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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.  Protein structure in context: The molecular landscape of angiogenesis 
A team of students, educators, and researchers has developed new materials to teach cell signaling within its cellular context. Two non-traditional modalities are employed: physical models, to explore the atomic details of several of the proteins in the angiogenesis signaling cascade, and illustrations of the proteins in their cellular environment, to give an intuitive understanding of the cellular context of the pathway. The experiences of the team underscore the utility of these types of materials as an effective mode for fostering students’ understanding of the molecular world, and the scientific method used to define it.
doi:10.1002/bmb.20706
PMCID: PMC4074543  PMID: 23868376
visual learning; VEGF signaling; protein structure; angiogenesis
4.  The Effect of Lithospermic Acid, an Antioxidant, on Development of Diabetic Retinopathy in Spontaneously Obese Diabetic Rats 
PLoS ONE  2014;9(6):e98232.
Background
Lithospermic acid B (LAB), an active component isolated from Salvia miltiorrhiza radix, has been reported to have antioxidant effects. We examined the effects of LAB on the prevention of diabetic retinopathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of type 2 diabetes.
Methods and Findings
LAB (10 or 20 mg/kg) or normal saline were given orally once daily to 24-week-old male OLETF rats for 52 weeks. At the end of treatment, fundoscopic findings, vascular endothelial growth factor (VEGF) expression in the eyeball, VEGF levels in the ocular fluid, and any structural abnormalities in the retina were assessed. Glucose metabolism, serum levels of high-sensitivity C-reactive protein (hsCRP), monocyte chemotactic protein-1 (MCP1), and tumor necrosis factor-alpha (TNFα) and urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels were also measured. Treatment with LAB prevented vascular leakage and basement membrane thickening in retinal capillaries in a dose-dependent manner. Insulin resistance and glucose intolerance were significantly improved by LAB treatment. The levels of serum hsCRP, MCP1, TNFα, and urinary 8-OHdG were lower in the LAB-treated OLETF rats than in the controls.
Conclusions
Treatment with LAB had a preventive effect on the development of diabetic retinopathy in this animal model, probably because of its antioxidative effects and anti-inflammatory effects.
doi:10.1371/journal.pone.0098232
PMCID: PMC4048190  PMID: 24905410
5.  Roles of gap junctions, connexins, and pannexins in epilepsy 
Enhanced gap junctional communication (GJC) between neurons is considered a major factor underlying the neuronal synchrony driving seizure activity. In addition, the hippocampal sharp wave ripple complexes, associated with learning and seizures, are diminished by GJC blocking agents. Although gap junctional blocking drugs inhibit experimental seizures, they all have other non-specific actions. Besides interneuronal GJC between dendrites, inter-axonal and inter-glial GJC is also considered important for seizure generation. Interestingly, in most studies of cerebral tissue from animal seizure models and from human patients with epilepsy, there is up-regulation of glial, but not neuronal gap junctional mRNA and protein. Significant changes in the expression and post-translational modification of the astrocytic connexin Cx43, and Panx1 were observed in an in vitro Co++ seizure model, further supporting a role for glia in seizure-genesis, although the reasons for this remain unclear. Further suggesting an involvement of astrocytic GJC in epilepsy, is the fact that the expression of astrocytic Cx mRNAs (Cxs 30 and 43) is several fold higher than that of neuronal Cx mRNAs (Cxs 36 and 45), and the number of glial cells outnumber neuronal cells in mammalian hippocampal and cortical tissue. Pannexin expression is also increased in both animal and human epileptic tissues. Specific Cx43 mimetic peptides, Gap 27 and SLS, inhibit the docking of astrocytic connexin Cx43 proteins from forming intercellular gap junctions (GJs), diminishing spontaneous seizures. Besides GJs, Cx membrane hemichannels in glia and Panx membrane channels in neurons and glia are also inhibited by traditional gap junctional pharmacological blockers. Although there is no doubt that connexin-based GJs and hemichannels, and pannexin-based membrane channels are related to epilepsy, the specific details of how they are involved and how we can modulate their function for therapeutic purposes remain to be elucidated.
doi:10.3389/fphys.2014.00172
PMCID: PMC4019879  PMID: 24847276
gap junctions; connexins; pannexins; epilepsy; neurons; glia; animal models; human cerebral tissue
6.  Accelerated Edge-Preserving Image Restoration Without Boundary Artifacts 
To reduce blur in noisy images, regularized image restoration methods have been proposed that use non-quadratic regularizers (like l1 regularization or total-variation) that suppress noise while preserving edges in the image. Most of these methods assume a circulant blur (periodic convolution with a blurring kernel) that can lead to wraparound artifacts along the boundaries of the image due to the implied periodicity of the circulant model. Using a non-circulant model could prevent these artifacts at the cost of increased computational complexity.
In this work we propose to use a circulant blur model combined with a masking operator that prevents wraparound artifacts. The resulting model is non-circulant, so we propose an efficient algorithm using variable splitting and augmented Lagrangian (AL) strategies. Our variable splitting scheme, when combined with the AL framework and alternating minimization, leads to simple linear systems that can be solved non-iteratively using FFTs, eliminating the need for more expensive CG-type solvers. The proposed method can also efficiently tackle a variety of convex regularizers including edge-preserving (e.g., total-variation) and sparsity promoting (e.g., l1 norm) regularizers. Simulation results show fast convergence of the proposed method, along with improved image quality at the boundaries where the circulant model is inaccurate.
doi:10.1109/TIP.2013.2244218
PMCID: PMC3609946  PMID: 23372080
Image restoration; Non-Circulant System; Edge-preserving Regularization; Variable splitting; Augmented Lagrangian
7.  Frequency, Characteristics and Management of Adolescent Inpatient Aggression 
Abstract
Background
Inpatient aggression is a serious challenge in pediatric psychiatry.
Methods
A chart review study in adolescent psychiatric inpatients consecutively admitted over 24 months was conducted, to describe aggressive events requiring an intervention (AERI) and to characterize their management. AERIs were identified based on specific institutional event forms and/or documentation of as-needed (STAT/PRN) medication administration for aggression, both recorded by nursing staff.
Results
Among 408 adolescent inpatients (age: 15.2±1.6 years, 43.9% male), 1349 AERIs were recorded, with ≥1 AERI occurring in 28.4% (n=116; AERI+). However, the frequency of AERIs was highly skewed (median 4, range: 1–258). In a logistical regression model, the primary diagnosis at discharge of disruptive behavior disorders and bipolar disorders, history of previous inpatient treatment, length of hospitalization, and absence of a specific precipitant prior to admission were significantly associated with AERIs (R2=0.32; p<0.0001). The first line treatment of patients with AERIs (AERI+) was pharmacological in nature (95.6%). Seclusion or restraint (SRU) was used at least once in 59.4% of the AERI+ subgroup (i.e., in 16.9% of all patients; median within-group SRU frequency: 3). Treatment and discharge characteristics indicated a poorer prognosis in the AERI+ (discharge to residential care AERI+: 22.8%, AERI−: 5.6%, p<0.001) and a greater need for psychotropic polypharmacy (median number of psychotropic medications AERI+: 2; AERI−: 1, p<0.001).
Conclusions
Despite high rates of pharmacological interventions, SRU continue to be used in adolescent inpatient care. As both of these approaches lack a clear evidence base, and as adolescents with clinically significant inpatient aggression have increased illness acuity/severity and service needs, structured research into the most appropriate inpatient aggression management is sorely needed.
doi:10.1089/cap.2012.0116
PMCID: PMC3657279  PMID: 23647136
8.  Genotype-Phenotype studies of VCP-associated Inclusion Body Myopathy with Paget Disease of Bone and/or Frontotemporal Dementia 
Clinical genetics  2012;83(5):422-431.
VCP disease associated with Inclusion body myopathy, Paget disease of the bone and frontotemporal dementia is a progressive autosomal dominant disorder caused by mutations in Valosin containing protein gene. To establish genotype-phenotype correlations we analyzed clinical and biochemical markers from a database of 190 members in 27 families harboring ten missense mutations. Individuals were grouped into three categories: symptomatic, presymptomatic carriers and non-carriers. The symptomatic families were further divided into ten groups based on their VCP mutations. There was marked intra and inter-familial variation; and significant genotype-phenotype correlations were difficult because of small numbers. Nevertheless when comparing the two most common mutations, R155C mutation was found to be more severe, with earlier onset of myopathy and Paget (p=0.03).
Survival analysis of all subjects revealed an average life span after diagnosis of myopathy and Paget of 18 and 19 years respectively, and after dementia only 6 years. R155C had a reduced survival compared to the R155H mutation (p=0.03). We identified amyotrophic lateral sclerosis (ALS) in thirteen individuals (8.9%) and Parkinson’s disease in five individuals (3%); however there was no genotypic correlation. This study represents the largest dataset of patients with VCP disease and expands our understanding of natural history and provides genotype-phenotype correlations in this unique disease.
doi:10.1111/cge.12000
PMCID: PMC3618576  PMID: 22909335
amyotrophic lateral sclerosis; frontotemporal dementia; genotype-phenotype; inclusion body myopathy; Paget’s disease of bone; valosin containing protein
9.  Relationship of Resting Brain Hyperconnectivity and Schizophrenia-like Symptoms Produced by the NMDA receptor Antagonist Ketamine in Humans 
Molecular psychiatry  2013;18(11):10.1038/mp.2012.194.
N-methyl-D-aspartate glutamate receptor (NMDA-R) antagonists produce schizophrenia-like positive and negative symptoms in healthy human subjects. Preclinical research suggests that NMDA-R antagonists interfere with the function of gamma-aminobutyric acid (GABA) neurons and alter brain oscillations. These changes have been hypothesized to contribute to psychosis. In this investigation, we evaluated the hypothesis that the NMDA-R antagonist ketamine produces alterations in cortical functional connectivity during rest that are related to symptoms. We administered ketamine to a primary sample of twenty-two subjects and to an additional, partially overlapping, sample of twelve subjects. Symptoms before and after the experimental session were rated with the Positive and Negative Symptom Scale (PANSS). In the primary sample, functional connectivity was measured via functional magnetic resonance imaging almost immediately after infusion began. In the additional sample, this assessment was repeated after 45 minutes of continuous ketamine infusion. Global, enhanced functional connectivity was observed at both timepoints and this hyperconnectivity was related to symptoms in a region-specific manner. This study supports the hypothesis that pathological increases in resting brain functional connectivity contribute to the emergence of positive and negative symptoms associated with schizophrenia.
doi:10.1038/mp.2012.194
PMCID: PMC3646075  PMID: 23337947
N-methyl-D-aspartate; gamma-aminobutyric acid; ketamine; functional connectivity; psychosis; schizophrenia
10.  The heparanase/syndecan-1 axis in cancer: mechanisms and therapies 
The FEBS journal  2013;280(10):2294-2306.
Heparanase is an endoglucuronidase that cleaves heparan sulfate chains of proteoglycans. In many malignancies, high heparanase expression and activity correlate with an aggressive tumor phenotype. A major consequence of heparanase action in cancer is a robust up-regulation of growth factor expression and increased shedding of syndecan-1, a transmembrane heparan sulfate proteoglycan. Substantial evidence indicates that heparanase and syndecan-1 work together to drive growth factor signaling and regulate cell behaviors that enhance tumor growth, dissemination, angiogenesis and osteolysis. Pre-clinical and clinical studies have demonstrated that therapies targeting the heparanase/syndecan-1 axis hold promise in blocking the aggressive behavior of cancer.
doi:10.1111/febs.12168
PMCID: PMC3651779  PMID: 23374281
Heparanase, syndecan-1; heparan sulfates; hepatocyte growth factor; vascular endothelial growth factor; extracellular regulated kinase; heparin mimics
11.  Unraveling the paradox of cardiac tamponade: case presentation and discussion of physiology 
BMJ Case Reports  2012;2012:bcr0920114792.
A 53-year-old man on warfarin for postoperative pulmonary embolism presented with chest pain and was found to be in cardiac tamponade due to an atraumatic haemopericardium. Findings of tamponade and a novel approach to the pathophysiology of pericardial disease to explain these finding are presented.
doi:10.1136/bcr.09.2011.4792
PMCID: PMC3339179  PMID: 22604764
12.  Dll4-Notch Signalling Blockade Synergizes Combined Ultrasound-Stimulated Microbubble and Radiation Therapy in Human Colon Cancer Xenografts 
PLoS ONE  2014;9(4):e93888.
Tumour vasculature acts as an essential lifeline for tumour progression and facilitates metastatic spread. Novel vascular targeting strategies aiming to sustain vascular shutdown could potentially induce substantial damage, resulting in a significant tumour growth delay. We investigated the combination of two novel complementary vascular targeting agents with radiation therapy in a strategy aiming to sustain vascular disruption. Experiments were carried out with delta-like ligand 4 (Dll4) blockade (angiogenesis deregulator) treatment administered in combination with a radiation-based vascular destruction treatment in a highly aggressive well-perfused colon cancer tumour line implanted in female athymic nude mice. Tumours were treated with permutations of radiation, ultrasound-stimulated microbubbles (USMB) and Dll4 monoclonal antibody (mAb). Tumour vascular response was assessed with three-dimensional power Doppler ultrasound to measure active flow and immunohistochemistry. Tumour response was assessed with histochemical assays and longitudinal measurements of tumour volume. Our results suggest a significant tumour response in animals treated with USMB combined with radiation, and Dll4 mAb, leading to a synergistic tumour growth delay of up to 24 days. This is likely linked to rapid cell death within the tumour and a sustained tumour vascular shutdown. We conclude that the triple combination treatments cause a vascular shutdown followed by a sustained inhibition of angiogenesis and tumour cell death, leading to a rapid tumour vascular-based ‘collapse’ and a significant tumour growth delay.
doi:10.1371/journal.pone.0093888
PMCID: PMC3988033  PMID: 24736631
13.  Toxicity of Bevacizumab in Combination with Chemotherapy in Older Patients 
The Oncologist  2013;18(4):408-414.
Heart disease is more common in those who do not receive bevacizumab. Older patients who receive bevacizumab with chemotherapy have higher odds of developing a grade 3–5 toxicity compared with those who receive chemotherapy alone.
Learning Objectives
Compare characteristics of older patients that receive bevacizumab plus chemotherapy to those treated with chemotherapy alone for advanced NSCLC and CRC.Compare outcomes between older patients treated with bevacizumab plus chemotherapy to chemotherapy alone for advanced NSCLC and CRC.Describe toxicities in older patients treated with bevacizumab plus chemotherapy for advanced NSCLC and CRC.
Background.
Bevacizumab leads to improved survival for patients with metastatic colorectal cancer (CRC) or non-small cell lung cancer (NSCLC) when added to chemotherapy. Little is known about factors associated with receipt of bevacizumab, or whether bevacizamab is associated with increased toxicity when added to chemotherapy.
Patients and Methods.
We conducted a prospective study of patients aged ≥65 years, which evaluated the association between geriatric assessment (GA) metrics and chemotherapy toxicity. We examined differences in characteristics and outcomes of patients with CRC and NSCLC cancers who received bevacizumab with chemotherapy versus chemotherapy alone.
Results.
From a total of 207 patients, 27 (13%) received bevacizumab plus chemotherapy and 180 (87%) received chemotherapy alone. Groups were similar in sociodemographic and cancer characteristics. There were no baseline differences in GA domains except that patients with heart disease were less likely to receive bevacizumab (4% vs. 26%, p = .01). Seventy-eight percent of patients who had bevacizumab had grade 3–5 toxicity compared to only 57% who received chemotherapy alone (p = .06). Patients receiving bevacizumab were more likely to develop grade 3 hypertension than those who received chemotherapy alone (15% vs. 2%, p < .01). In multivariable analysis, factors associated with grade 3 or more toxicity included: bevacizumab (OR: 2.86, p = .04), CRC (OR: 2.54, p < .01), and baseline anemia (OR: 2.58, p = .03).
Conclusion.
Heart disease was more common in those who did not receive bevacizumab. Older patients who receive bevacizumab with chemotherapy have a higher odds of developing a grade 3–5 toxicity compared with those who receive chemotherapy alone.
doi:10.1634/theoncologist.2012-0351
PMCID: PMC3639527  PMID: 23576485
Chemotherapy; Geriatric assessment; Bevacizumab; Drug toxicity; Health services for the aged
14.  Genomes of Two Clinical Isolates of Mycobacterium tuberculosis from Odisha, India 
Genome Announcements  2014;2(2):e00199-14.
We report whole-genome sequences of two clinical isolates of Mycobacterium tuberculosis isolated from patients in Odisha, India. The sequence analysis revealed that these isolates are of an ancestral type and might represent some of the “pristine” isolates in India that have not admixed with other lineages.
doi:10.1128/genomeA.00199-14
PMCID: PMC3961728  PMID: 24652981
15.  A FAST MAJORIZE MINIMIZE ALGORITHM FOR HIGHER DEGREE TOTAL VARIATION REGULARIZATION 
The main focus of this paper is to introduce a computationally efficient algorithm for solving image recovery problems, regularized by the recently introduced higher degree total variation (HDTV) penalties. The anisotropic HDTV penalty is the fully separable L1 semi-norm of the directional image derivatives; the use of this penalty is seen to considerably improve image quality in biomedical inverse problems. We introduce a novel majorize minimize algorithm to solve the HDTV optimization problem, thus considerably speeding it over the previous implementation. Specifically, comparisons with previous iterative reweighted algorithm show an approximate ten fold speedup. The new algorithm enables us to obtain reconstructions that are free of patchy artifacts exhibited by classical TV schemes, while being comparable to state of the art total variation regularization schemes in run time.
doi:10.1109/ISBI.2013.6556478
PMCID: PMC3960000  PMID: 24663389
Higher degree total variation; majorize minimize; compressed sensing
16.  Epidemiology of human noroviruses and updates on vaccine development 
Purpose of review
Noroviruses (NoVs) are the most common cause of epidemic and sporadic cases of acute gastroenteritis worldwide. This review summarizes recent NoV disease burden estimates, epidemiology findings and provides an update on virus-like particle (VLP) vaccine studies.
Recent findings
NoVs are the leading cause of food-borne gastroenteritis and are replacing rotavirus as the predominant gastrointestinal pathogen in pediatric populations. Genogroup II, genotype 4 NoVs (GII.4) remain the dominant genotype worldwide. Increased NoV activity reported in late 2012 was associated with the emergence of a new GII.4 variant called Sydney 2012. New studies suggest that human NoVs can bind a larger range of histoblood group antigens, a susceptibility factor for NoV illness, thus expanding the susceptible population pool for infection. Intranasal immunization with a monovalent GI NoV VLP vaccine showed proof-of-concept efficacy. Studies using intramuscular immunization with a bivalent formulation including GII.4 VLPs are now underway.
Summary
The importance of NoVs as a major gastrointestinal pathogen underscores the need for well tolerated and effective vaccines. Results of VLP vaccine trials appear promising. However, the rapid evolution of NoV genotypes through antigenic drift and changing glycan specificities provide new challenges to epidemiology studies and vaccine trials.
doi:10.1097/MOG.0000000000000022
PMCID: PMC3955997  PMID: 24232370
histoblood group antigens; norovirus epidemiology; variants; virus-like particle vaccines
17.  Feeding Practices and NEC 
Clinics in perinatology  2013;40(1):1-10.
Synopsis
Necrotizing enterocolitis (NEC) is a multifactorial disorder that primarily affects premature infants. Human milk as compared to formula reduces the incidence of NEC. Feeding practices such as minimal enteral nutrition (versus complete fasting) before progressive advancement of feeds, early introduction of feeds (before day 4 of life as compared to later), and a more rapid advancement of feeds (30–35 ml/kg/day as compared to 15–20 ml/kg/day) do not increase the incidence of NEC in preterm infants. There is no evidence supporting continuous over intermittent tube feedings in preterm infants. In a feed-intolerant preterm infant without any other clinical and radiological evidence of NEC, minimal enteral nutrition rather than complete suspension of enteral feeding may be an alternative. Human milk-based fortifier as compared to bovine-based fortifier may reduce the incidence of NEC but additional studies are required.
doi:10.1016/j.clp.2012.12.001
PMCID: PMC3576724  PMID: 23415260
Necrotizing enterocolitis; Feeding Methods; Enteral Nutrition; Premature Infant
18.  Accelerated Regularized Estimation of MR Coil Sensitivities Using Augmented Lagrangian Methods 
Several magnetic resonance (MR) parallel imaging techniques require explicit estimates of the receive coil sensitivity profiles. These estimates must be accurate over both the object and its surrounding regions to avoid generating artifacts in the reconstructed images. Regularized estimation methods that involve minimizing a cost function containing both a data-fit term and a regularization term provide robust sensitivity estimates. However, these methods can be computationally expensive when dealing with large problems. In this paper, we propose an iterative algorithm based on variable splitting and the augmented Lagrangian method that estimates the coil sensitivity profile by minimizing a quadratic cost function. Our method, ADMM–Circ, reformulates the finite differencing matrix in the regularization term to enable exact alternating minimization steps. We also present a faster variant of this algorithm using intermediate updating of the associated Lagrange multipliers. Numerical experiments with simulated and real data sets indicate that our proposed method converges approximately twice as fast as the preconditioned conjugate gradient method (PCG) over the entire field-of-view. These concepts may accelerate other quadratic optimization problems.
doi:10.1109/TMI.2012.2229711
PMCID: PMC3595372  PMID: 23192524
Augmented Lagrangian; coil sensitivity; finite differences; parallel imaging; quadratic minimization
19.  A Brief Overview of the Role of Drugs and Novel Methodologies on the Stability and Growth of Hair Follicles: An Approach Towards Hair Regeneration 
doi:10.7860/JCDR/2014/7341.3881
PMCID: PMC3939579  PMID: 24596810
Hair regeneration; Stem Cell Therapy; Follicular unit transplantation; Hair transplantation; Mesotherapy
20.  Best Practices for the Development, Scale-up, and Post-approval Change Control of IR and MR Dosage Forms in the Current Quality-by-Design Paradigm 
AAPS PharmSciTech  2014;15(3):665-693.
In this whitepaper, the Manufacturing Technical Committee of the Product Quality Research Institute provides information on the common, best practices in use today in the development of high-quality chemistry, manufacturing and controls documentation. Important topics reviewed include International Conference on Harmonization, in vitro–in vivo correlation considerations, quality-by-design approaches, process analytical technologies and current scale-up, and process control and validation practices. It is the hope and intent that this whitepaper will engender expanded dialog on this important subject by the pharmaceutical industry and its regulatory bodies.
doi:10.1208/s12249-014-0087-x
PMCID: PMC4037495  PMID: 24578237
CMC; ICH; IVIVC; PAT; QbD
21.  Reduced L-Carnitine Transport in Aortic Endothelial Cells from Spontaneously Hypertensive Rats 
PLoS ONE  2014;9(2):e90339.
Impaired L-carnitine uptake correlates with higher blood pressure in adult men, and L-carnitine restores endothelial function in aortic rings from spontaneously hypertensive rat (SHR). Thus, endothelial dysfunction in hypertension could result from lower L-carnitine transport in this cell type. L-Carnitine transport is mainly mediated by novel organic cation transporters 1 (Octn1, Na+-independent) and 2 (Octn2, Na+-dependent); however, their kinetic properties and potential consequences in hypertension are unknown. We hypothesize that L-carnitine transport kinetic properties will be altered in aortic endothelium from spontaneously hypertensive rats (SHR). L-Carnitine transport was measured at different extracellular pH (pHo 5.5–8.5) in the absence or presence of sodium in rat aortic endothelial cells (RAECs) from non-hypertensive Wistar-Kyoto (WKY) rats and SHR. Octn1 and Octn2 mRNA relative expression was also determined. Dilation of endothelium-intact or denuded aortic rings in response to calcitonine gene related peptide (CGRP, 0.1–100 nmol/L) was measured (myography) in the absence or presence of L-carnitine. Total L-carnitine transport was lower in cells from SHR compared with WKY rats, an effect due to reduced Na+-dependent (Na+dep) compared with Na+-independent (Na+indep) transport components. Saturable L-carnitine transport kinetics show maximal velocity (Vmax), without changes in apparent Km for Na+indep transport in SHR compared with WKY rats. Total and Na+dep component of transport were increased, but Na+indep transport was reduced by extracellular alkalization in WKY rats. However, alkalization reduced total and Na+indep transport in cells from SHR. Octn2 mRNA was higher than Octn-1 mRNA expression in cells from both conditions. Dilation of artery rings in response to CGRP was reduced in vessels from SHR compared with WKY rats. CGRP effect was endothelium-dependent and restored by L-carnitine. All together these results suggest that reduced L-carnitine transport (likely via Na+-dependent Octn2) could limit this compound's potential beneficial effects in RAECs from SHR.
doi:10.1371/journal.pone.0090339
PMCID: PMC3938671  PMID: 24587332
22.  Attenuation of L-Type Ca2+ Channel Expression and Vasomotor Response in the Aorta with Age in Both Wistar-Kyoto and Spontaneously Hypertensive Rats 
PLoS ONE  2014;9(2):e88975.
Age-related vascular diseases are induced by vascular dysfunction, which involves changes in the vasomotor response. The voltage-dependent L-type calcium channel (VDCC) protein is involved in the regulation of vessel function (contraction/relaxation action). In the present study, we evaluated age-related vasomotor function and expression of the signal-related target proteins, including VDCC, using thoracic aorta from both 8- and 40-week old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). In contraction experiments using aortic rings, vasomotor responses of both phenylephrine-induced contraction and acetylcholine-induced relaxation were significantly attenuated with age in SHR, whereas WKY did not lose activity with age. Contraction induced by angiotensin II was impaired only for the 40-week old SHR among all the rat groups tested, although enhanced AT1R/reduced AT2R expression with age was observed for both WKY and SHR. In contrast, a vasomotor responsiveness to Bay K 8644 (a VDCC agonist) at the initial contraction phase was significantly attenuated in both 40-week WKY and SHR with significant reduction of VDCC protein expression. The reduced VDCC expression in 40-week old rats significantly lowered the relaxation activity of VDCC blockers, such as verapamil and Trp-His, but did not affect that of nifedipine. Taken together, we provided the first evidence that aging caused a reduction of VDCC expression in rat aorta, irrespective of the rat strain, along with diminishment of the therapeutic potential of VDCC blockers.
doi:10.1371/journal.pone.0088975
PMCID: PMC3923070  PMID: 24533163
23.  Detection and characterisation of rotaviruses from children less than 5 years hospitalised with acute gastroenteritis in Nagercoil 
Group A rotavirus continues to be the major cause of severe gastroenteritis in young children in developing countries. In this study, we report the prevalence and genotype of rotaviruses identified from children <5 years of age hospitalised with acute gastroenteritis from Nagercoil, Tamil Nadu from 2007-2010. From the 139 children included in the study, 71 samples (51%) were positive by ELISA and 65 samples were positive by PCR-based methods. G1P[8] (44.6%) was the most commonly identified genotype. In addition, we report detection of rotavirus in two of three CSF samples from children with seizures.
doi:10.4103/0255-0857.108727
PMCID: PMC3919954  PMID: 23508433
Cerebrospinal fluid; genotyping; Nagercoil; rotavirus
24.  Comparative RNAi Screens in C. elegans and C. briggsae Reveal the Impact of Developmental System Drift on Gene Function 
PLoS Genetics  2014;10(2):e1004077.
Although two related species may have extremely similar phenotypes, the genetic networks underpinning this conserved biology may have diverged substantially since they last shared a common ancestor. This is termed Developmental System Drift (DSD) and reflects the plasticity of genetic networks. One consequence of DSD is that some orthologous genes will have evolved different in vivo functions in two such phenotypically similar, related species and will therefore have different loss of function phenotypes. Here we report an RNAi screen in C. elegans and C. briggsae to identify such cases. We screened 1333 genes in both species and identified 91 orthologues that have different RNAi phenotypes. Intriguingly, we find that recently evolved genes of unknown function have the fastest evolving in vivo functions and, in several cases, we identify the molecular events driving these changes. We thus find that DSD has a major impact on the evolution of gene function and we anticipate that the C. briggsae RNAi library reported here will drive future studies on comparative functional genomics screens in these nematodes.
Author Summary
Although two related species may appear similar, the genetic pathways that underpin this shared biology may have drifted and changed. This phenomenon is known as Developmental System Drift (DSD). One consequence of DSD is that equivalent genes may play different roles in phenotypically similar, related species but there have been no systematic studies to examine this. How often do genes have different functions in similar species? Are certain genes more likely to change functions? Finally, what are the molecular changes that drive this? Here, we compare the effects of reducing the levels of over 1300 different genes in two species of nematode worm. These worms are very similar— they live in the same ecological niche, and have near-identical development and behavior. We find that over 25% of conserved genes have different functions in these two species, showing that DSD has a major impact on how gene function evolves. Intriguingly, we find that genes that have arisen recently are most likely to change functions and that this is often driven by changes in their expression. This is the first systematic comparison of loss of function phenotypes in related species and sheds light on how genetic pathways rewire during DSD.
doi:10.1371/journal.pgen.1004077
PMCID: PMC3916228  PMID: 24516395
25.  Neurodevelopmental Impairment Following Neonatal Hyperoxia in the Mouse 
Neurobiology of disease  2012;50:69-75.
Extremely premature infants are often exposed to supra-physiologic concentrations of oxygen, and frequently have hypoxemic episodes. These preterm infants are at high risk (~40%) for neurodevelopmental impairment (NDI) even in the absence of obvious intracranial pathology such as intraventricular hemorrhage or periventricular leukomalacia. The etiology for NDI has not been determined, and there are no animal models to simulate neurodevelopmental outcomes of prematurity. Our objectives were to develop and characterize a mouse model to determine long-term effects of chronic hypoxia or hyperoxia exposure on neurodevelopment. Newborn C57BL/6 mice were exposed to hypoxia (12% O2) or hyperoxia (85% O2) from postnatal day 1 to 14 and then returned to air. At 12–14 weeks of age, neurobehavioral assessment (Water Maze test, Novel Object Recognition test, Open Field test, Elevated Plus Maze, and Rotarod test) was performed, followed by MRI and brain histology. Neurobehavioral testing revealed that hyperoxia-exposed mice did poorly on the water maze and novel object recognition tests compared to air-exposed mice. MRI demonstrated smaller hippocampi in hyperoxia- and hypoxia-exposed mice with a greater reduction in hyperoxia-exposed mice, including a smaller cerebellum in hyperoxia-exposed mice. Brain histology showed reduced CA1 and CA3 and increased dentate gyral width in hippocampus. In conclusion, neonatal hyperoxia in mice leads to abnormal neurobehavior, primarily deficits in spatial and recognition memory, associated with smaller hippocampal sizes, similar to findings in ex-preterm infants. This animal model may be useful to determine mechanisms underlying developmental programming of NDI in preterm infants, and for evaluation of therapeutic strategies.
doi:10.1016/j.nbd.2012.10.005
PMCID: PMC3534920  PMID: 23064437
Disease models; animal; Infant; premature; Developmental programming; Hippocampus; Cerebellum; Oxidative stress

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