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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
4.  Transcriptomic Approaches to Neural Repair 
The Journal of Neuroscience  2015;35(41):13860-13867.
Understanding why adult CNS neurons fail to regenerate their axons following injury remains a central challenge of neuroscience research. A more complete appreciation of the biological mechanisms shaping the injured nervous system is a crucial prerequisite for the development of robust therapies to promote neural repair. Historically, the identification of regeneration associated signaling pathways has been impeded by the limitations of available genetic and molecular tools. As we progress into an era in which the high-throughput interrogation of gene expression is commonplace and our knowledge base of interactome data is rapidly expanding, we can now begin to assemble a more comprehensive view of the complex biology governing axon regeneration. Here, we highlight current and ongoing work featuring transcriptomic approaches toward the discovery of novel molecular mechanisms that can be manipulated to promote neural repair.
SIGNIFICANCE STATEMENT Transcriptional profiling is a powerful technique with broad applications in the field of neuroscience. Recent advances such as single-cell transcriptomics, CNS cell type-specific and developmental stage-specific expression libraries are rapidly enhancing the power of transcriptomics for neuroscience applications. However, extracting biologically meaningful information from large transcriptomic datasets remains a formidable challenge. This mini-symposium will highlight current work using transcriptomic approaches to identify regulatory networks in the injured nervous system. We will discuss analytical strategies for transcriptomics data, the significance of noncoding RNA networks, and the utility of multiomic data integration. Though the studies featured here specifically focus on neural repair, the approaches highlighted in this mini-symposium will be of broad interest and utility to neuroscientists working in diverse areas of the field.
doi:10.1523/JNEUROSCI.2599-15.2015
PMCID: PMC4604224  PMID: 26468186
5.  A Role for Molecular Chaperone Hsc70 in Reovirus Outer Capsid Disassembly* 
The Journal of biological chemistry  2007;282(16):12210-12219.
After crossing the cellular membrane barrier during cell entry, most animal viruses must undergo further disassembly before initiating viral gene expression. In many cases, these disassembly mechanisms remain poorly defined. For this report, we examined a final step in disassembly of the mammalian reovirus outer capsid: cytoplasmic release of the central, δ fragment of membrane penetration protein μ1 to yield the transcriptionally active viral core particle. An in vitro assay with reticulocyte lysate recapitulated the release of intact δ molecules. Requirements for activity in this system were shown to include a protein factor, ATP, and Mg2+ and K+ ions, consistent with involvement of a molecular chaperone such as Hsc70. Immunodepletion of Hsc70 and Hsp70 impaired δ release, which was then rescued by addition of purified Hsc70. Hsc70 was associated with released δ molecules not only in the lysate but also during cell entry. We conclude that Hsc70 plays a defined role in reovirus outer capsid disassembly, during or soon after membrane penetration, to prepare the entering particle for gene expression and replication.
doi:10.1074/jbc.M610258200
PMCID: PMC4822165  PMID: 17284448
6.  A Case of Vascular Malformation of the Neck 
The Indian Journal of Surgery  2014;77(Suppl 1):72-74.
Vascular malformations are rare congenital vascular anomalies composed of inappropriately connected vasculature. They are usually present at birth, are progressive, infiltrative and require intervention. Vascular malformations need to be differentiated from haemangiomas which are congenital vascular neoplasms. We present a case of vascular malformation in a 6-year old child who presented with a progressive swelling in the neck and was treated by surgical excision. This case is being presented because of its peculiar clinical presentation.
doi:10.1007/s12262-014-1142-2
PMCID: PMC4425787  PMID: 25972650
Vascular anomaly; Vascular malformation; Haemangioma; Neck swelling
7.  OBESITY, BODY FAT DISTRIBUTION, AND RISK OF BREAST CANCER SUBTYPES IN AFRICAN AMERICAN WOMEN PARTICIPATING IN THE AMBER CONSORTIUM 
Purpose
African American (AA) women are more likely than white women to be obese and to be diagnosed with ER- and triple negative (TN) breast cancer, but few studies have evaluated the impact of obesity and body fat distribution on breast cancer subtypes in AA women. We evaluated these associations in the AMBER Consortium by pooling data from four large studies.
Methods
Cases were categorized according to hormone receptor status as ER+, ER-, and TN (ER-, PR-, and HER2-) based on pathology data. A total of 2,104 ER+ cases, 1,070 ER- cases (including 491 TN cases), and 12,060 controls were included. Odds ratios (OR) and 95% confidence intervals (CI) were computed using logistic regression, taking into account breast cancer risk factors.
Results
In postmenopausal women, higher recent (most proximal value to diagnosis/index date) BMI was associated with increased risk of ER+ cancer (OR: 1.31; 95% CI: 1.02–1.67 for BMI≥35 vs <25 kg/m2) and with decreased risk of TN tumors (OR: 0.60; 95% CI: 0.39–0.93 for BMI≥35 vs. <25). High young adult BMI was associated with decreased premenopausal ER+ cancer and all subtypes of postmenopausal cancer, and high recent waist-to-hip ratio (WHR) with increased risk of pre-menopausal ER+ tumors (OR: 1.35; 95% CI: 1.01–1.80) and all tumor subtypes combined in postmenopausal women (OR: 1.26; 95% CI: 1.02–1.56).
Conclusions
The impact of general and central obesity varies by menopausal status and hormone receptor subtype in AA women. Our findings imply different mechanisms for associations of adiposity with TN and ER+ breast cancers.
doi:10.1007/s10549-015-3353-z
PMCID: PMC4440799  PMID: 25809092
Obesity; breast cancer subtypes; triple negative; African Americans; waist-to-hip ratio
8.  Distinct cathepsins control necrotic cell death mediated by pyroptosis inducers and lysosome-destabilizing agents 
Cell Cycle  2015;14(7):964-972.
Necrotic cell death triggers a range of biological responses including a strong adaptive immune response, yet we know little about the cellular pathways that control necrotic cell death. Inhibitor studies suggest that proteases, and in particular cathepsins, drive necrotic cell death. The cathepsin B-selective inhibitor CA-074-Me blocks all forms of programmed necrosis by an unknown mechanism. We found that cathepsin B deficiency does not prevent induction of pyroptosis and lysosome-mediated necrosis suggesting that CA-074-Me blocks necrotic cell death by targeting cathepsins other than cathepsin B. A single cathepsin, cathepsin C, drives necrotic cell death mediated by the lysosome-destabilizing agent Leu-Leu-OMe (LLOMe). Here we present evidence that cathepsin C-deficiency and CA-074-Me block LLOMe killing in a distinct and cell type-specific fashion. Cathepsin C-deficiency and CA-074-Me block LLOMe killing of all myeloid cells, except for neutrophils. Cathepsin C-deficiency, but not CA-074-Me, blocks LLOMe killing of neutrophils suggesting that CA-074-Me does not target cathepsin C directly, consistent with inhibitor studies using recombinant cathepsin C. Unlike other cathepsins, cathepsin C lacks endoproteolytic activity, and requires activation by other lysosomal proteases, such as cathepsin D. Consistent with this theory, we found that lysosomotropic agents and cathepsin D downregulation by siRNA block LLOMe-mediated necrosis. Our findings indicate that a proteolytic cascade, involving cathepsins C and D, controls LLOMe-mediated necrosis. In contrast, cathepsins C and D were not required for pyroptotic cell death suggesting that distinct cathepsins control pyroptosis and lysosome-mediated necrosis.
doi:10.4161/15384101.2014.991194
PMCID: PMC4614982  PMID: 25830414
Caspase-1; inflammasome; lysosome rupture; necrosis; pyroptosis
9.  Detection of Tuberculosis in HIV Co-infected Individuals: Use of Multiple ELISA Responses to 38kDa, Lipoarabinomannan and ESAT– 6 of M. tuberculosis 
Introduction
There is a constant search for more sensitive and specific laboratory markers for tuberculosis (TB) infection. The early detection of TB in HIV co infected individuals is a diagnostic challenge. This is further compounded in those harbouring extrapulmonary disease.
Aim
To evaluate the use of multiple Enzyme Linked Immunosorbent Assays (ELISA) quantifying antibody responses to 38kDa, LAM and ESAT-6 M.tb antigens in detection of TB in patients with TB and HIV-TB co-infection.
Materials and Methods
This is a cross-sectional study carried out in Hyderabad, India. Patient groups included 124 HIV-TB {62 with pulmonary TB (PTB) and 62 with extrapulmonary TB (ETB)}, 39 TB, 56 HIV and 57 healthy subjects (HS). A combination of anti 38kDa and LAM ELISAs measuring IgG, IgM and IgA levels and another ELISA measuring anti ESAT-6 combined antibody levels of IgG, IgM and IgA were evaluated. One-way ANOVA was performed to compare antibody responses among groups. To assess the efficacy of multiple ELISAs in detecting TB, concomitant seropositivity of an individual for all four ELISAs were evaluated for sensitivity and specificity.
Results
A single ELISA carried out to detect TB in HIV patients showed a sensitivity ranging from 39% to 72%. The sensitivities of concomitant evaluation of multiple ELISAs were 92% for any single, 72% for any two, 44% for any three and 14% for any four. Based on the specificities, a simple algorithm for TB detection can be deduced. When four ELISAs are positive (specificity 100%) in a patient-confirmed TB; when three ELISAs are positive (specificity 98%) – probably TB; when two ELISAs are positive (specificity 95%) – possibly TB; and when one ELISA is positive (specificity 70%) – suspicion of TB.
Conclusion
The present study establishes the value of combining two or more M.tb antigen based ELISAs to enhance the sensitivity and specificity of TB detection in patients with tuberculosis as well as in those co-infected with HIV.
doi:10.7860/JCDR/2016/16559.7322
PMCID: PMC4800549  PMID: 27042484
Extrapulmonary TB; HIV-TB diagnosis; Pulmonary TB; TB test sensitivity
10.  Implant – Supported Full Mouth Rehabilitation: A Guided Surgical and Prosthetic Protocol 
doi:10.7860/JCDR/2016/17467.7264
PMCID: PMC4800668  PMID: 27042601
CAD - CAM restorations; Dental implants; Flapless implant surgery; Surgical template; Stereolithography
11.  Scapular winging in a patient with Arnold–Chiari malformation type 1 and syringomyelia 
BMJ Case Reports  2014;2014:bcr2014203571.
doi:10.1136/bcr-2014-203571
PMCID: PMC3975549  PMID: 24686803
12.  Comparison of Matching Pursuit Algorithm with Other Signal Processing Techniques for Computation of the Time-Frequency Power Spectrum of Brain Signals 
The Journal of Neuroscience  2016;36(12):3399-3408.
Signals recorded from the brain often show rhythmic patterns at different frequencies, which are tightly coupled to the external stimuli as well as the internal state of the subject. In addition, these signals have very transient structures related to spiking or sudden onset of a stimulus, which have durations not exceeding tens of milliseconds. Further, brain signals are highly nonstationary because both behavioral state and external stimuli can change on a short time scale. It is therefore essential to study brain signals using techniques that can represent both rhythmic and transient components of the signal, something not always possible using standard signal processing techniques such as short time fourier transform, multitaper method, wavelet transform, or Hilbert transform. In this review, we describe a multiscale decomposition technique based on an over-complete dictionary called matching pursuit (MP), and show that it is able to capture both a sharp stimulus-onset transient and a sustained gamma rhythm in local field potential recorded from the primary visual cortex. We compare the performance of MP with other techniques and discuss its advantages and limitations. Data and codes for generating all time-frequency power spectra are provided.
doi:10.1523/JNEUROSCI.3633-15.2016
PMCID: PMC4804002  PMID: 27013668
13.  Endosomal Proteolysis of the Ebola Virus Glycoprotein Is Necessary for Infection 
Science (New York, N.Y.)  2005;308(5728):1643-1645.
Ebola virus (EboV) causes rapidly fatal hemorrhagic fever in humans and there is currently no effective treatment. We found that the infection of African green monkey kidney (Vero) cells by vesicular stomatitis viruses bearing the EboV glycoprotein (GP) requires the activity of endosomal cysteine proteases. Using selective protease inhibitors and protease-deficient cell lines, we identified an essential role for cathepsin B (CatB) and an accessory role for cathepsin L (CatL) in EboV GP-dependent entry. Biochemical studies demonstrate that CatB and CatL mediate entry by carrying out proteolysis of the EboV GP subunit GP1 and support a multistep mechanism that explains the relative contributions of these enzymes to infection. CatB and CatB/CatL inhibitors diminish the multiplication of infectious EboV-Zaire in cultured cells and may merit investigation as anti-EboV drugs.
doi:10.1126/science.1110656
PMCID: PMC4797943  PMID: 15831716
15.  Epstein-Barr Virus and Its Association with Oral Hairy Leukoplakia: A Short Review 
In immunocompromised subjects, Epstein-Barr virus (EBV) infection of terminally differentiated oral keratinocytes may result in subclinical productive infection of the virus in the stratum spinosum and in the stratum granulosum with shedding of infectious virions into the oral fluid in the desquamating cells. In a minority of cases this productive infection with dysregulation of the cell cycle of terminally differentiated epithelial cells may manifest as oral hairy leukoplakia. This is a white, hyperkeratotic, benign lesion of low morbidity, affecting primarily the lateral border of the tongue. Factors that determine whether productive EBV replication within the oral epithelium will cause oral hairy leukoplakia include the fitness of local immune responses, the profile of EBV gene expression, and local environmental factors.
doi:10.1155/2016/4941783
PMCID: PMC4800082  PMID: 27047546
16.  A Retrospective Study of the Pattern of Sexually Transmitted Infections in Males: Viral Infections in Emerging Trend 
Introduction
Sexually transmitted infections (STIs) continue to be a major public health problem with significant burden on the society even after so many health care programmes being organized by the governmental and non-governmental organizations and awareness created among general public about STIs. Male patients are common visitors to STI clinic than females who are generally traced as a contact in our society.
Aim
The aim of this study was to give an overview of the pattern of STIs among males at a tertiary care teaching hospital over a period of 5 years.
Materials and Methods
A retrospective chart review of the data collected from the clinical records of all male patients, who had attended the STI clinic of Chengalpattu Medical College Hospital, Chengalpattu, Tamil Nadu, for various complaints during the 5 year period from 2010 to 2014 was carried out. All male patients with confirmed STIs were included in the study and those patients without any evidence of STIs either clinically or serologically were excluded from the study.
Results
Out of the 4454 male cases who had attended the STI clinic, 175 (3.93%) patients had STIs. Genital wart accounted for the maximum number among the STIs with 61 cases (34.86%), followed by genital herpes 56 (32%), urethral discharge 19(10.86%), non-herpetic genital ulcerative diseases 17(9.71%) and serological test for syphilis (RPR) was reactive in 22 (12.57%) patients. HIV was positive in 68 (1.53%) among the total 4454 male patients attended the clinic.
Conclusion
Viral STIs occur significantly more than the bacterial STIs because of its incurable and recurrent nature. Health programmes should be still more focused on creating awareness about the minor STIs and to remove the stigma so that the patients attend the proper health care facilities in the early stage itself for treatment thereby, complications and further transmission of the STIs can be avoided.
doi:10.7860/JCDR/2016/16142.7138
PMCID: PMC4740688  PMID: 26894160
Genital wart; Genital herpes; Syndromic management; Stigma
17.  Cardiovascular Risk and Acute Coronary Syndrome in Giant Cell Arteritis: A Population Based Retrospective Cohort Study 
Arthritis care & research  2015;67(3):396-402.
Objective
We assessed the occurrence of acute coronary syndrome (ACS) in patients with giant cell arteritis (GCA) compared to subjects without GCA.
Methods
We retrospectively reviewed a population-based incidence cohort of Olmsted County, Minnesota residents with GCA diagnosed in 1950-2009. We compared this cohort with a cohort of patients without GCA of similar age, sex and calendar year from the same population.
Results
The study included 245 patients with GCA and 245 non-GCA subjects. Mean Framingham cardiovascular risk score was 30% (SD 19%) in GCA and 34% (SD 23%) in non-GCA (p=0.096) at incidence/index date. Diabetes mellitus was significantly less common in GCA than non-GCA at index date. Mean high-density lipoprotein was higher and triglycerides were lower and fewer patients were using lipid-lowering medications in the GCA cohort compared to the non-GCA at index date. During follow-up, no difference between the two cohorts was noted in overall rate of ACS events [hazard ratio (HR) 0.74; 95% confidence interval (CI) 0.44, 1.26]. Overall thrombosis in myocardial infarction (TIMI) scores were similar in both cohorts. Revascularization procedures were done less frequently in GCA than non-GCA subjects (19% vs. 50%; p=0.015). Post ACS hospital length of stays and complications were similar in both cohorts.
Conclusion
Multiple cardiovascular risk factors are more favorable at incidence of GCA. There is no overall increased risk of acute coronary syndromes in patients with GCA.
doi:10.1002/acr.22416
PMCID: PMC4310813  PMID: 25074472
Acute Coronary Syndrome; Giant Cell Arteritis
18.  HIV-Associated Oral Mucosal Melanin Hyperpigmentation: A Clinical Study in a South African Population Sample 
AIDS Research and Treatment  2016;2016:8389214.
Objective. The aim of the study was to determine the prevalence of HIV-associated oral mucosal melanin hyperpigmentation (HIV-OMH) in a specific population of HIV-seropositive South Africans and to analyse the associations between HIV-OMH clinical features and the demographic and immunological characteristics of the study cohort. Material and Methods. This cross-sectional study included 200 HIV-seropositive Black subjects. The collected data comprised age, gender, CD4+ T cell count, viral load, systemic disease, medications, oral site affected by HIV-OMH, extent (localized or generalized), intensity of the pigmentation (dark or light), and smoking and snuff use. Results. Overall, 18.5% of the study cohort had HIV-OMH. Twenty-two and a half percent had OMH that could not with confidence be attributed to HIV infection, and 59% did not have any OMH. There was a significant but weak association between smoking and the presence of HIV-OMH. Conclusions. The prevalence of HIV-OMH in the study population was 18.5%, the gingiva being the most commonly affected site. It appears that the CD4+ T cell count does not play any role in the biopathology of HIV-OMH.
doi:10.1155/2016/8389214
PMCID: PMC4783540  PMID: 27006825
19.  Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies 
mBio  2016;7(1):e02154-15.
ABSTRACT
The filovirus surface glycoprotein (GP) mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS) that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics.
IMPORTANCE
Ebola virus uses its glycoprotein (GP) to enter new host cells. During entry, GP must be cleaved by human enzymes in order for receptor binding to occur. Here, we provide the crystal structure of the cleaved form of Ebola virus GP. We demonstrate that cleavage exposes a site at the top of GP and that this site binds the critical domain C of the receptor, termed Niemann-Pick C1 (NPC1). We perform mutagenesis to find parts of the site essential for binding NPC1 and map distinct roles for an upper, charged crest and lower, hydrophobic trough in cleaved GP. We find that this 3-dimensional site is conserved across the filovirus family and that antibody directed against this site is able to bind cleaved GP from every filovirus tested and neutralize viruses bearing those GPs.
doi:10.1128/mBio.02154-15
PMCID: PMC4791852  PMID: 26908579
20.  Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway 
mBio  2016;7(1):e01857-15.
ABSTRACT
Ebola virus (EBOV) makes extensive and intricate use of host factors in the cellular endosomal/lysosomal pathway to release its genome into the cytoplasm and initiate infection. Following viral internalization into endosomes, host cysteine proteases cleave the EBOV fusion glycoprotein (GP) to unmask the binding site for its intracellular receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). GP-NPC1 interaction is required for viral entry. Despite these and other recent discoveries, late events in EBOV entry following GP-NPC1 binding and culminating in GP-catalyzed fusion between viral and cellular lipid bilayers remain enigmatic. A mechanistic understanding of EBOV membrane fusion has been hampered by the failure of previous efforts to reconstitute fusion in vitro or at the cell surface. This report describes an assay to monitor initial steps directly in EBOV membrane fusion—triggering of GP and virus-cell lipid mixing—by single virions in live cells. Fusogenic triggering of GP occurs predominantly in Rab7-positive (Rab7+) endosomes, absolutely requires interaction between proteolytically primed GP and NPC1, and is blocked by key GP-specific neutralizing antibodies with therapeutic potential. Unexpectedly, cysteine protease inhibitors do not inhibit lipid mixing by virions bearing precleaved GP, even though they completely block cytoplasmic entry by these viruses, as shown previously. These results point to distinct cellular requirements for different steps in EBOV membrane fusion and suggest a model in which host cysteine proteases are dispensable for GP fusion triggering after NPC1 binding but are required for the formation of fusion pores that permit genome delivery.
IMPORTANCE
Ebola virus (EBOV) causes outbreaks of highly lethal disease for which no approved vaccines or treatments exist. Recent work has elucidated key molecular features of the complex EBOV entry process, including stepwise interactions with multiple host factors. However, there is a critical gap in our understanding of events that surround the final membrane fusion step which persists due to the paucity of direct and extensive investigation of EBOV fusion. Here, we report a real-time assay for EBOV glycoprotein fusion triggering and use it to define its cellular location and requirements. We also uncover an unexpected requirement for host proteases at a step after fusion triggering that may reflect their role in formation of fusion pores for genome delivery.
doi:10.1128/mBio.01857-15
PMCID: PMC4752599  PMID: 26861015
21.  Clinical Aspects: Focusing on Key Unique Organ-Specific Issues of Renal Transplantation 
Kidney transplantation is the treatment of choice for patients with end-stage renal disease because of improved patient survival and quality of life as compared with dialysis. Successful transplantation requires the prompt recognition and appropriate management of both the immediate posttransplant surgical and medical complications as well as subsequent issues like recurrent disease and chronic rejection that affect long-term graft survival. Guidelines for understanding and managing some of the more important early and late kidney-specific transplant problems, including urologic complications, delayed graft function, acute and chronic rejection, BK polyoma virus infection, and recurrent glomerular disease, are reviewed.
Kidney transplant patients present a complex set of medical issues that require intensive management. These include both immediate posttransplant complications (e.g., delayed graft function) and subsequent issues (e.g., recurrent disease).
doi:10.1101/cshperspect.a015644
PMCID: PMC3904099  PMID: 24370927
22.  The Nuclear DNA Sensor IFI16 Acts as a Restriction Factor for Human Papillomavirus Replication through Epigenetic Modifications of the Viral Promoters 
Journal of Virology  2015;89(15):7506-7520.
ABSTRACT
The human interferon-inducible IFI16 protein, an innate immune sensor of intracellular DNA, was recently demonstrated to act as a restriction factor for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) infection by inhibiting both viral-DNA replication and transcription. Through the use of two distinct cellular models, this study provides strong evidence in support of the notion that IFI16 can also restrict human papillomavirus 18 (HPV18) replication. In the first model, an immortalized keratinocyte cell line (NIKS) was used, in which the IFI16 protein was knocked down through the use of small interfering RNA (siRNA) technology and overexpressed following transduction with the adenovirus IFI16 (AdVIFI16) vector. The second model consisted of U2OS cells transfected by electroporation with HPV18 minicircles. In differentiated IFI16-silenced NIKS-HPV18 cells, viral-load values were significantly increased compared with differentiated control cells. Consistent with this, IFI16 overexpression severely impaired HPV18 replication in both NIKS and U2OS cells, thus confirming its antiviral restriction activity. In addition to the inhibition of viral replication, IFI16 was also able to reduce viral transcription, as demonstrated by viral-gene expression analysis in U2OS cells carrying episomal HPV18 minicircles and HeLa cells. We also provide evidence that IFI16 promotes the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin at both early and late promoters, thus reducing both viral replication and transcription. Altogether, these results argue that IFI16 restricts chromatinized HPV DNA through epigenetic modifications and plays a broad surveillance role against viral DNA in the nucleus that is not restricted to herpesviruses.
IMPORTANCE Intrinsic immunity is mediated by cellular restriction factors that are constitutively expressed and active even before a pathogen enters the cell. The host nuclear factor IFI16 acts as a sensor of foreign DNA and an antiviral restriction factor, as recently demonstrated by our group for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1). Here, we provide the first evidence that IFI16 inhibits HPV18 replication by repressing viral-gene expression and replication. This antiviral restriction activity was observed in immortalized keratinocytes transfected with the religated genomes and in U2OS cells transfected with HPV18 minicircles, suggesting that it is not cell type specific. We also show that IFI16 promotes the assembly of heterochromatin on HPV DNA. These changes in viral chromatin structure lead to the generation of a repressive state at both early and late HPV18 promoters, thus implicating the protein in the epigenetic regulation of HPV gene expression and replication.
doi:10.1128/JVI.00013-15
PMCID: PMC4505635  PMID: 25972554
23.  Activated Nrf2 Interacts with Kaposi's Sarcoma-Associated Herpesvirus Latency Protein LANA-1 and Host Protein KAP1 To Mediate Global Lytic Gene Repression 
Journal of Virology  2015;89(15):7874-7892.
ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. We have previously shown that KSHV utilizes the host transcription factor Nrf2 to aid in infection of endothelial cells and oncogenesis. Here, we investigate the role of Nrf2 in PEL and PEL-derived cell lines and show that KSHV latency induces Nrf2 protein levels and transcriptional activity through the COX-2/PGE2/EP4/PKCζ axis. Next-generation sequencing of KSHV transcripts in the PEL-derived BCBL-1 cell line revealed that knockdown of this activated Nrf2 results in global elevation of lytic genes. Nrf2 inhibition by the chemical brusatol also induces lytic gene expression. Both Nrf2 knockdown and brusatol-mediated inhibition induced KSHV lytic reactivation in BCBL-1 cells. In a series of follow-up experiments, we characterized the mechanism of Nrf2-mediated regulation of KSHV lytic repression during latency. Biochemical assays showed that Nrf2 interacted with KSHV latency-associated nuclear antigen 1 (LANA-1) and the host transcriptional repressor KAP1, which together have been shown to repress lytic gene expression. Promoter studies showed that although Nrf2 alone induces the open reading frame 50 (ORF50) promoter, its association with LANA-1 and KAP1 abrogates this effect. Interestingly, LANA-1 is crucial for efficient KAP1/Nrf2 association, while Nrf2 is essential for LANA-1 and KAP1 recruitment to the ORF50 promoter and its repression. Overall, these results suggest that activated Nrf2, LANA-1, and KAP1 assemble on the ORF50 promoter in a temporal fashion. Initially, Nrf2 binds to and activates the ORF50 promoter during early de novo infection, an effect that is exploited during latency by LANA-1-mediated recruitment of the host transcriptional repressor KAP1 on Nrf2. Cell death assays further showed that Nrf2 and KAP1 knockdown induce significant cell death in PEL cell lines. Our studies suggest that Nrf2 modulation through available oral agents is a promising therapeutic approach in the treatment of KSHV-associated malignancies.
IMPORTANCE KS and PEL are aggressive KSHV-associated malignancies with moderately effective, highly toxic chemotherapies. Other than ganciclovir and alpha interferon (IFN-α) prophylaxis, no KSHV-associated chemotherapy targets the underlying infection, a major oncogenic force. Hence, drugs that selectively target KSHV infection are necessary to eradicate the malignancy while sparing healthy cells. We recently showed that KSHV infection of endothelial cells activates the transcription factor Nrf2 to promote an environment conducive to infection and oncogenesis. Nrf2 is modulated through several well-tolerated oral agents and may be an important target in KSHV biology. Here, we investigate the role of Nrf2 in PEL and demonstrate that Nrf2 plays an important role in KSHV gene expression, lytic reactivation, and cell survival by interacting with the host transcriptional repressor KAP1 and the viral latency-associated protein LANA-1 to mediate global lytic gene repression and thus cell survival. Hence, targeting Nrf2 with available therapies is a viable approach in the treatment of KSHV malignancies.
doi:10.1128/JVI.00895-15
PMCID: PMC4505678  PMID: 25995248
24.  Persistence of CTL clones targeting melanocyte differentiation antigens was insufficient to mediate significant melanoma regression in humans 
Purpose
Adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) can mediate durable cancer regression in selected patients with metastatic melanoma. However, the tumor antigens associated with these favorable responses remain unclear. We hypothesized that a clinical strategy involving the iterative adoptive transfer of selected autologous antigen specific T cell clones could help systematically define immunologic targets associated with successful cancer therapy, without the interpretative ambiguity of transferring polyclonal populations. Here, we evaluated the clinical efficacy of CD8+ T cell clones specific for the melanocyte differentiation antigens (MDA), gp100 and MART-1, respectively.
Experimental Design
We conducted two consecutive phase II clinical trials involving the adoptive transfer of highly selected autologous antigen specific CD8+ T cell clones against gp100 and MART-1, respectively. Fifteen HLA-A2+ treatment-refractory metastatic melanoma patients received highly avid MDA specific CD8+ T cell clones specific for either gp100 (n=10) or MART-1 (n=5) with or without intravenous interleukin-2 after a lymphodepleting myeloablative preparative regimen.
Results
Of the fifteen treated patients, we observed immune mediated targeting of skin melanocytes in eleven patients (73%) and clonal engraftment in eight patients (53%) after cell transfer. There were only transient minor tumor regressions observed, but no objective tumor responses based upon RECIST criteria.
Conclusions
Despite successful clonal repopulation and evidence of in vivo antigen targeting, the poor therapeutic efficacy after the adoptive transfer of autologous MDA specific T cells raises significant concerns regarding future immunotherapy efforts targeting this class of tumor antigens.
doi:10.1158/1078-0432.CCR-14-2208
PMCID: PMC4315711  PMID: 25424856
Immunotherapy; CTL clones; melanocyte differentiation antigens; metastatic melanoma
25.  Loose Plant Architecture1 (LPA1) determines lamina joint bending by suppressing auxin signalling that interacts with C-22-hydroxylated and 6-deoxo brassinosteroids in rice 
Journal of Experimental Botany  2016;67(6):1883-1895.
Highlight
LPA1 suppresses auxin signalling that interacts with C-22-hydroxylated and 6-deoxo brassinosteroids, which regulates lamina inclination independently of OsBRI1.
Lamina inclination is a key agronomical character that determines plant architecture and is sensitive to auxin and brassinosteroids (BRs). Loose Plant Architecture1 (LPA1) in rice (Oryza sativa) and its Arabidopsis homologues (SGR5/AtIDD15) have been reported to control plant architecture and auxin homeostasis. This study explores the role of LPA1 in determining lamina inclination in rice. LPA1 acts as a positive regulator to suppress lamina bending. Genetic and biochemical data indicate that LPA1 suppresses the auxin signalling that interacts with C-22-hydroxylated and 6-deoxo BRs, which regulates lamina inclination independently of OsBRI1. Mutant lpa1 plants are hypersensitive to indole-3-acetic acid (IAA) during the lamina inclination response, which is suppressed by the brassinazole (Brz) inhibitor of C-22 hydroxylase involved in BR synthesis. A strong synergic effect is detected between lpa1 and d2 (the defective mutant for catalysis of C-23-hydroxylated BRs) during IAA-mediated lamina inclination. No significant interaction between LPA1 and OsBRI1 was identified. The lpa1 mutant is sensitive to C-22-hydroxylated and 6-deoxo BRs in the d61-1 (rice BRI1 mutant) background. We present evidence verifying that two independent pathways function via either BRs or BRI1 to determine IAA-mediated lamina inclination in rice. RNA sequencing analysis and qRT-PCR indicate that LPA1 influences the expression of three OsPIN genes (OsPIN1a, OsPIN1c and OsPIN3a), which suggests that auxin flux might be an important factor in LPA1-mediated lamina inclination in rice.
doi:10.1093/jxb/erw002
PMCID: PMC4783368  PMID: 26826218
Auxin; C-22-hydroxylated BRs; lamina inclination; lamina joints; Loose Plant Architecture1 (LPA1); OsPINs; plant architecture; rice (Oryza sativa); 6-deoxos BRs.

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