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author:("ramanis, S.")
1.  Rapid flow cytometric susceptibility testing of Candida albicans. 
Journal of Clinical Microbiology  1997;35(9):2320-2324.
A rapid flow cytometric assay for in vitro antifungal drug susceptibility testing was developed by adapting the proposed reference method for broth macrodilution testing of yeasts. Membrane permeability changes caused by the antifungal agent were measured by flow cytometry using propidium iodide, a nucleic acid-binding fluorochrome largely excluded by the intact cell membrane. We determined the in vitro susceptibility of 31 Candida albicans isolates and two quality control strains (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) to amphotericin B and fluconazole. Amphotericin B MICs ranged from 0.03 to 2.0 microg/ml, while fluconazole MICs ranged from 0.125 to 128 microg/ml. This method results in clear-cut endpoints that were reproducible. Four-hour incubation was required for fluconazole, whereas a 2-h incubation was sufficient for amphotericin B to provide MICs comparable to the reference macrodilution method developed by the National Committee for Clinical Laboratory Standards Subcommittee on Antifungal Susceptibility Tests. Results of these studies show that flow cytometry provides a rapid and sensitive in vitro method for antifungal susceptibility testing of C. albicans.
PMCID: PMC229962  PMID: 9276410
2.  Ultrasound-guided needle aspiration of amoebic liver abscess. 
Postgraduate Medical Journal  1993;69(811):381-383.
This prospective study was carried out on 200 patients with clinically, ultrasonographically and serologically confirmed amoebic liver abscess. The role of ultrasound-guided needle aspiration in addition to medications was evaluated compared to drug treatment alone. Both the groups were monitored clinically and sonographically for up to 6 months after diagnosis. The initial response (after 15 days) was better in the aspirated group (P < 0.05) but resolution of abscess after 6 months were similar. There was a more rapid clinical response in the aspirated group, particularly in those with larger (> 6 cm) abscesses and there were no complications. Percutaneous ultrasound-guided needle aspiration is a safe diagnostic and therapeutic approach which enhances clinical recovery, accelerates resolution, especially in large abscesses, and prevents complications.
PMCID: PMC2399819  PMID: 8346134
3.  Polypharmacy and Potentially Inappropriate Medication Use among Older Adults with Cancer Undergoing Chemotherapy: Impact on Chemotherapy-Related Toxicity and Hospitalization During Treatment 
Polypharmacy and potentially inappropriate medication (PIM) use are understudied among older adults with cancer undergoing chemotherapy. The current study’s aims were to evaluate in this population: 1) the prevalence of polypharmacy and PIM use; and 2) the association between these and chemotherapy-related adverse events.
Methods
This was a secondary analysis of prospectively collected data of adults age ≥65 years with cancer undergoing chemotherapy. Measures included: the number of daily medications (i.e, polypharmacy); PIM use based on 3 indices [Beers, Zhan, and Drugs to Avoid in the Elderly (DAE) criteria], as well as use of 6 “high-risk” medication classes for adverse drug events (i.e., anticoagulants, antiplatelet agents, opioids, insulin, oral hypoglycemics and antiarrhythmics). Using multivariate logistic regression, the relations were evaluated between these criteria and 1) Grade 3-5 chemotherapy-related toxicity; and 2) hospitalization during chemotherapy.
Results
The patients (N=500; mean age, 73 years, 61% Stage IV disease) took a mean of 5 daily medications (±4; range, 0-23). PIM use among patients was common (up to 29% using Beers criteria). No association was found between the number of daily medications and either toxicity (0-3 medications as reference: 4-9, OR=1.34, 95% CI: 0.92-1.97; ≥10, OR=0.82, 95% CI: 0.45-1.49), or hospitalization (0-3 medications as reference, ≥4, OR=1.34, 95%CI: 0.82-2.18, p=0.24). There was also no association between PIM use and toxicity (p=0.93) or hospitalization (p=0.98). No medication class was associated with either outcome.
Conclusions
Polypharmacy and PIM use were common but werenot associated with chemotherapy-related toxicity or hospitalization in older adults with cancer.
doi:10.1111/jgs.12942
PMCID: PMC4134360  PMID: 25041361
Polypharmacy; Cancer; Elderly; Chemotherapy; Toxicity
4.  Gene Expression in Experimental Aortic Coarctation and Repair: Candidate Genes for Therapeutic Intervention? 
PLoS ONE  2015;10(7):e0133356.
Coarctation of the aorta (CoA) is a constriction of the proximal descending thoracic aorta and is one of the most common congenital cardiovascular defects. Treatments for CoA improve life expectancy, but morbidity persists, particularly due to the development of chronic hypertension (HTN). Identifying the mechanisms of morbidity is difficult in humans due to confounding variables such as age at repair, follow-up duration, coarctation severity and concurrent anomalies. We previously developed an experimental model that replicates aortic pathology in humans with CoA without these confounding variables, and mimics correction at various times using dissolvable suture. Here we present the most comprehensive description of differentially expressed genes (DEGs) to date from the pathology of CoA, which were obtained using this model. Aortic samples (n=4/group) from the ascending aorta that experiences elevated blood pressure (BP) from induction of CoA, and restoration of normal BP after its correction, were analyzed by gene expression microarray, and enriched genes were converted to human orthologues. 51 DEGs with >6 fold-change (FC) were used to determine enriched Gene Ontology terms, altered pathways, and association with National Library of Medicine Medical Subject Headers (MeSH) IDs for HTN, cardiovascular disease (CVD) and CoA. The results generated 18 pathways, 4 of which (cell cycle, immune system, hemostasis and metabolism) were shared with MeSH ID’s for HTN and CVD, and individual genes were associated with the CoA MeSH ID. A thorough literature search further uncovered association with contractile, cytoskeletal and regulatory proteins related to excitation-contraction coupling and metabolism that may explain the structural and functional changes observed in our experimental model, and ultimately help to unravel the mechanisms responsible for persistent morbidity after treatment for CoA.
doi:10.1371/journal.pone.0133356
PMCID: PMC4514739  PMID: 26207811
5.  HIV/AIDS in Older Women: Unique Challenges, Unmet Needs 
As persons living with HIV/AIDS live longer, both the prevalence and incidence of HIV infection in older women is expected to increase, and this review presents a model and review of the extant literature on older women with HIV/AIDS in the United States. Older women are rarely addressed in the discourse about HIV risk and prevention, and their concerns are often missed by risk reduction programs that typically target men and younger adults. Societal biases around aging can compound factors such as stigma and disclosure for older women. Primary care providers are often not recommending routine HIV testing to older women, or addressing the impact of age related physiological changes on risk and sexual health. Many older women may be starting new relationships and the role of relational variables that are specific to this group of women are key in understanding prevention and treatment. Empirical research focused on the needs of older women, and recognition of the diverse composition and needs of this group is needed to inform prevention, intervention and best practices with this population of women.
doi:10.1080/08964289.2014.893983
PMCID: PMC4152459  PMID: 25090361
HIV; older women; sexuality; risk; stigma
6.  Using ‘appreciative inquiry’ in India to improve infection control practices in maternity care: a qualitative study 
Global Health Action  2015;8:10.3402/gha.v8.26693.
Background
Infections acquired during childbirth are a common cause of maternal and perinatal mortality and morbidity. Changing provider behaviour and organisational settings within the health system is key to reducing the spread of infection.
Objective
To explore the opinions of health personnel on health system factors related to infection control and their perceptions of change in a sample of hospital maternity units.
Design
An organisational change process called ‘appreciative inquiry’ (AI) was introduced in three maternity units of hospitals in Gujarat, India. AI is a change process that builds on recognition of positive actions, behaviours, and attitudes. In-depth interviews were conducted with health personnel to elicit information on the environment within which they work, including physical and organisational factors, motivation, awareness, practices, perceptions of their role, and other health system factors related to infection control activities. Data were obtained from three hospitals which implemented AI and another three not involved in the intervention.
Results
Challenges which emerged included management processes (e.g. decision-making and problem-solving modalities), human resource shortages, and physical infrastructure (e.g. space, water, and electricity supplies). AI was perceived as having a positive influence on infection control practices. Respondents also said that management processes improved although some hospitals had already undergone an accreditation process which could have influenced the changes described. Participants reported that team relationships had been strengthened due to AI.
Conclusion
Technical knowledge is often emphasised in health care settings and less attention is paid to factors such as team relationships, leadership, and problem solving. AI can contribute to improving infection control by catalysing and creating forums for team building, shared decision making and problem solving in an enabling environment.
doi:10.3402/gha.v8.26693
PMCID: PMC4483369  PMID: 26119249
India; infection control; sepsis; maternal health; maternity services; appreciative inquiry
7.  A pair of RNA binding proteins controls networks of splicing events contributing to specialization of neural cell types 
Molecular cell  2014;54(6):946-959.
SUMMARY
Alternative splicing is important for the development and function of the nervous system, but little is known about the differences in alternative splicing between distinct types of neurons. Furthermore, the factors that control cell-type-specific splicing and the physiological roles of these alternative isoforms are unclear. By monitoring alternative splicing at single cell resolution in Caenorhabditis elegans, we demonstrate that splicing patterns in different neurons are often distinct and highly regulated. We identify two conserved RNA binding proteins, UNC-75/CELF and EXC-7/Hu/ELAV, which regulate overlapping networks of splicing events in GABAergic and cholinergic neurons. We use the UNC-75 exon network to discover regulators of synaptic transmission and to identify unique roles for isoforms of UNC-64/Syntaxin, a protein required for synaptic vesicle fusion. Our results indicate that combinatorial regulation of alternative splicing in distinct neurons provides a mechanism to specialize metazoan nervous systems.
doi:10.1016/j.molcel.2014.05.004
PMCID: PMC4096705  PMID: 24910101
8.  Core outcome measures in perioperative and anaesthetic care 
Trials  2015;16(Suppl 1):P2.
doi:10.1186/1745-6215-16-S1-P2
PMCID: PMC4460931
9.  Monte Carlo SURE-Based Parameter Selection for Parallel Magnetic Resonance Imaging Reconstruction 
Purpose
Regularizing parallel MRI reconstruction significantly improves image quality but requires tuning parameter selection. We propose a Monte Carlo method for automatic parameter selection based on Stein’s unbiased risk estimate (SURE) that minimizes the multi-channel k-space mean squared error (MSE). We automatically tune parameters for image reconstruction methods that preserve the undersampled acquired data, which cannot be accomplished using existing techniques.
Theory
We derive a weighted MSE criterion appropriate for data-preserving regularized parallel imaging reconstruction and the corresponding weighted SURE. We describe a Monte Carlo approximation of the weighted SURE that uses two evaluations of the reconstruction method per candidate parameter value.
Methods
We reconstruct images using the sparsity-promoting methods DESIGN and L1-SPIRiT. We validate Monte Carlo SURE against the weighted MSE. We select the regularization parameter using these methods for various noise levels and undersampling factors and compare the results to those using MSE-optimal parameters.
Results
Our method selects nearly MSE-optimal regularization parameters for both DESIGN and L1-SPIRiT over a range of noise levels and undersampling factors.
Conclusion
The proposed method automatically provides nearly MSE-optimal choices of regularization parameters for data-preserving nonlinear parallel MRI reconstruction methods.
doi:10.1002/mrm.24840
PMCID: PMC3858446  PMID: 23821331
parallel imaging reconstruction; regularization parameter selection; Stein’s unbiased risk estimate (SURE); Monte Carlo methods
10.  Generalized Higher Degree Total Variation (HDTV) Regularization 
We introduce a family of novel image regularization penalties called generalized higher degree total variation (HDTV). These penalties further extend our previously introduced HDTV penalties, which generalize the popular total variation (TV) penalty to incorporate higher degree image derivatives. We show that many of the proposed second degree extensions of TV are special cases or are closely approximated by a generalized HDTV penalty. Additionally, we propose a novel fast alternating minimization algorithm for solving image recovery problems with HDTV and generalized HDTV regularization. The new algorithm enjoys a ten-fold speed up compared to the iteratively reweighted majorize minimize algorithm proposed in a previous work. Numerical experiments on 3D magnetic resonance images and 3D microscopy images show that HDTV and generalized HDTV improve the image quality significantly compared with TV.
doi:10.1109/TIP.2014.2315156
PMCID: PMC4411246  PMID: 24710832
11.  Design and methodology of SNAP-1: a Sprint National Anaesthesia Project to measure patient reported outcome after anaesthesia 
Background
Patient satisfaction is an important metric of health-care quality. Accidental awareness under general anaesthesia (AAGA) is a serious complication of anaesthesia care which may go unrecognised in the immediate perioperative period but leads to long-term psychological harm for affected patients. The SNAP-1 study aimed to measure patient satisfaction with anaesthesia care and the incidence of AAGA, reported on direct questioning within 24 h of surgery, in a large multicentre cohort. A secondary aim of SNAP-1 was to test the effectiveness of a new network of Quality Audit and Research Coordinators in NHS anaesthetic departments, to achieve widespread study participation and high patient recruitment rates. This manuscript describes the study methodology.
Methods
SNAP-1 was a prospective observational cohort study. The study protocol was approved by the National Research Ethics Service. All UK NHS hospitals with anaesthetic departments were invited to participate. Adult patients undergoing any type of non-obstetric surgery were recruited in participating hospitals on 13th and 14th May 2014. Demographic data were collected by anaesthetists providing perioperative care. Patients were then approached within 24 h of surgery to complete two questionnaires—the Bauer patient satisfaction questionnaire (to measure patient reported outcome) and the modified Brice questionnaire (to detect possible accidental awareness). Completion of postoperative questionnaires was taken as evidence of implied consent. Results were recorded on a standard patient case report form, and local investigators entered anonymised data into an electronic database for later analysis by the core research team.
Results
Preliminary analyses indicate that over 15,000 patients were recruited across the UK, making SNAP-1 the largest NIHR portfolio-adopted study in anaesthesia to date. Both descriptive and analytic epidemiological analyses will be used to answer specific questions about the patient perception of anaesthesia care overall and in surgical sub-specialties and to determine the incidence of AAGA.
Conclusions
The SNAP-1 study recruited a large number of UK hospitals and thousands of perioperative patients using newly established networks in the UK anaesthetic profession. The results will provide benchmarking information to aid interpretation of patient satisfaction data and also determine the incidence of AAGA reported on a single postoperative visit.
Electronic supplementary material
The online version of this article (doi:10.1186/s13741-015-0011-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13741-015-0011-2
PMCID: PMC4422533  PMID: 25949810
Patient-reported outcome; Patient satisfaction; Anesthesia awareness; Epidemiology; Cohort study
12.  Ceftaroline fosamil for the treatment of community-acquired bacterial pneumonia in the intensive care unit 
The Clinical Assessment Program and Teflaro® Utilization Registry (CAPTURE) is a multicenter study evaluating the clinical use of ceftaroline fosamil in patients with community-acquired bacterial pneumonia (CABP) or acute bacterial skin and skin structure infection. Data were collected between August 2011 and February 2013, from 398 evaluable patients receiving treatment at 33 sites in the USA. This manuscript presents data collected from patients with CABP who received care in an intensive care unit (ICU) or in general medical wards (35% and 64% of evaluable patients, respectively). The majority of ICU and general medical ward patients had underlying comorbidities (78% and 74%, respectively), with structural lung disease being the most common (42% in the ICU and 40% in general medical wards). Patients admitted to the ICU had a longer duration of stay, a longer duration of symptoms before treatment, and a longer duration of ceftaroline fosamil therapy than did general medical ward patients. Most patients treated in the ICU and in general medical wards were given ceftaroline fosamil as second-line therapy (87% and 80%, respectively). The overall rate of clinical success for patients treated with ceftaroline fosamil was 68% in the ICU and 85% in the general medical wards. Clinical success for patients receiving ceftaroline fosamil as a second-line agent was 84% in the ICU and 86% in general medical wards. These findings indicate that ceftaroline fosamil is a viable treatment option for CABP, both in the ICU and in general medical wards.
doi:10.2147/TCRM.S75191
PMCID: PMC4397928  PMID: 25897240
CAPTURE; registry
13.  Determination of the 50% Human Infectious Dose for Norwalk Virus 
The Journal of Infectious Diseases  2013;209(7):1016-1022.
Background. Noroviruses are the most common cause of gastroenteritis in the United States. An understanding of the infectious dose of these viruses is important for risk assessment studies.
Methods. Healthy adults were enrolled in a randomized, double-blind, placebo-controlled evaluation of different dosages of Norwalk virus. Eligible subjects were monitored for clinical gastroenteritis, and infection status was determined. The presence of virus in vomitus was also assessed.
Results. Fifty-seven persons were enrolled; 8 received placebo and an additional 8 persons were considered to be nonsusceptible on the basis of being secretor negative. Twenty-one persons were infected (all blood group O or A), and 67% of those infected developed viral gastroenteritis. The 50% human infectious dose was calculated to be 3.3 reverse transcription polymerase chain reaction units (approximately 1320 genomic equivalents [gEq]) for secretor-positive blood group O or A persons and 7.0 (approximately 2800 gEq) for all secretor-positive persons. The time of illness onset was inversely correlated with inoculum dose. The maximal concentration of virus shedding was higher for persons with gastroenteritis. Norwalk virus was identified in 15 of 27 (56%) vomitus samples at a median concentration of 41 000 gEq/mL.
Conclusions. The 50% human infectious dose measured is higher than previous estimates and similar to that of other RNA viruses.
Clinical Trials Registration NCT00138476.
doi:10.1093/infdis/jit620
PMCID: PMC3952671  PMID: 24253285
norovirus; infectious dose; virus shedding; serology; secretor
14.  Hierarchical O(N) computation of small-angle scattering profiles and their associated derivatives 
Journal of Applied Crystallography  2014;47(Pt 2):755-761.
Structure refinement protocols based on small-angle X-ray scattering iteratively adjust the posited positions of atoms in a structure via a gradient-based constrained optimization to ensure that experimental scattering profile data are matched. This requires computation of the profile and its gradient with respect to atomic coordinates at every step. The fast algorithms proposed in this article reduce these to a cost that is linear in the number of atoms for any specified accuracy.
The need for fast approximate algorithms for Debye summation arises in computations performed in crystallography, small/wide-angle X-ray scattering and small-angle neutron scattering. When integrated into structure refinement protocols these algorithms can provide significant speed up over direct all-atom-to-all-atom computation. However, these protocols often employ an iterative gradient-based optimization procedure, which then requires derivatives of the profile with respect to atomic coordinates. This article presents an accurate, O(N) cost algorithm for the computation of scattering profile derivatives. The results reported here show orders of magnitude improvement in computational efficiency, while maintaining the prescribed accuracy. This opens the possibility to efficiently integrate small-angle scattering data into the structure determination and refinement of macromolecular systems.
doi:10.1107/S1600576714004671
PMCID: PMC3970052  PMID: 24701198
small-angle scattering; wide-angle scattering; Jacobian; gradients
15.  The Effect of Aromatase Inhibition on the Cognitive Function of Older Patients with Breast Cancer 
Clinical breast cancer  2013;14(2):132-140.
Purpose
To evaluate the association between aromatase inhibitor (AI) therapy and cognitive function (over a 6-month time period) in a cohort of patients age ≥ 60 compared with an age-matched healthy control group, and to evaluate changes in regional cerebral metabolism as measured by positron emission tomography (PET) scans of the brain done in a subset of the patient cohort.
Patients and Methods
Thirty-five patients (32 evaluable) and 35 healthy controls were recruited to this study. Patients with breast cancer completed a neuropsychological battery, self-reported memory questionnaire, and geriatric assessment prior to initiation of AI therapy and again 6 months later. Age-matched healthy control participants completed the same assessments at the same time points as the patient group.
Results
No significant decline in cognitive function was seen among individuals receiving an AI from pre-treatment to 6 months later compared with healthy controls. In the PET cohort over the same period, both standardized volume of interest (sVOI) and statistical parametric mapping (SPM) analyses detected specific changes in metabolic activity between baseline and follow-up uniquely in the AI patients, uniquely, most significantly in medial temporal lobes.
Conclusion
While patients undergoing AI treatment demonstrated few changes in neuropsychologic performance compared with healthy controls over a 6-month period during this interval, regionally specific changes in cerebral metabolic activity were identified in the patient group. Additional longitudinal follow-up is needed to understand the potential clinical implications of these findings.
doi:10.1016/j.clbc.2013.10.010
PMCID: PMC4103787  PMID: 24291380
cognitive function; older adults; breast cancer; aromatase inhibitors; PET scan
16.  The Comparative Evaluation of the Translucency of Crowns Fabricated with Three Different All-Ceramic Materials: An in Vitro Study 
Introduction: All-ceramic crowns with different core materials of different strength and aesthetics are available in recent years. The aesthetics of the crown depends mainly on the shade and translucency. Clinician should be aware of the quality and characteristics of these materials so that they will be able to opt for good material for successful clinical use.
Aim and Objective: The aim of this study was to evaluate and compare the translucency of crowns fabricated with three different commercially available all-ceramic materials viz. Alumina - CAD-CAM Procera, Lithium disilicate - Pressable IPS e.max Press, Zirconia - CAD-CAM Lava.
Materials and Methods: All-ceramic crowns (5 per each group and total of 15 samples) were made of Alumina – CAD-CAM Procera (Group I), Lithium disilicate – Pressable IPS e.max Press (Group II), Zirconia – CAD-CAM Lava (Group III) and veneered with their respective layering ceramic. Evaluation for the Translucency (CR=Yb/Yw) over the White (Yw) and Black (Yb) backgrounds at the Incisal, Middle, Cervical, Mesial and Distal thirds of each crown were done using the Spectrophotometer. The results obtained were statistically analyzed by Paired t-test (p<0.05) and Analysis of Variance (p<0.05) for the test of significance among the groups.
Results: Significant differences in the contrast ratios were obtained among the three Groups (p<0.001). In this study, Group II Lithium disilicate–Pressable IPS e.max Press showed higher translucency (0.54). Group III Zirconia – CAD-CAM Lava showed the least translucency (0.75) and the translucency of Group I Alumina – CAD-CAM Procera (0.7) was in between both the groups.
Conclusion: Translucency of material gives fair idea to clinician for the choice of material in different zones during replacement and suitability for restoration in aesthetic zone. Selection of all ceramic system depends on the translucency needed for successful prosthesis of artificial tooth so that it mimics patient’s natural dentition. The qualitative measurement of translucency will give the evidence for the clinicians during selection of high or low value translucent tooth for successful replacement. Lithium disilicate – Pressable IPS e.max Press is having better translucency in comparison with other two materials in our study.
doi:10.7860/JCDR/2015/12069.5559
PMCID: PMC4378803  PMID: 25859521
Alumina-CAD-CAM procera; Aesthetics; Contrast ratio; Lithium disilicate-pressable IPS e.max press; Metal free ceramics; Spectrophotometer; Transmitted light; Zirconia - CAD-CAM Lava
17.  Chemotherapy stimulates syndecan-1 shedding: A potentially negative effect of treatment that may promote tumor relapse 
In patients with multiple myeloma, the heparan sulfate proteoglycan syndecan-1 (CD138) is shed from the surface of tumor cells and accumulates in the serum and within the extracellular matrix of the bone marrow where it promotes tumor growth and metastasis. In the present study we discovered that commonly used anti-myeloma drugs stimulate syndecan-1 shedding both in vitro and in animals bearing myeloma tumors. Enhanced shedding is accompanied by increased syndecan-1 synthesis prior to drug induced tumor cell death. Addition of a caspase inhibitor blocks the drug-induced shedding of syndecan-1 in vitro indicating that shedding is linked to the onset of apoptosis. ADAMs inhibitors or siRNA targeting ADAMs blocked drug-induced shedding suggesting that up regulation or activation of ADAMs is responsible for cleaving syndecan-1 from the tumor cell surface. These results reveal that myeloma chemotherapy stimulates synthesis and shedding of syndecan-1, a potentially negative side effect that may lead to accumulation of high levels of syndecan-1 to establish a microenvironment that nurtures relapse and promotes tumor progression. Interestingly, we also found that chemotherapeutic drugs stimulated syndecan-1 shedding from pancreatic cancer cells as well, indicating that drug-induced shedding of syndecan-1 may occur in many cancer types. Overall, our results indicate that use of metalloproteinase inhibitors (to inhibit syndecan-1 shedding) in combination with chemotherapy may represent a novel therapeutic strategy to prevent re-establishment of a microenvironment conducive for tumor relapse.
doi:10.1016/j.matbio.2013.10.005
PMCID: PMC4377822  PMID: 24145151
Chemotherapy; multiple myeloma; shedding; shed syndecan-1; batimastat; hepatocyte growth factor
18.  Delta-like 4 mRNA is regulated by adjacent natural antisense transcripts 
Vascular Cell  2015;7:3.
Background
Recent evidence suggests that a majority of RNAs in the genome do not code for proteins. They are located in the sense (S) or antisense (AS) orientation and, to date, the functional significance of these non-coding RNAs (ncRNAs) is poorly understood. Here, we examined the relationship between S and AS transcripts in the regulation of a key angiogenesis gene, Delta-like 4 (Dll4).
Methods
Rapid Amplification of cDNA Ends (RACE) method was used to identify natural antisense transcripts in the Dll4 gene locus in murine and human endothelial cells, referred to as Dll4 Anti-Sense (Dll4-AS). Messenger RNA (mRNA) levels of Dll4 and Dll4-AS were quantified by real-time PCR. The function of Dll4-AS was investigated by overexpression and knocking down of Dll4-AS.
Results
Dll4-AS comprises of three isoforms that map proximal to the Dll4 promoter region. Expression patterns of Dll4-AS isoforms vary among different endothelial cell lines, but are always congruent with those of Dll4. A dual promoter element in the Dll4 locus has been identified that controls the expression of both transcripts. Both Dll4-AS and Dll4 are sensitive to cellular density in that higher cellular density favors their expression. Exogenous Dll4 stimuli such as VEGF, FGF and Notch signaling inhibitor altered both DLL4-AS and DLL4 expression suggesting co-regulation of the transcripts. Also, knocking down of Dll4-AS results in down-regulation of Dll4 expression. As a consequence, endothelial cell proliferation and migration increases in vitro, and sprout formation increases. The regulation of Dll4 by Dll4-AS was also conserved in vivo.
Conclusion
A novel form of non-coding RNA-mediated regulation at the Dll4 locus contributes to vascular developmental processes such as cell proliferation, migration and sprouting.
Electronic supplementary material
The online version of this article (doi:10.1186/s13221-015-0028-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s13221-015-0028-9
PMCID: PMC4409748  PMID: 25914805
Non-coding RNA; Delta-like4; Vascular; Hemangiomas
19.  SIRT2 regulates ciliogenesis and contributes to abnormal centrosome amplification caused by loss of polycystin-1 
Human Molecular Genetics  2013;23(6):1644-1655.
The mechanisms underlying many of the human disease phenotypes associated with ciliary dysfunction and abnormal centrosome amplification have yet to be fully elucidated. Here, we present for the first time that SIRT2, a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, regulates ciliogenesis and centrosome amplification. Overexpression of SIRT2 in renal epithelial cells appeared to disrupt cilia formation, causing decreased numbers of cells with cilia and decreased cilia length, while inhibition of SIRT2 activity by nicotinamide treatment or knockdown of SIRT2 with siRNA was shown to block cilia disassembly during the cell cycle. Overexpression of SIRT2 in zebrafish decreased cilia numbers in Kupffer's vesicle, while morpholino knock down of SIRT2 increased cilia length. Aberrant centrosome amplification and polyploidy were seen with overexpression of SIRT2 in mouse inner medullary collecting duct 3 cells, similar to that observed following Pkd1 knockdown. SIRT2 was up-regulated in both Pkd1 mutant and knockdown cells. Depletion of SIRT2 prevented the abnormal centrosome amplification and polyploidy associated with loss of polycystin-1 (PC1) alone. Thus, we conclude that the aberrant centrosome amplification and polyploidy in Pkd1 mutant or depleted cells was mediated through overexpression of SIRT2. Our results suggest a novel function of SIRT2 in cilia dynamics and centrosome function, and in ciliopathy-associated disease progression.
doi:10.1093/hmg/ddt556
PMCID: PMC3929098  PMID: 24203696
20.  A Rare Case of Malignant Fibrous Histiocytoma/ Pleomorphic Undifferentiated Sarcoma of the Kidney 
Malignant Fibrous Histiocytoma (MFH) usually arises from the extremities with the retroperitoneum being the second most common site. We present the case report of a 58-year-old man presenting with fever and was detected to have a renal mass on evaluation. He underwent a radical nephrectomy with a preoperative diagnosis of renal cell carcinoma. But the final histopathological report was MFH. Primary renal MFH is extremely rare and is usually diagnosed on histopathology with the aid of immunohistochemistry.
doi:10.7860/JCDR/2015/9460.5493
PMCID: PMC4347127  PMID: 25738036
CD 68; Kidney; Radical nephrectomy
21.  Heat-Passing Framework for Robust Interpretation of Data in Networks 
PLoS ONE  2015;10(2):e0116121.
Researchers are regularly interested in interpreting the multipartite structure of data entities according to their functional relationships. Data is often heterogeneous with intricately hidden inner structure. With limited prior knowledge, researchers are likely to confront the problem of transforming this data into knowledge. We develop a new framework, called heat-passing, which exploits intrinsic similarity relationships within noisy and incomplete raw data, and constructs a meaningful map of the data. The proposed framework is able to rank, cluster, and visualize the data all at once. The novelty of this framework is derived from an analogy between the process of data interpretation and that of heat transfer, in which all data points contribute simultaneously and globally to reveal intrinsic similarities between regions of data, meaningful coordinates for embedding the data, and exemplar data points that lie at optimal positions for heat transfer. We demonstrate the effectiveness of the heat-passing framework for robustly partitioning the complex networks, analyzing the globin family of proteins and determining conformational states of macromolecules in the presence of high levels of noise. The results indicate that the methodology is able to reveal functionally consistent relationships in a robust fashion with no reference to prior knowledge. The heat-passing framework is very general and has the potential for applications to a broad range of research fields, for example, biological networks, social networks and semantic analysis of documents.
doi:10.1371/journal.pone.0116121
PMCID: PMC4323200  PMID: 25668316
22.  Absence of Genetic Differences among G10P[11] Rotaviruses Associated with Asymptomatic and Symptomatic Neonatal Infections in Vellore, India 
Journal of Virology  2014;88(16):9060-9071.
ABSTRACT
Rotaviruses (RVs) are leading causes of severe diarrhea and vomiting in infants and young children. RVs with G10P[11] genotype specificity have been associated with symptomatic and asymptomatic neonatal infections in Vellore, India. To identify possible viral genetic determinants responsible for differences in symptomology, the genome sequences of G10P[11] RVs in stool samples of 19 neonates with symptomatic infections and 20 neonates with asymptomatic infections were determined by Sanger and next-generation sequencing. The data showed that all 39 viruses had identical genotype constellations (G10-P[11]-I2-R2-C2-M2-A1-N1-T1-E2-H3), the same as those of the previously characterized symptomatic N155 Vellore isolate. The data also showed that the RNA and deduced protein sequences of all the Vellore G10P[11] viruses were nearly identical; no nucleotide or amino acid differences were found that correlated with symptomatic versus asymptomatic infection. Next-generation sequencing data revealed that some stool samples, both from neonates with symptomatic infections and from neonates with asymptomatic infections, also contained one or more positive-strand RNA viruses (Aichi virus, astrovirus, or salivirus/klassevirus) suspected of being potential causes of pediatric gastroenteritis. However, none of the positive-strand RNA viruses could be causally associated with the development of symptoms. These results indicate that the diversity of clinical symptoms in Vellore neonates does not result from genetic differences among G10P[11] RVs; instead, other undefined factors appear to influence whether neonates develop gastrointestinal disease symptoms.
IMPORTANCE Rotavirus (RV) strains have been identified that preferentially replicate in neonates, in some cases, without causing gastrointestinal disease. Surveillance studies have established that G10P[11] RVs are a major cause of neonatal infection in Vellore, India, with half of infected neonates exhibiting symptoms. We used Sanger and next-generation sequencing technologies to contrast G10P[11] RVs recovered from symptomatic and asymptomatic neonates. Remarkably, the data showed that the RNA genomes of the viruses were virtually indistinguishable and lacked any differences that could explain the diversity of clinical outcomes among infected Vellore neonates. The sequencing results also indicated that some symptomatic and some asymptomatic Vellore neonates were infected with other enteric viruses (Aichi virus, astrovirus, salvirus/klassevirus); however, none could be correlated with the presence of symptoms in neonates. Together, our findings suggest that other poorly defined factors, not connected to the genetic makeup of the Vellore G10P[11] viruses, influence whether neonates develop gastrointestinal disease symptoms.
doi:10.1128/JVI.01417-14
PMCID: PMC4136299  PMID: 24899175
23.  Amino Acid Profile in Oral Submucous Fibrosis: A High Performance Liquid Chromatography (HPLC) Study 
Background: Collagen is a significant structural protein, the integrity of which is essential to be maintained for proper homeostasis. Oral submucous fibrosis (OSMF), being a collagen metabolic disorder, may be subject to changes in amino acid profiling.
Aim: The present study was attempted to evaluate the amino acid profile to assess its feasibility as a biological marker in OSMF.
Materials and Methods: The study group comprised of 13 patents with OSMF and the normal group comprised of 13 normal patients without associated habits or systemic disorders. Venous blood was collected from the antecubital vein, plasma was separated and the plasma was then subjected to high profile liquid chromatographic analysis.
Results: The assay levels of threonine, alanine and tyrosine did not yield any significant results. The decreased assay levels of valine, Isoleucine and the increased assay level of methionine and glycine observed in group II yielded significant results in correlation with the control group. The decreased assay level seen in phenylalanine in group II and III in correlation with group IV is statistically significant.
Conclusion: A few amino acids have been identified which can be used as biological markers for the severity of the disease such as valine, methionine and phenyl alanine. Large scale studies are required to elucidate the potential of these biological markers.
doi:10.7860/JCDR/2014/10201.5290
PMCID: PMC4316336  PMID: 25654030
Amino acids; Collagen; OSMF; Plasma
24.  Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome-blocking complexes 
Aging Cell  2014;14(1):35-48.
Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington's, accrues aggregates in muscle 2–6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer's disease models, nematodes with muscle or pan-neuronal Aβ1–42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72–86% (P < 10−6). In worms expressing Aβ1–42, knockdown of cram-1 reduced β-amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10−6). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly beneficial for early reproduction, but ultimately impair aggregate clearance and limit longevity.
doi:10.1111/acel.12296
PMCID: PMC4326912  PMID: 25510159
Alzheimer (disease); C. elegans; Huntington (disease); neurodegeneration; (protein) aggregation; proteasome
25.  Seroepidemiology of Norovirus-Associated Travelers’ Diarrhea 
Journal of travel medicine  2014;21(1):6-11.
Background
Noroviruses (NoVs) are the most common cause of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and were recently identified as a leading cause of travelers’ diarrhea (TD) in U.S. and European travelers to Mexico, Guatemala and India.
Methods
Serum and diarrheic stool samples were collected from 75 US student travelers to Cuernavaca, Mexico, who developed TD. NoV RNA was detected in acute diarrheic stool samples using RT-PCR. Serology assays were performed using GI.1 Norwalk virus (NV) and GII.4 Houston virus (HOV) virus-like particles (VLP) to measure serum levels of IgA and IgG by Dissociation-Enhanced Lanthanide Fluorescent Immunoassay (DELFIA); serum IgM was measured by capture ELISA, and the 50% antibody blocking titer (BT50) was determined by a carbohydrate-blocking assay.
Results
NoV infection was identified in 12 (16%; 9 GI-NoV and 3 GII-NoV) of 75 travelers by either RT-PCR or ≥4-fold rise in antibody titer. Significantly more individuals had detectable pre-existing IgA antibodies against HOV (62/75, 83%) than against NV (49/75, 65%) (p=0.025) VLPs. A significant difference was observed between NV- and HOV-specific preexisting IgA antibody levels (p=0.0037), IgG (p=0.003) and BT50 (p=<0.0001). None of the NoV-infected TD travelers had BT50 >200, a level that has been described previously as a possible correlate of protection.
Conclusions
We found that GI-NoVs are commonly associated with TD cases identified in U.S. adults traveling to Mexico, and seroprevalence rates and geometric mean antibody levels to a GI-NoV were lower than to a GII-NoV strain.
doi:10.1111/jtm.12092
PMCID: PMC3904865  PMID: 24383649

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