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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 3.2.1.1), 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.
doi:10.1590/S1517-838220110004000022
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.
doi:10.1128/JVI.01065-10
PMCID: PMC3004349  PMID: 20943963
3.  Photodynamic inactivation of biofilm: taking a lightly colored approach to stubborn infection 
Microbial biofilms are responsible for a variety of microbial infections in different parts of the body, such as urinary tract infections, catheter infections, middle-ear infections, gingivitis, caries, periodontitis, orthopedic implants, and so on. The microbial biofilm cells have properties and gene expression patterns distinct from planktonic cells, including phenotypic variations in enzymic activity, cell wall composition and surface structure, which increase the resistance to antibiotics and other antimicrobial treatments. There is consequently an urgent need for new approaches to attack biofilm-associated microorganisms, and antimicrobial photodynamic therapy (aPDT) may be a promising candidate. aPDT involves the combination of a nontoxic dye and low-intensity visible light which, in the presence of oxygen, produces cytotoxic reactive oxygen species. It has been demonstrated that many biofilms are susceptible to aPDT, particularly in dental disease. This review will focus on aspects of aPDT that are designed to increase efficiency against biofilms modalities to enhance penetration of photosensitizer into biofilm, and a combination of aPDT with biofilm-disrupting agents.
doi:10.1586/14787210.2013.811861
PMCID: PMC4336791  PMID: 23879608
antimicrobial photodynamic therapy; biofilm; dental infection; extracellular polysaccharide; microbial resistance; multidrug efflux pump; persister cells; photosensitizers
4.  Axonal transport of TDP-43 mRNA granules in neurons is impaired by ALS-causing mutations 
Neuron  2014;81(3):536-543.
Summary
The RNA binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43.
doi:10.1016/j.neuron.2013.12.018
PMCID: PMC3939050  PMID: 24507191
5.  Home injury risks to young children in Karachi, Pakistan: a pilot study 
Archives of disease in childhood  2013;98(11):881-886.
Objective
To pilot an in-home unintentional injury hazard assessment tool and to quantify potential injury risks for young children in a low-income urban setting.
Methods
Two low-income neighbourhoods in Karachi, Pakistan, were mapped, and families with at least one child between the ages of 12 and 59 months were identified. Using existing available home injury risk information, an in-home injury risk assessment tool was drafted and tailored to the local setting. Home injury assessments were done in June–July 2010 after obtaining informed consent.
Results
Approximately 75.4% of mothers were educated through at least grade 12. The main risks identified were stoves within the reach of the child (n=279, 55.5%), presence of open buckets in the bathroom (n=240, 47.7%) within the reach of the child, and pedestal fans accessible to the child (n=242, 48.1%). In terms of safety equipment, a first-aid box with any basic item was present in 70% of households, but only 4.8% of households had a fire extinguisher in the kitchen.
Conclusions
This was the first time that an in-home, all-unintentional injury risk assessment tool was tailored and applied in the context of a low-income community in Pakistan. There was a significant burden of hazards present in the homes in these communities, representing an important opportunity for injury prevention. This pilot may have future relevance to other LMICs where child injury prevention is a critical need.
doi:10.1136/archdischild-2013-303907
PMCID: PMC4316730  PMID: 23995075
6.  Restoration of Glutamine Synthetase Activity, Nitric Oxide Levels and Amelioration of Oxidative Stress by Propolis in Kainic Acid Mediated Excitotoxicity 
Background
Propolis has been proposed to be protective on neurodegenerative disorders. To understand the neuroprotective effects of honeybee propolis, glutamine synthetase (GS) activity, nitric oxide (NO), thiobarbituric acid reactive substances (TBARS) and total antioxidant status (TAS) were studied in different brain regions-cerebral cortex (CC), cerebellum (CB) and brain stem (BS) of rats supplemented with propolis and subjected to kainic acid (KA) mediated excitotoxicity.
Materials and Methods
Male Sprague-Dawley rats were divided into four groups; Control group and KA group received vehicle and saline. Propolis group and propolis + KA group were orally administered with propolis (150mg/kg body weight), five times every 12 hours. KA group and propolis + KA group were injected subcutaneously with kainic acid (15mg/kg body weight) and were sacrificed after 2 hrs and CC, CB and BS were separated homogenized and used for estimation of GS activity, NO, TBARS, and TAS concentrations by colorimetric methods. Results were analyzed by one-way ANOVA, reported as mean + SD from 6 animals, and p<0.05 considered statistically significant.
Results
NO was increased (p< 0.001) and GS activity was decreased (p< 0.001) in KA treated group compared to control group as well as propolis + KA treated group. TBARS was decreased and TAS was increased (p< 0.001) in propolis + KA treated group compared KA treated group.
Conclusion
This study clearly demonstrated the restoration of GS activity, NO levels and decreased oxidative stress by propolis in kainic acid mediated excitotoxicity. Hence the propolis can be a possible potential candidate (protective agent) against excitotoxicity and neurodegenerative disorders.
PMCID: PMC4202657  PMID: 25435633
Nitric oxide; Glutamine Synthetase; Oxidative Stress; Excitotoxicity; Propolis; Rat Brain
7.  Quantitative tandem mass-spectrometry of skin tissue reveals putative psoriatic arthritis biomarkers 
Clinical Proteomics  2015;12(1):1.
Background
Psoriatic arthritis (PsA) is a distinct inflammatory arthritis occurring in 30% of psoriasis patients. There is a high prevalence of undiagnosed PsA in psoriasis patients; therefore, identifying soluble biomarkers for PsA could help in screening psoriasis patients for appropriate referral to a rheumatologist. Potential PsA biomarkers likely originate in sites of inflammation, such as the skin, and subsequently enter systemic circulation. Our goal was to identify candidate PsA biomarkers by comparing the proteome of skin biopsies obtained from patients with PsA to that from patients with psoriasis without PsA.
Methods
Skin biopsies were obtained from involved and uninvolved skin of 10 PsA and 10 age/gender-matched psoriasis patients without PsA (PsC). Using strong cation exchange chromatography, followed by label-free quantitative tandem mass spectrometry, we characterized the proteomes of pooled skin samples. Extracted ion current intensities were used to calculate protein abundance ratios, and these were utilized to identify differentially regulated proteins.
Results
Forty-seven proteins were elevated in PsA-derived skin compared to PsC-derived skin. Selected reaction monitoring assays were developed to quantify these potential PsA markers in individual skin samples, and 8 markers were confirmed in an independent sample set. ITGB5 and POSTN were measured in serum samples from 33 PsA and 15 PsC patients, using enzyme-linked immunosorbent assays. ITGB5 was significantly elevated in PsA serum (P < 0.01), and POSTN showed a trend. ITGB5 and POSTN correlated significantly in both patient groups (r = 0.472, P < 0.001).
Conclusion
Proteomic analysis of PsA and PsC skin identified eight new candidate biomarkers. These markers need to be validated with a larger and independent cohort, in order to delineate their clinical utility in PsA patients. These proteins may also uncover unknown aspects of PsA pathobiology.
Electronic supplementary material
The online version of this article (doi:10.1186/1559-0275-12-1) contains supplementary material, which is available to authorized users.
doi:10.1186/1559-0275-12-1
PMCID: PMC4304122
Psoriatic arthritis; Cutaneous psoriasis; Proteomics; Mass spectrometry; Biomarker
8.  Caveolin-1 Regulates Genomic Action of the Glucocorticoid Receptor in Neural Stem Cells 
Molecular and Cellular Biology  2014;34(14):2611-2623.
While glucocorticoids (GCs) are used clinically to treat many conditions, their neonatal and prenatal usage is increasingly controversial due to reports of delayed adverse outcomes, especially their effects on brain development. Such alterations may reflect the impact of GCs on neural progenitor/stem cell (NPSC) function. We previously demonstrated that the lipid raft protein caveolin-1 (Cav-1) was required for rapid GC signaling in embryonic mouse NPSCs operating through plasma membrane-bound glucocorticoid receptors (GRs). We show here that genomic GR signaling in NPSCs requires Cav-1. Loss of Cav-1 impacts the transcriptional response of many GR target genes (e.g., the serum- and glucocorticoid-regulated kinase 1 gene) that are likely to mediate the antiproliferative effects of GCs. Microarray analysis of wild-type C57 or Cav-1-deficient NPSCs identified approximately 100 genes that are differentially regulated by GC treatment. These changes in hormone responsiveness in Cav-1 knockout NPSCs are associated with the loss of GC-regulated phosphorylation of GR at serine 211 but not at serine 226. Chromatin recruitment of total GR to regulatory regions of target genes such as Fkbp-5, RhoJ, and Sgk-1, as well as p211-GR recruitment to Sgk-1, are compromised in Cav-1 knockout NPSCs. Cav-1 is therefore a multifunctional regulator of GR in NPSCs influencing both rapid and genomic action of the receptor to impact cell proliferation.
doi:10.1128/MCB.01121-13
PMCID: PMC4097667  PMID: 24777604
9.  Diesel biodegradation capacities of indigenous bacterial species isolated from diesel contaminated soil 
Petroleum based products are the major source of energy for industries and daily life. Leaks and accidental spills occur regularly during the exploration, production, refining, transport, and storage of petroleum and petroleum products. In the present study we isolated the bacteria from diesel contaminated soil and screened them for diesel biodegradation capacity. One monoculture isolate identified by 16S rRNA gene sequence analysis to be Acinetobacter baumannii was further studied for diesel oil biodegradation. The effects of various culture parameters (pH, temperature, NaCl concentrations, initial hydrocarbon concentration, initial inoculum size, role of chemical surfactant, and role of carbon and nitrogen sources) on biodegradation of diesel oil were evaluated. Optimal diesel oil biodegradation by A. baumanii occurred at initial pH 7, 35°C and initial hydrocarbon concentration at 4%. The biodegradation products under optimal cultural conditions were analyzed by GC-MS. The present study suggests that A. baumannii can be used for effective degradation of diesel oil from industrial effluents contaminated with diesel oil.
doi:10.1186/s40201-014-0142-2
PMCID: PMC4271493  PMID: 25530870
Acinetobacter baumannii; Diesel oil; Parameter optimization; Biodegradation; GC-MS analysis
10.  Imaging Self-assembly Dependent Spatial Distribution of Small Molecules in Cellular Environment 
Self-assembly of small molecules, as a more common phenomenon than one previously thought, can be either beneficial or detrimental to cells. Despite its profound biological implications, how the self-assembly of small molecules behave in cellular environment is largely unknown and barely explored. This work studies four fluorescent molecules that consist of the same peptidic backbone (e.g., Phe-Phe-Lys) and enzyme trigger (e.g., a phosphotyrosine residue), but bear different fluorophores on the side chain of the lysine residue of the peptidic motif. These molecules, however, exhibit different ability of self-assembly before and after enzymatic transformation (e.g., dephosphorylation). Fluorescent imaging reveals that self-assembly directly affects the distribution of these small molecules in cellular environment. Moreover, cell viability tests suggest that the states and the location of the molecular assemblies in the cellular environment control the phenotypes of the cells. For example, the molecular nanofibers of one of the small molecules apparently stabilize actin filaments and alleviate the insult of an F-actin toxin (e.g., latrunculin A). Combining fluorescent imaging and enzyme-instructed self-assembly of small peptidic molecules, this work not only demonstrates that self-assembly as a key factor for dictating the spatial distribution of small molecules in cellular environment. In addition, it illustrates a useful approach, based on enzyme-instructed self-assembly of small molecules, to modulate spatiotemporal profiles of small molecules in cellular environment, which allows the use of the emergent properties of small molecules to control the fate of cells.
doi:10.1021/la403457c
PMCID: PMC3895920  PMID: 24266765
11.  Nations’ Income Inequality Predicts Ambivalence in Stereotype Content: How Societies Mind the Gap 
Income inequality undermines societies: the more inequality, the more health problems, social tensions, and the lower social mobility, trust, life expectancy. Given people’s tendency to legitimate existing social arrangements, the Stereotype Content Model (SCM) argues that ambivalence—perceiving many groups as either warm or competent, but not both—may help maintain socio-economic disparities. The association between stereotype ambivalence and income inequality in 37 cross-national samples from Europe, the Americas, Oceania, Asia, and Africa investigates how groups’ overall warmth-competence, status-competence, and competition-warmth correlations vary across societies, and whether these variations associate with income inequality (Gini index). More unequal societies report more ambivalent stereotypes, while more equal ones dislike competitive groups and do not necessarily respect them as competent. Unequal societies may need ambivalence for system stability: income inequality compensates groups with partially positive social images.
doi:10.1111/bjso.12005
PMCID: PMC3855559  PMID: 23039178
ambivalence; stereotype; income inequality; warmth; competence
12.  Racial Disparities in Red Meat and Poultry Intake and Breast Cancer Risk 
Cancer causes & control : CCC  2013;24(12):10.1007/s10552-013-0299-5.
Purpose
Research on the role of red meat and poultry consumption in breast carcinogenesis is inconclusive, but the evidence in African American (AA) women is lacking. The association between consuming meat and breast cancer risk was examined in the Women’s Circle of Health Study involving 803 AA cases, 889 AA controls, 755 Caucasian cases, and 701 Caucasian controls.
Methods
Dietary information was collected using a Food Frequency Questionnaire. Odds ratios (OR) and 95% confidence intervals (CI) were obtained from logistic regression models adjusting for potential covariates.
Results
Comparing the fourth vs. the first quartile, among Caucasian women, processed meat (OR=1.48; 95% CI: 1.07–2.04), unprocessed red meat (OR=1.40; 95% CI: 1.01–1.94) and poultry intakes (OR=1.42; 95% CI: 1.01–1.99) increased breast cancer risk. Risk associated with poultry intake was more dominant in premenopausal women (OR=2.33; 95% CI: 1.44–3.77) and for women with ER- tumors (OR=2.55; 95% CI: 1.29–5.03) in the Caucasian group. Associations in AA women were mostly null except for a significant increased risk trend with processed meat consumption for ER+ tumors (OR=1.36; 95% CI: 0.94–1.97, p trend=0.04).
Conclusions
Overall, associations between breast cancer risk and consumption of red meat and poultry were of different magnitude in AA and Caucasian women, with further differences noted by menopausal and hormone receptor status in Caucasian women. This is the first study to examine racial differences in meat and breast cancer risk, and represents some of the first evidence in AA women.
doi:10.1007/s10552-013-0299-5
PMCID: PMC3873343  PMID: 24091794
Meat; African American; Breast Cancer; Poultry; Race; Estrogen receptor
13.  Body size in early life and breast cancer risk in African American and European American women 
Cancer causes & control : CCC  2013;24(12):10.1007/s10552-013-0302-1.
Purpose
There is growing evidence that body size in early life influences lifetime breast cancer risk, but little is known for African American (AA) women.
Methods
We evaluated body size during childhood and young adulthood and breast cancer risk among 1,751 cases [979 AA and 772 European American (EA)] and 1,673 controls (958 AA and 715 EA) in the Women’s Circle of Health Study. Odds ratio (OR) and 95% confidence intervals (CI) were computed using logistic regression models while adjusting for potential covariates.
Results
Among AA women, being shorter at 7–8 y compared to peers was associated with increased postmenopausal breast cancer risk (OR: 1.68, 95% CI: 1.02–2.74), and being heavier at menarche with decreased postmenopausal breast cancer risk, although of borderline significance (OR: 0.45, 95% CI: 0.20–1.02). For EA women, being shorter from childhood through adolescence, particularly at menarche, was associated with reduced premenopausal breast cancer risk (OR: 0.55, 95% CI: 0.31–0.98). After excluding hormone replacement therapy users, an inverse association with postmenopausal breast cancer was found among EA women reporting to be heavier than their peers at menarche (OR: 0.18, 95% CI: 0.04–0.79). The inverse relationship between BMI at age 20 and breast cancer risk was stronger and only statistically significant in EA women. No clear association with weight gain since age 20 was found.
Conclusions
Findings suggest that the impact of childhood height on breast cancer risk may differ for EA and AA women and confirm the inverse association previously reported in EA populations with adolescent body fatness, in AA women.
doi:10.1007/s10552-013-0302-1
PMCID: PMC3886722  PMID: 24113797
Breast cancer; African American; childhood; adolescence; young adult; BMI
14.  Thiocyanate potentiates antimicrobial photodynamic therapy: In situ generation of the sulfur trioxide radical anion by singlet oxygen 
Antimicrobial photodynamic therapy (PDT) is used for the eradication of pathogenic microbial cells and involves the light excitation of dyes in the presence of O2, yielding reactive oxygen species including the hydroxyl radical (•OH) and singlet oxygen (1O2). In order to chemically enhance PDT by the formation of longer-lived radical species, we asked whether thiocyanate (SCN−) could potentiate the methylene blue (MB) and light-mediated killing of the gram-positive Staphylococcus aureus and the gram-negative Escherichia coli. SCN− enhanced PDT (10 μM MB, 5J/cm2 660 nm hv) killing in a concentration-dependent manner of S. aureus by 2.5 log10 to a maximum of 4.2 log10 at 10 mM (P < 0.001) and increased killing of E. coli by 3.6 log10 to a maximum of 5.0 log10 at 10 mM (P < 0.01). We determined that SCN− rapidly depleted O2 from an irradiated MB system, reacting exclusively with 1O2, without quenching the MB excited triplet state. SCN− reacted with 1O2, producing a sulfur trioxide radical anion (a sulfur-centered radical demonstrated by EPR spin trapping). We found that MB-PDT of SCN− in solution produced both sulfite and cyanide anions, and that addition of each of these salts separately enhanced MB-PDT killing of bacteria. We were unable to detect EPR signals of •OH, which, together with kinetic data, strongly suggests that MB, known to produce •OH and 1O2, may, under the conditions used, preferentially form 1O2.
doi:10.1016/j.freeradbiomed.2013.08.162
PMCID: PMC3889203  PMID: 23969112
Thiocyanate; Antimicrobial photodynamic inactivation; Sulfite; Cyanide; ESR spin trapping; Gram-positive bacteria; Gram-negative bacteria
15.  Obtaining a good lip roll in congenital, secondary and traumatic cleft lip repairs 
The present article includes a video designed to show the reader/viewer how to obtain a better lip roll in primary and secondary cleft lips as well as in traumatic cleft lips. The key is to not damage the delicate glands and fat in the lip roll. The actual surgery demonstrated in the video is a cleft lip redo with an effaced lip roll.
PMCID: PMC3910528  PMID: 24497768
Cleft lip; Lip roll; White roll
16.  Integrative functional genomic analyses implicate specific molecular pathways and circuits in autism 
Cell  2013;155(5):1008-1021.
Genetic studies have identified dozens of autism spectrum disorder (ASD) susceptibility genes, raising two critical questions: 1) do these genetic loci converge on specific biological processes, and 2) where does the phenotypic specificity of ASD arise, given its genetic overlap with intellectual disability (ID)? To address this, we mapped ASD and ID risk genes onto co-expression networks representing developmental trajectories and transcriptional profiles representing fetal and adult cortical laminae. ASD genes tightly coalesce in modules that implicate distinct biological functions during human cortical development, including early transcriptional regulation and synaptic development. Bioinformatic analyses suggest translational regulation by FMRP and transcriptional co-regulation by common transcription factors connect these processes. At a circuit level, ASD genes are enriched in superficial cortical layers and glutamatergic projection neurons. Furthermore, we show that the patterns of ASD and ID risk genes are distinct, providing a novel biological framework for investigating the pathophysiology of ASD.
doi:10.1016/j.cell.2013.10.031
PMCID: PMC3934107  PMID: 24267887
gene networks; systems biology; exome; rare variants; Intellectual disability; human cortical development; gene expression; FMRP; Satb1; MEF2; RNA-seq
17.  Hyaluronic Acid-Based Hydrogels Containing Covalently Integrated Drug Depots: Implication for Controlling Inflammation in Mechanically Stressed Tissues 
Biomacromolecules  2013;14(11):10.1021/bm4011276.
Synthetic hydrogels containing covalently-integrated soft and deformable drug depots capable of releasing therapeutic molecules in response to mechanical forces are attractive candidates for the treatment of degenerated tissues that are normally load bearing. Herein, radically crosslinkable block copolymer micelles (xBCM) assembled from an amphiphilic block copolymer consisting of hydrophilic poly(acrylic acid) (PAA) partially modified with 2-hydroxyethyl acrylate, and hydrophobic poly(n-butyl acryclate) (PnBA) were employed as the drug depots and the microscopic crosslinkers for the preparation of hyaluronic acid (HA)-based, hydrogels. HA hydrogels containing covalently integrated micelles (HAxBCM) were prepared by radical polymerization of glycidyl methacrylate (GMA)-modified HA (HAGMA) in the presence of xBCMs. When micelles prepared from the parent PAA-b-PnBA without any polymerizable double bonds were used, hydrogels containing physically entrapped micelles (HApBCM) were obtained. The addition of xBCMs to a HAGMA precursor solution accelerated the gelation kinetics and altered the hydrogel mechanical properties. The resultant HAxBCM gels exhibit an elastic modulus of 847 ± 43 Pa and a compressive modulus of 9.2 ± 0.7 kPa. Diffusion analysis of Nile Red (NR)-labeled xBCMs employing fluorescence correlation spectroscopy confirmed the covalent immobilization of xBCMs in HA networks. Covalent integration of dexamethasone (DEX)-loaded xBCMs in HA gels significantly reduced the initial burst release and provided sustained release over a prolonged period. Importantly, DEX release from HAxBCM gels was accelerated by intermittently-applied external compression in a strain-dependent manner. Culturing macrophages in the presence of DEX-releasing HAxBCM gels significantly reduced cellular production of inflammatory cytokines. Incorporating mechano-responsive modules in synthetic matrices offers a novel strategy to harvest mechanical stress present in the healing wounds to initiate tissue repair.
doi:10.1021/bm4011276
PMCID: PMC3856199  PMID: 24093583
Block copolymer micelles; Mechano-responsive; Hyaluronic acid; Hydrogels; Anti-inflammatory; Drug delivery
18.  Identification of Serum MicroRNA Signatures for Diagnosis of Mild Traumatic Brain Injury in a Closed Head Injury Model 
PLoS ONE  2014;9(11):e112019.
Wars in Iraq and Afghanistan have highlighted the problems of diagnosis and treatment of mild traumatic brain injury (mTBI). MTBI is a heterogeneous injury that may lead to the development of neurological and behavioral disorders. In the absence of specific diagnostic markers, mTBI is often unnoticed or misdiagnosed. In this study, mice were induced with increasing levels of mTBI and microRNA (miRNA) changes in the serum were determined. MTBI was induced by varying weight and fall height of the impactor rod resulting in four different severity grades of the mTBI. Injuries were characterized as mild by assessing with the neurobehavioral severity scale-revised (NSS-R) at day 1 post injury. Open field locomotion and acoustic startle response showed behavioral and sensory motor deficits in 3 of the 4 injury groups at day 1 post injury. All of the animals recovered after day 1 with no significant neurobehavioral alteration by day 30 post injury. Serum microRNA (miRNA) profiles clearly differentiated injured from uninjured animals. Overall, the number of miRNAs that were significantly modulated in injured animals over the sham controls increased with the severity of the injury. Thirteen miRNAs were found to identify mTBI regardless of its severity within the mild spectrum of injury. Bioinformatics analyses revealed that the more severe brain injuries were associated with a greater number of miRNAs involved in brain related functions. The evaluation of serum miRNA may help to identify the severity of brain injury and the risk of developing adverse effects after TBI.
doi:10.1371/journal.pone.0112019
PMCID: PMC4224512  PMID: 25379886
19.  IFI16 Restricts HSV-1 Replication by Accumulating on the HSV-1 Genome, Repressing HSV-1 Gene Expression, and Directly or Indirectly Modulating Histone Modifications 
PLoS Pathogens  2014;10(11):e1004503.
Interferon-γ inducible factor 16 (IFI16) is a multifunctional nuclear protein involved in transcriptional regulation, induction of interferon-β (IFN-β), and activation of the inflammasome response. It interacts with the sugar-phosphate backbone of dsDNA and modulates viral and cellular transcription through largely undetermined mechanisms. IFI16 is a restriction factor for human cytomegalovirus (HCMV) and herpes simplex virus (HSV-1), though the mechanisms of HSV-1 restriction are not yet understood. Here, we show that IFI16 has a profound effect on HSV-1 replication in human foreskin fibroblasts, osteosarcoma cells, and breast epithelial cancer cells. IFI16 knockdown increased HSV-1 yield 6-fold and IFI16 overexpression reduced viral yield by over 5-fold. Importantly, HSV-1 gene expression, including the immediate early proteins, ICP0 and ICP4, the early proteins, ICP8 and TK, and the late proteins gB and Us11, was reduced in the presence of IFI16. Depletion of the inflammasome adaptor protein, ASC, or the IFN-inducing transcription factor, IRF-3, did not affect viral yield. ChIP studies demonstrated the presence of IFI16 bound to HSV-1 promoters in osteosarcoma (U2OS) cells and fibroblasts. Using CRISPR gene editing technology, we generated U2OS cells with permanent deletion of IFI16 protein expression. ChIP analysis of these cells and wild-type (wt) U2OS demonstrated increased association of RNA polymerase II, TATA binding protein (TBP) and Oct1 transcription factors with viral promoters in the absence of IFI16 at different times post infection. Although IFI16 did not alter the total histone occupancy at viral or cellular promoters, its absence promoted markers of active chromatin and decreased those of repressive chromatin with viral and cellular gene promoters. Collectively, these studies for the first time demonstrate that IFI16 prevents association of important transcriptional activators with wt HSV-1 promoters and suggest potential mechanisms of IFI16 restriction of wt HSV-1 replication and a direct or indirect role for IFI16 in histone modification.
Author Summary
HSV-1, a ubiquitous human pathogen that establishes a life-long infection, has evolved several mechanisms to evade host immune detection and responses. However, it is still subject to regulation by cellular factors. Recently, a host nuclear protein, IFI16, was shown to be involved in the innate defense response to HSV-1 infection. Here, we provide the first evidence that IFI16 inhibits wild-type HSV-1 replication by repressing viral gene expression independent of its roles in the immune response. We show that IFI16 binds the HSV-1 genome at the transcription start sites of several HSV-1 genes. Using a permanently IFI16-negative cell line that we generated, we demonstrate that IFI16 reduces the association of important transcription factors. IFI16 also promotes global histone modifications by increasing the markers of repressive chromatin and decreasing the markers for activating chromatin on viral and cellular genes. These insights into the role of IFI16 in HSV-1 biology suggest that stabilization of IFI16 is an attractive avenue for antiviral drug development.
doi:10.1371/journal.ppat.1004503
PMCID: PMC4223080  PMID: 25375629
22.  Novel mechanisms of tube-size regulation revealed by the Drosophila trachea 
Cell and tissue research  2013;354(2):10.1007/s00441-013-1673-z.
The size of various tubes within tubular organs such as the lung, vascular system and kidney must be finely tuned for the optimal delivery of gases, nutrients, waste and cells within the entire organism. Aberrant tube sizes lead to devastating human illnesses, such as polycystic kidney disease, fibrocystic breast disease, pancreatic cystic neoplasm and thyroid nodules. However, the underlying mechanisms that are responsible for tube-size regulation have yet to be fully understood. Therefore, no effective treatments are available for disorders caused by tube-size defects. Recently, the Drosophila tracheal system has emerged as an excellent in vivo model to explore the fundamental mechanisms of tube-size regulation. Here, we discuss the role of the apical luminal matrix, cell polarity and signaling pathways in regulating tube size in Drosophila trachea. Previous studies of the Drosophila tracheal system have provided general insights into epithelial tube morphogenesis. Mechanisms that regulate tube size in Drosophila trachea could be well conserved in mammalian tubular organs. This knowledge should greatly aid our understanding of tubular organogenesis in vertebrates and potentially lead to new avenues for the treatment of human disease caused by tube-size defects.
doi:10.1007/s00441-013-1673-z
PMCID: PMC3812325  PMID: 23824100
Trachea; Tube size; Luminal matrix; Apical secretion; Cell polarity; Endocytosis; Drosophila
23.  D. Hominis or Another Species? 
doi:10.4103/0019-5154.143539
PMCID: PMC4248504  PMID: 25484397
24.  Antimicrobial strategies centered around reactive oxygen species - bactericidal antibiotics, photodynamic therapy and beyond 
FEMS microbiology reviews  2013;37(6):955-989.
Reactive oxygen species (ROS) can attack a diverse range of targets to exert antimicrobial activity, which accounts for their versatility in mediating host defense against a broad range of pathogens. Most ROS are formed by the partial reduction of molecular oxygen. Four major ROS are recognized comprising: superoxide (O2•−), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and singlet oxygen (1O2), but they display very different kinetics and levels of activity. The effects of O2•− and H2O2 are less acute than those of •OH and 1O2, since the former are much less reactive and can be detoxified by endogenous antioxidants (both enzymatic and non-enzymatic) that are induced by oxidative stress. In contrast, no enzyme can detoxify •OH or 1O2, making them extremely toxic and acutely lethal. The present review will highlight the various methods of ROS formation and their mechanism of action. Antioxidant defenses against ROS in microbial cells and the use of ROS by antimicrobial host defense systems are covered. Antimicrobial approaches primarily utilizing ROS comprise both bactericidal antibiotics, and non-pharmacological methods such as photodynamic therapy, titanium dioxide photocatalysis, cold plasma and medicinal honey. A brief final section covers, reactive nitrogen species, and related therapeutics, such as acidified nitrite and nitric oxide releasing nanoparticles.
doi:10.1111/1574-6976.12026
PMCID: PMC3791156  PMID: 23802986
25.  Kaposi's Sarcoma-Associated Herpesvirus Induces Nrf2 during De Novo Infection of Endothelial Cells to Create a Microenvironment Conducive to Infection 
PLoS Pathogens  2014;10(10):e1004460.
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma. KSHV induces reactive oxygen species (ROS) early during infection of human dermal microvascular endothelial (HMVEC-d) cells that are critical for virus entry. One of the downstream targets of ROS is nuclear factor E2-related factor 2 (Nrf2), a transcription factor with important anti-oxidative functions. Here, we show that KS skin lesions have high Nrf2 activity compared to healthy skin tissue. Within 30 minutes of de novo KSHV infection of HMVEC-d cells, we observed Nrf2 activation through ROS-mediated dissociation from its inhibitor Keap1, Ser-40 phosphorylation, and subsequent nuclear translocation. KSHV binding and consequent signaling through Src, PI3-K and PKC-ζ were also important for Nrf2 stability, phosphorylation and transcriptional activity. Although Nrf2 was dispensable for ROS homeostasis, it was essential for the induction of COX-2, VEGF-A, VEGF-D, Bcl-2, NQO1, GCS, HO1, TKT, TALDO and G6PD gene expression in KSHV-infected HMVEC-d cells. The COX-2 product PGE2 induced Nrf2 activity through paracrine and autocrine signaling, creating a feed-forward loop between COX-2 and Nrf2. vFLIP, a product of KSHV latent gene ORF71, induced Nrf2 and its target genes NQO1 and HO1. Activated Nrf2 colocalized with the KSHV genome as well as with the latency protein LANA-1. Nrf2 knockdown enhanced ORF73 expression while reducing ORF50 and other lytic gene expression without affecting KSHV entry or genome nuclear delivery. Collectively, these studies for the first time demonstrate that during de novo infection, KSHV induces Nrf2 through intricate mechanisms involving multiple signal molecules, which is important for its ability to manipulate host and viral genes, creating a microenvironment conducive to KSHV infection. Thus, Nrf2 is a potential attractive target to intervene in KSHV infection and the associated maladies.
Author Summary
KSHV infection of endothelial cells in vivo causes Kaposi's sarcoma and understanding the steps involved in de novo KSHV infection of these cells and the consequences is important to develop therapies to counter KSHV pathogenesis. Infection of endothelial cells in vitro is preceded by the induction of a network of host signaling agents that are necessary for virus entry, gene expression and establishment of latency. Our previous studies have implicated reactive oxygen species (ROS) as part of this network. In the current study, we show that ROS activate Nrf2, a master transcriptional regulator of genes involved in ROS homeostasis, apoptosis, glucose metabolism and angiogenesis. Besides ROS, KSHV utilizes additional aspects of host signaling to induce Nrf2 activity. We also observed that infection of endothelial cells deficient in Nrf2 resulted in downregulation of multiple genes important in KSHV pathogenesis, such as COX-2 and VEGF, and affected proper expression of two hallmark KSHV genes, lytic ORF50 and latent ORF73. Taken together, this study is the first to demonstrate the importance of Nrf2 during de novo KSHV infection of endothelial cells, and establishes Nrf2 as an attractive therapeutic target to control KSHV infection, establishment of latency and the associated cancers.
doi:10.1371/journal.ppat.1004460
PMCID: PMC4207826  PMID: 25340789

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