strains. It was also observed that the maximum production was found on the 3rd day (i.e. 72 hr) and characterization of crude enzyme revealed that optimum activity was at pH 7 and 37°C.
PMCID: PMC3768710  PMID: 24031769
amylase; Bacillus licheniformis; enzyme; extracellular; glucose
2.  NFAT and CREB Regulate Kaposi's Sarcoma-Associated Herpesvirus-Induced Cyclooxygenase 2 (COX-2)▿ † 
Journal of Virology  2010;84(24):12733-12753.
COX-2 has been implicated in Kaposi's sarcoma-associated herpesvirus (KSHV) latency and pathogenesis (A. George Paul, N. Sharma-Walia, N. Kerur, C. White, and B. Chandran, Cancer Res. 70:3697-3708, 2010; P. P. Naranatt, H. H. Krishnan, S. R. Svojanovsky, C. Bloomer, S. Mathur, and B. Chandran, Cancer Res. 64:72-84, 2004; N. Sharma-Walia, A. G. Paul, V. Bottero, S. Sadagopan, M. V. Veettil, N. Kerur, and B. Chandran, PLoS Pathog. 6:e1000777, 2010; N. Sharma-Walia, H. Raghu, S. Sadagopan, R. Sivakumar, M. V. Veettil, P. P. Naranatt, M. M. Smith, and B. Chandran, J. Virol. 80:6534-6552, 2006). However, the precise regulatory mechanisms involved in COX-2 induction during KSHV infection have never been explored. Here, we identified cis-acting elements involved in the transcriptional regulation of COX-2 upon KSHV de novo infection. Promoter analysis using human COX-2 promoter deletion and mutation reporter constructs revealed that nuclear factor of activated T cells (NFAT) and the cyclic AMP (cAMP) response element (CRE) modulate KSHV-mediated transcriptional regulation of COX-2. Along with multiple KSHV-induced signaling pathways, infection-induced prostaglandin E2 (PGE2) also augmented COX-2 transcription. Infection of endothelial cells markedly induced COX-2 expression via a cyclosporine A-sensitive, calcineurin/NFAT-dependent pathway. KSHV infection increased intracellular cAMP levels and activated protein kinase A (PKA), which phosphorylated the CRE-binding protein (CREB) at serine 133, which probably led to interaction with CRE in the COX-2 promoter, thereby enhancing COX-2 transcription. PKA selective inhibitor H-89 pretreatment strongly inhibited CREB serine 133, indicating the involvement of a cAMP-PKA-CREB-CRE loop in COX-2 transcriptional regulation. In contrast to phosphatidylinositol 3-kinase and protein kinase C, inhibition of FAK and Src effectively reduced KSHV infection-induced COX-2 transcription and protein levels. Collectively, our study indicates that mediation of COX-2 transcription upon KSHV infection is a paradigm of a complex regulatory milieu involving the interplay of multiple signal cascades and transcription factors. Intervention at each step of COX-2/PGE2 induction can be used as a potential therapeutic target to treat KSHV-associated neoplasm and control inflammatory sequels of KSHV infection.
PMCID: PMC3004349  PMID: 20943963
4.  Gene expression analysis of novel genes in the prefrontal cortex of major depressive disorder subjects 
Dysregulation of the glutamatergic system has been implicated not only in the treatment of major depressive disorder (MDD), but also in the excitotoxic effects of stress and anxiety on the prefrontal cortex, which may precede the onset of a depressive episode. Our previous studies demonstrate marked deficits in prominent postsynaptic proteins involved in glutamate neurotransmission in the prefrontal cortex (PFC), Brodmann’s area 10 (BA 10) from subjects diagnosed with major depressive disorder (MDD). In the same group of subjects we have identified deficits in expression and phosphorylation level of key components of the mammalian target of rapamycin (mTOR) signalling pathway, known to regulate translation initiation. Based on our previous findings, we have postulated that glutamate-dependent dysregulation of mTOR- initiated protein synthesis in the PFC may underlie the pathology of MDD. The aim of this study was to use the NanoString nCounter System to perform analysis of genes coding for glutamate transporters, glutamate metabolizing enzymes, neurotrophic factors and other intracellular signaling markers involved in glutamate signaling that were not previously investigated by our group in the PFC BA10 from subjects with MDD. We have analyzed a total of 200 genes from 16 subjects with MDD and 16 healthy controls. These are part of the same cohort used in our previous studies. Setting our cutoff p-value ≤ 0.01, marked upregulation of genes coding for mitochondrial glutamate carrier (GC1; p=0.0015), neuropilin 1 (NRP-1; p=0.0019), glutamate receptor ionotropic N-methyl-D-aspartate-associated protein 1 (GRINA; p=0.0060), and fibroblast growth factor receptor 1 (FGFR-1; p=0.010) was identified. No significant differences in expression of the remaining 196 genes were observed between MDD subjects and controls. While upregulation of FGFR-1 has been previously shown in MDD; abnormalities in GC-1, GRINA, and NRP-1 have not been reported. Therefore, this postmortem study identifies GC1, GRINA, and NRP-1 as novel factors associated with MDD; however, future studies will be needed to address the significance of these genes in the pathophysiology of depression and antidepressant activity.
PMCID: PMC4089971  PMID: 23261523
prefrontal cortex; major depressive disorder; postmortem; gene expression; digital PCR
5.  Liposomal nanoparticles encapsulating iloprost exhibit enhanced vasodilation in pulmonary arteries 
Prostacyclin analogues are standard therapeutic options for vasoconstrictive diseases, including pulmonary hypertension and Raynaud’s phenomenon. Although effective, these treatment strategies are expensive and have several side effects. To improve drug efficiency, we tested liposomal nanoparticles as carrier systems. In this study, we synthesized liposomal nanoparticles tailored for the prostacyclin analogue iloprost and evaluated their pharmacologic efficacy on mouse intrapulmonary arteries, using a wire myograph. The use of cationic lipids, stearylamine, or 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP) in liposomes promoted iloprost encapsulation to at least 50%. The addition of cholesterol modestly reduced iloprost encapsulation. The liposomal nanoparticle formulations were tested for toxicity and pharmacologic efficacy in vivo and ex vivo, respectively. The liposomes did not affect the viability of human pulmonary artery smooth muscle cells. Compared with an equivalent concentration of free iloprost, four out of the six polymer-coated liposomal formulations exhibited significantly enhanced vasodilation of mouse pulmonary arteries. Iloprost that was encapsulated in liposomes containing the polymer polyethylene glycol exhibited concentration-dependent relaxation of arteries. Strikingly, half the concentration of iloprost in liposomes elicited similar pharmacologic efficacy as nonencapsulated iloprost. Cationic liposomes can encapsulate iloprost with high efficacy and can serve as potential iloprost carriers to improve its therapeutic efficacy.
PMCID: PMC4094575  PMID: 25045260
prostacyclin; cationic liposomes; pulmonary hypertension; wire myograph
Cancer causes & control : CCC  2013;24(7):1427-1436.
Consumption of foods high in sugar promotes insulin production, which has been linked to endometrial carcinogenesis. We evaluated the impact of dietary intake of sugary foods and beverages, as well as added sugar and total sugar on endometrial cancer risk in a population-based case-control study, including 424 cases and 398 controls. Participants completed an interview and food frequency questionnaire, and provided self-recorded waist and hip measurements. Women in the highest quartile of added sugar intake had significantly increased endometrial cancer risk (OR=1.84, 95% CI: 1.16–2.92). Among women with waist-to-hip ratio ≥0.85, risk was significantly higher for the highest vs. lowest tertile of added sugar intakes (OR=2.50, 95% CI: 1.38–4.52). The association with added sugar also became stronger when analyses were restricted to never users of hormone replacement therapy (OR: 2.03; 95% CI: 1.27–3.26, for highest vs. lowest tertile). There was little evidence of effect modification by body mass index or physical activity. Given the high prevalence of intake of sugary foods and drinks in Western populations, additional research is warranted to confirm our findings on endometrial cancer.
PMCID: PMC3683350  PMID: 23657460
Endometrial neoplasms; diet; sugar; sugary foods; sugary drinks; added sugars
7.  Self-assembled liposomal nanoparticles in photodynamic therapy 
European journal of nanomedicine  2013;5(3):10.1515/ejnm-2013-0010.
Photodynamic therapy (PDT) employs the combination of non-toxic photosensitizers (PS) together with harmless visible light of the appropriate wavelength to produce reactive oxygen species that kill unwanted cells. Because many PS are hydrophobic molecules prone to aggregation, numerous drug delivery vehicles have been tested to solubilize these molecules, render them biocompatible and enhance the ease of administration after intravenous injection. The recent rise in nanotechnology has markedly expanded the range of these nanoparticulate delivery vehicles beyond the well-established liposomes and micelles. Self-assembled nanoparticles are formed by judicious choice of monomer building blocks that spontaneously form a well-oriented 3-dimensional structure that incorporates the PS when subjected to the appropriate conditions. This self-assembly process is governed by a subtle interplay of forces on the molecular level. This review will cover the state of the art in the preparation and use of self-assembled liposomal nanoparticles within the context of PDT.
PMCID: PMC3857307  PMID: 24348377
liposomes; nanoparticles; nanotechnology; photodynamic therapy; self-assembly
9.  Population Distribution Analyses Reveal a Hierarchy of Molecular Players Underlying Parallel Endocytic Pathways 
PLoS ONE  2014;9(6):e100554.
Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis.
PMCID: PMC4074053  PMID: 24971745
10.  A Proteolytic Cascade Controls Lysosome Rupture and Necrotic Cell Death Mediated by Lysosome-Destabilizing Adjuvants 
PLoS ONE  2014;9(6):e95032.
Recent studies have linked necrotic cell death and proteolysis of inflammatory proteins to the adaptive immune response mediated by the lysosome-destabilizing adjuvants, alum and Leu-Leu-OMe (LLOMe). However, the mechanism by which lysosome-destabilizing agents trigger necrosis and proteolysis of inflammatory proteins is poorly understood. The proteasome is a cellular complex that has been shown to regulate both necrotic cell death and proteolysis of inflammatory proteins. We found that the peptide aldehyde proteasome inhibitors, MG115 and MG132, block lysosome rupture, degradation of inflammatory proteins and necrotic cell death mediated by the lysosome-destabilizing peptide LLOMe. However, non-aldehyde proteasome inhibitors failed to prevent LLOMe-induced cell death suggesting that aldehyde proteasome inhibitors triggered a pleotropic effect. We have previously shown that cathepsin C controls lysosome rupture, necrotic cell death and the adaptive immune response mediated by LLOMe. Using recombinant cathepsin C, we found that aldehyde proteasome inhibitors directly block cathepsin C, which presumably prevents LLOMe toxicity. The cathepsin B inhibitor CA-074-Me also blocks lysosome rupture and necrotic cell death mediated by a wide range of necrosis inducers, including LLOMe. Using cathepsin-deficient cells and recombinant cathepsins, we demonstrate that the cathepsins B and C are not required for the CA-074-Me block of necrotic cell death. Taken together, our findings demonstrate that lysosome-destabilizing adjuvants trigger an early proteolytic cascade, involving cathepsin C and a CA-074-Me-dependent protease. Identification of these early events leading to lysosome rupture will be crucial in our understanding of processes controlling necrotic cell death and immune responses mediated by lysosome-destabilizing adjuvants.
PMCID: PMC4043491  PMID: 24893007
Archives of virology  2013;158(6):1425-1432.
The International Committee on Taxonomy of Viruses (ICTV) organizes the classification of viruses into taxa, but is not responsible for the nomenclature for taxa members. International experts groups, such as the ICTV Study Groups, recommend the classification and naming of viruses and their strains, variants, and isolates. The ICTV Filoviridae Study Group has recently introduced an updated classification and nomenclature for filoviruses. Subsequently, and together with numerous other filovirus experts, a consistent nomenclature for their natural genetic variants and isolates was developed that aims at simplifying the retrieval of sequence data from electronic databases. This is a first important step toward a viral genome annotation standard as sought by the US National Center for Biotechnology Information (NCBI). Here, this work is extended to include filoviruses obtained in the laboratory by artificial selection through passage in laboratory hosts. The previously developed template for natural filovirus genetic variant naming ( ///-) is retained, but it is proposed to adapt the type of information added to each field for laboratory animal-adapted variants. For instance, the full-length designation of an Ebola virus Mayinga variant adapted at the State Research Center for Virology and Biotechnology “Vector” to cause disease in guinea pigs after seven passages would be akin to “Ebola virus VECTOR/C.porcellus-lab/COD/1976/Mayinga-GPA-P7”. As was proposed for the names of natural filovirus variants, we suggest using the full-length designation in databases, as well as in the method section of publications. Shortened designations (such as “EBOV VECTOR/C.por/COD/76/May-GPA-P7”) and abbreviations (such as “EBOV/May-GPA-P7”) could be used in the remainder of the text depending on how critical it is to convey information contained in the full-length name. “EBOV” would suffice if only one EBOV strain/variant/isolate is addressed.
PMCID: PMC3669655  PMID: 23358612
cuevavirus; Ebola; Ebola virus; ebolavirus; filovirid; Filoviridae; filovirus; genome annotation; ICTV; International Committee on Taxonomy of Viruses; Lloviu virus; Marburg virus; marburgvirus; mononegavirad; Mononegavirales; mononegavirus; virus classification; virus isolate; virus nomenclature; virus strain; virus taxonomy; virus variant
12.  Neural Precursor Cells Cultured at Physiologically Relevant Oxygen Tensions Have a Survival Advantage Following Transplantation 
Cell transplantation strategies typically introduce a stress challenge at the time of transplantation as the cells are switched from 20% to 3% O2 (the average in adult organs). This study found that culture of neural precursor cells (NPCs) at 3% rather than 20% O2 approximately doubles survival in the immediate post-transplantation phase. Furthermore, NPC fate was affected by culture at low, physiological O2 tensions (3%), with particularly marked effects on the oligodendrocyte lineage, both in vitro and in vivo. It is proposed that careful consideration of physiological oxygen environments, and particularly changes in oxygen tension, has relevance for the practical approaches to cellular therapies.
Traditionally, in vitro stem cell systems have used oxygen tensions that are far removed from the in vivo situation. This is particularly true for the central nervous system, where oxygen (O2) levels range from 8% at the pia to 0.5% in the midbrain, whereas cells are usually cultured in a 20% O2 environment. Cell transplantation strategies therefore typically introduce a stress challenge at the time of transplantation as the cells are switched from 20% to 3% O2 (the average in adult organs). We have modeled the oxygen stress that occurs during transplantation, demonstrating that in vitro transfer of neonatal rat cortical neural precursor cells (NPCs) from a 20% to a 3% O2 environment results in significant cell death, whereas maintenance at 3% O2 is protective. This survival benefit translates to the in vivo environment, where culture of NPCs at 3% rather than 20% O2 approximately doubles survival in the immediate post-transplantation phase. Furthermore, NPC fate is affected by culture at low, physiological O2 tensions (3%), with particularly marked effects on the oligodendrocyte lineage, both in vitro and in vivo. We propose that careful consideration of physiological oxygen environments, and particularly changes in oxygen tension, has relevance for the practical approaches to cellular therapies.
PMCID: PMC3673758  PMID: 23677643
Hypoxia; Neural stem cell; Cell transplantation; Oligodendrocytes
13.  A Hairy Tail not a Fairy Tale – Rapunzel Syndrome 
The Indian Journal of Surgery  2011;75(Suppl 1):80-81.
Rapunzel syndrome is an unusual and rare type of trichobezoar. Bezoars can be classified according to the primary constituent, as trichobezoar (hair), phytobezoar (plant material) or miscellaneous (pharmacobezoar, lactobezoar, fungal agglomeration and foreign bodies). When a long tail of hair strands extends from the main mass in the stomach along the small intestine and beyond it is known as Rapunzel syndrome. Here we are reporting a case of Rapunzel syndrome with a very long tail who was managed successfully. And reviewing the literature on the pathophysiology and management of these patients. These patients commonly present with obstructive symptoms and needs a high index of suspicion especially, in young female patients who have alopecia circumscripta and underlying psychiatric disorders. Early diagnosis and treatment is required to prevent complications due to this condition. Currently surgical management of this condition is the treatment of choice.
PMCID: PMC3693240  PMID: 24426521
Bezoars/complications; Bezoars/diagnosis; Bezoars/psychology; Bezoars/surgery; Female; Laparotomy
14.  Prevalence of Diarrhea-Associated Virulence Genes and Genetic Diversity in Escherichia coli Isolates from Fecal Material of Various Animal Hosts 
Applied and Environmental Microbiology  2013;79(23):7371-7380.
In order to assess the health risk associated with a given source of fecal contamination using bacterial source tracking (BST), it is important to know the occurrence of potential pathogens as a function of host. Escherichia coli isolates (n = 593) from the feces of diverse animals were screened for various virulence genes: stx1 and stx2 (Shiga toxin-producing E. coli [STEC]), eae and EAF (enteropathogenic E. coli [EPEC]), STh, STp, and LT (enterotoxigenic E. coli [ETEC]), and ipaH (enteroinvasive E. coli [EIEC]). Eleven hosts were positive for only the eae (10.11%) gene, representing atypical EPEC, while two hosts were positive for both eae and EAF (1.3%), representing typical EPEC. stx1, stx2, or both stx1 and stx2 were present in 1 (0.1%,) 10 (5.56%), and 2 (1.51%) hosts, respectively, and confirmed as non-O157 by using a E. coli O157 rfb (rfbO157) TaqMan assay. STh and STp were carried by 2 hosts (2.33%) and 1 host (0.33%), respectively, while none of the hosts were positive for LT and ipaH. The repetitive element palindromic PCR (rep-PCR) fingerprint analysis identified 221 unique fingerprints with a Shannon diversity index of 2.67. Multivariate analysis of variance revealed that majority of the isolates clustered according to the year of sampling. The higher prevalence of atypical EPEC and non-O157 STEC observed in different animal hosts indicates that they can be a reservoir of these pathogens with the potential to contaminate surface water and impact human health. Therefore, we suggest that E. coli from these sources must be included while constructing known source fingerprint libraries for tracking purposes. However, the observed genetic diversity and temporal variation need to be considered since these factors can influence the accuracy of BST results.
PMCID: PMC3837721  PMID: 24056456
15.  Production of recombinant antibodies using bacteriophages 
Recombinant antibody fragments such as Fab, scFv, diabodies, triabodies, single domain antibodies and minibodies have recently emerged as potential alternatives to monoclonal antibodies, which can be engineered using phage display technology. These antibodies match the strengths of conventionally produced monoclonal antibodies and offer advantages for the development of immunodiagnostic kits and assays. These fragments not only retain the specificity of the whole monoclonal antibodies but also easy to express and produce in prokaryotic expression system. Further, these antibody fragments are genetically stable, less expensive, easy to modify in response to viral mutations and safer than monoclonal antibodies for use in diagnostic and therapeutic applications. This review describes the potential of antibody fragments generated using phage display and their use as diagnostic reagents.
PMCID: PMC4029287  PMID: 24883194
antibody fragments; bacteriophage; diagnostics; phage display; therapeutics
16.  Evaluation of immune responses by live infectious bursal disease vaccines to avoid vaccination failures 
Infectious Bursal Disease (IBD) is a viral, contagious immunosuppressive disease posing an important threat to the commercial poultry industry. Evolution of highly virulent strains of IBD virus warranted the need for detailed characterization of the immune responses offered by the currently available vaccines. Two extensively used live vaccines of varied attenuation levels – intermediate and intermediate plus – strains were analyzed for the induction of immune responses. Both the vaccines induced protective antibody titers with the onset, quicker and higher with the intermediate plus vaccine. The intermediate plus strain vaccinate was observed to induce higher levels of IFN-γ in the birds. These results were supported by immunophenotype analyses with an increase in CD8+ and simultaneous decrease in CD4+ cell population. Both vaccine strains conferred protective immunity against virulent challenge. The study warrants the use of intermediate plus vaccines in disease endemic regions and intermediate vaccines in non-endemic regions to prevent IBD infection.
PMCID: PMC4029291  PMID: 24883198
IFN-γ; immunophenotyping; infectious bursal disease; live vaccines
17.  Peak Wall Stress Predicts Expansion Rate in Descending Thoracic Aortic Aneurysms 
The Annals of thoracic surgery  2012;95(2):593-598.
Aortic diseases, including aortic aneurysms, are the 12th leading cause of death in the United States. The incidence of descending thoracic aortic aneurysms is estimated at 10.4 per 100,000 patient-years. Growing evidence suggests that stress measurements derived from structural analysis of aortic geometries predict clinical outcomes better than diameter alone.
Twenty-five patients undergoing clinical and radiologic surveillance for thoracic aortic aneurysms were retrospectively identified. Custom MATLAB algorithms were employed to extract aortic wall and intraluminal thrombus geometry from computed tomography angiography scans. The resulting reconstructions were loaded with 120 mm Hg of pressure using finite element analysis. Relationships among peak wall stress, aneurysm growth, and clinical outcome were examined.
The average patient age was 71.6 ± 10.0 years, and average follow-up time was 17.5 ± 9 months (range, 6 to 43). The mean initial aneurysm diameter was 47.8 ± 8.0 mm, and the final diameter was 52.1 ± 10.0 mm. Mean aneurysm growth rate was 2.9 ± 2.4 mm per year. A stronger correlation (r = 0.894) was found between peak wall stress and aneurysm growth rate than between maximal aortic diameter and growth rate (r = 0.531). Aneurysms undergoing surgical intervention had higher peak wall stresses than aneurysms undergoing continued surveillance (300 ± 75 kPa versus 229 ± 47 kPa, p = 0.01).
Computational peak wall stress in thoracic aortic aneurysms was found to be strongly correlated with aneurysm expansion rate. Aneurysms requiring surgical intervention had significantly higher peak wall stresses. Peak wall stress may better predict clinical outcome than maximal aneurysmal diameter, and therefore may guide clinical decision-making.
PMCID: PMC4037886  PMID: 23245445
18.  Conformational Properties of Peptides Corresponding to the Ebolavirus GP2 Membrane-Proximal External Region in the Presence of Micelle-Forming Surfactants and Lipids 
Biochemistry  2013;52(20):10.1021/bi400040v.
Ebola virus and Sudan virus are members of the family Filoviridae of nonsegmented negative-strand RNA viruses (‘filoviruses’) that cause severe hemorrhagic fever with fatality rates as high as 90%. Infection by filoviruses requires membrane fusion between the host and the virus; this process is facilitated by the two subunits of the envelope glycoprotein, GP1 (the surface subunit) and GP2 (the transmembrane subunit). The membrane proximal external region (MPER) is a Trp-rich segment that immediately precedes the transmembrane domain of GP2. In the analogous glycoprotein for HIV-1 (gp41), the MPER is critical for membrane fusion and is the target of several neutralizing antibodies. However, the role of the MPER in filovirus GP2 and its importance in membrane fusion has not been established. Here, we characterize the conformational properties of peptides representing the GP MPER segments of Ebola virus and Sudan virus in the presence of micelle-forming surfactants and lipids, at pH 7 and pH 4.6. Circular dichroism (CD) spectroscopy and tryptophan fluorescence indicate that the GP2 MPER peptides bind to micelles of sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC). Nuclear magnetic resonance (NMR) spectroscopy of the Sudan virus MPER peptide revealed that the residues 644–651 interact directly with DPC, and that this interaction enhances helical conformation of the peptide. The Sudan virus MPER peptide was found to moderately inhibit cell entry by a GP-pseudotyped vesicular stomatitis virus, but did not induce leakage of a fluorescent molecule from large unilammellar vesicle comprised of 1-palmitoyl-2-oleoylphostatidyl choline (POPC) or cause hemolysis. Taken together, this analysis suggests the filovirus GP MPER binds and inserts shallowly into lipid membranes.
PMCID: PMC3772975  PMID: 23650881
19.  Interactions between Exosomes from Breast Cancer Cells and Primary Mammary Epithelial Cells Leads to Generation of Reactive Oxygen Species Which Induce DNA Damage Response, Stabilization of p53 and Autophagy in Epithelial Cells 
PLoS ONE  2014;9(5):e97580.
Exosomes are nanovesicles originating from multivesicular bodies and are released by all cell types. They contain proteins, lipids, microRNAs, mRNAs and DNA fragments, which act as mediators of intercellular communications by inducing phenotypic changes in recipient cells. Tumor-derived exosomes have been shown to play critical roles in different stages of tumor development and metastasis of almost all types of cancer. One of the ways by which exosomes affect tumorigenesis is to manipulate the tumor microenvironments to create tumor permissive “niches”. Whether breast cancer cell secreted exosomes manipulate epithelial cells of the mammary duct to facilitate tumor development is not known. To address whether and how breast cancer cell secreted exosomes manipulate ductal epithelial cells we studied the interactions between exosomes isolated from conditioned media of 3 different breast cancer cell lines (MDA-MB-231, T47DA18 and MCF7), representing three different types of breast carcinomas, and normal human primary mammary epithelial cells (HMECs). Our studies show that exosomes released by breast cancer cell lines are taken up by HMECs, resulting in the induction of reactive oxygen species (ROS) and autophagy. Inhibition of ROS by N-acetyl-L-cysteine (NAC) led to abrogation of autophagy. HMEC-exosome interactions also induced the phosphorylation of ATM, H2AX and Chk1 indicating the induction of DNA damage repair (DDR) responses. Under these conditions, phosphorylation of p53 at serine 15 was also observed. Both DDR responses and phosphorylation of p53 induced by HMEC-exosome interactions were also inhibited by NAC. Furthermore, exosome induced autophagic HMECs were found to release breast cancer cell growth promoting factors. Taken together, our results suggest novel mechanisms by which breast cancer cell secreted exosomes manipulate HMECs to create a tumor permissive microenvironment.
PMCID: PMC4022578  PMID: 24831807
20.  Neuroprotection by a mitochondria-targeted drug in a Parkinson’s disease model 
Free radical biology & medicine  2010;49(11):1674-1684.
The objective of this study was to assess the neuroprotective effects of a mitochondria-targeted antioxidant, Mito-Q10, the coenzyme-Q analog attached to a triphenylphosphonium cation that targets the antioxidant to mitochondria, in experimental models of Parkinson’s disease (PD). Primary mesencephalic neuronal cells and cultured dopaminergic cells were treated with 1-methyl-4-phenylpyridinium (MPP+), an active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and mice were used for testing the efficacy of Mito-Q10. MPP+ treatment caused a dose-dependent loss of tyrosine hydroxylase and membrane potential and an increase in caspase-3 activation in dopaminergic cells, which were reversed by Mito-Q10. MPTP treatment induced a loss of striatal dopamine and its metabolites, inactivation of mitochondrial aconitase in the substantia nigra, and a loss of locomotor activity in mice. Treatment with Mito-Q10 significantly inhibited both MPP+- and MPTP-induced neurotoxicity in cell culture and mouse models. Collectively, these results indicate that mitochondrial targeting of antioxidants is a promising neuroprotective strategy in this preclinical mouse model of PD.
PMCID: PMC4020411  PMID: 20828611
MPTP; MPP+; Mito-Q10; Co-Q; Parkinson’s disease; Oxidants; Oxidative stress; Free radicals
21.  Growth and Replication of Infectious Bursal Disease Virus in the DF-1 Cell Line and Chicken Embryo Fibroblasts 
BioMed Research International  2014;2014:494835.
Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. To determine a suitable cell line for IBDV infection, replication, and growth kinetics of the virus, DF-1 cells and chicken embryo fibroblasts (CEF) were used. The population doubling per day (Pd/D) was found to be higher in DF-1 as compared to CEF cells. A suitable time of infection (TOI) was established for increased production of virus and greater infectivity titers. The DF-1 and CEF cells were found to be susceptible to infection by producing marked cytopathic effects (CPEs), and the growth curves of IBDV in DF-1 and CEF cells were evaluated by infectivity assay using tissue culture infectious dose (TCID50). The cytopathic effects of the virus in DF-1 and CEF cells were found to be similar, but higher viral titers were detected in the DF-1 cells as compared to CEF. Thus the DF-1 cell line had a higher growth potential and infectivity, which will be of advantage in vaccine production.
PMCID: PMC4053150  PMID: 24949455
22.  Cigarette smoking and risk of ovarian cancer: a pooled analysis of 21 case–control studies 
Cancer causes & control : CCC  2013;24(5):10.1007/s10552-013-0174-4.
The majority of previous studies have observed an increased risk of mucinous ovarian tumors associated with cigarette smoking, but the association with other histological types is unclear. In a large pooled analysis, we examined the risk of epithelial ovarian cancer associated with multiple measures of cigarette smoking with a focus on characterizing risks according to tumor behavior and histology.
We used data from 21 case–control studies of ovarian cancer (19,066 controls, 11,972 invasive and 2,752 borderline cases). Study-specific odds ratios (OR) and 95 % confidence intervals (CI) were obtained from logistic regression models and combined into a pooled odds ratio using a random effects model.
Current cigarette smoking increased the risk of invasive mucinous (OR = 1.31; 95 % CI: 1.03–1.65) and borderline mucinous ovarian tumors (OR = 1.83; 95 % CI: 1.39–2.41), while former smoking increased the risk of borderline serous ovarian tumors (OR = 1.30; 95 % CI: 1.12–1.50). For these histological types, consistent dose– response associations were observed. No convincing associations between smoking and risk of invasive serous and endometrioid ovarian cancer were observed, while our results provided some evidence of a decreased risk of invasive clear cell ovarian cancer.
Our results revealed marked differences in the risk profiles of histological types of ovarian cancer with regard to cigarette smoking, although the magnitude of the observed associations was modest. Our findings, which may reflect different etiologies of the histological types, add to the fact that ovarian cancer is a heterogeneous disease.
PMCID: PMC3818570  PMID: 23456270
Case–control studies; Histological type; Ovarian neoplasms; Smoking
23.  Kaposi's Sarcoma-Associated Herpesvirus-Positive Primary Effusion Lymphoma Tumor Formation in NOD/SCID Mice Is Inhibited by Neomycin and Neamine Blocking Angiogenin's Nuclear Translocation 
Journal of Virology  2013;87(21):11806-11820.
Angiogenin (ANG) is a 14-kDa multifunctional proangiogenic secreted protein whose expression level correlates with the aggressiveness of several tumors. We observed increased ANG expression and secretion in endothelial cells during de novo infection with Kaposi's sarcoma-associated herpesvirus (KSHV), in cells expressing only latency-associated nuclear antigen 1 (LANA-1) protein, and in KSHV latently infected primary effusion lymphoma (PEL) BCBL-1 and BC-3 cells. Inhibition of phospholipase Cγ (PLCγ) mediated ANG's nuclear translocation by neomycin, an aminoglycoside antibiotic (not G418-neomicin), resulted in reduced KSHV latent gene expression, increased lytic gene expression, and increased cell death of KSHV+ PEL and endothelial cells. ANG detection in significant levels in KS and PEL lesions highlights its importance in KSHV pathogenesis. To assess the in vivo antitumor activity of neomycin and neamine (a nontoxic derivative of neomycin), BCBL-1 cells were injected intraperitoneally into NOD/SCID mice. We observed significant extended survival of mice treated with neomycin or neamine. Markers of lymphoma establishment, such as increases in animal body weight, spleen size, tumor cell spleen infiltration, and ascites volume, were observed in nontreated animals and were significantly diminished by neomycin or neamine treatments. A significant decrease in LANA-1 expression, an increase in lytic gene expression, and an increase in cleaved caspase-3 were also observed in neomycin- or neamine-treated animal ascitic cells. These studies demonstrated that ANG played an essential role in KSHV latency maintenance and BCBL-1 cell survival in vivo, and targeting ANG function by neomycin/neamine to induce the apoptosis of cells latently infected with KSHV is an attractive therapeutic strategy against KSHV-associated malignancies.
PMCID: PMC3807355  PMID: 23986578
24.  Calcified pseudomeningocele of the lumbar spine: a review 
European Spine Journal  2012;22(Suppl 3):443-449.
In this article, we review the English literature of calcified pseudomeningoceles in the lumbar region.
A systematic review using the Medline Database using the varied nomenclature for pseudomeningoceles, as well as reviewing the reference lists of relevant article found.
We discuss the different pathological theories on formation of a pseudomeningocele, the formation of a calcified wall and the optimal management for this entity. To date, 17 cases have been described, of which 13 are reviewed here. Calcification of pseudomeningocele is a rare entity and in the lumbar spine this occurs postsurgically. The only predisposing factor is prior surgery to the lumbar spine. Computer tomography, magnetic resonance imaging (MRI) and MRI myelography in combination are the preoperative investigations of choice. The radiological work-up can be preoperatively diagnostic and is important in the surgical planning.
The treatment is surgicel removal and the decision to treat is based on patient symptoms and correlating these with imaging. There is an average reported follow-up of 1.7 years postoperatively for these patients and the reported outcome after surgery is good.
PMCID: PMC3641256  PMID: 23233216
Calcification; Postsurgical; Pseudomeningocele; Lumbar spine
25.  Age-related changes in tissue macrophages precede cardiac functional impairment 
Aging (Albany NY)  2014;6(5):399-413.
Cardiac tissue macrophages (cTMs) are abundant in the murine heart but the extent to which the cTM phenotype changes with age is unknown. This study characterizes aging-dependent phenotypic changes in cTM subsets. Using the Cx3cr1GFP/+ mouse reporter line where GFP marks cTMs, and the tissue macrophage marker Mrc1, we show that two major cardiac tissue macrophage subsets, Mrc1−GFPhi and Mrc1+GFPhi cTMs, are present in the young (<10 week old) mouse heart, and a third subset, Mrc1+GFPlo, comprises ~50% of total Mrc1+ cTMs from 30 weeks of age. Immunostaining and functional assays show that Mrc1+ cTMs are the principal myeloid sentinels in the mouse heart and that they retain proliferative capacity throughout life. Gene expression profiles of the two Mrc1+ subsets also reveal that Mrc1+GFPlo cTMs have a decreased number of immune response genes (Cx3cr1, Lpar6, CD9, Cxcr4, Itga6 and Tgfβr1), and an increased number of fibrogenic genes (Ltc4s, Retnla, Fgfr1, Mmp9 and Ccl24), consistent with a potential role for cTMs in cardiac fibrosis. These findings identify early age-dependent gene expression changes in cTMs, with significant implications for cardiac tissue injury responses and aging-associated cardiac fibrosis.
PMCID: PMC4069267  PMID: 24861132
Cardiac macrophages; inflammation; cardiac senescence; Cx3cr1; tissue macrophages; ageing

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