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1.  Cysteine Residues Impact the Stability and Micelle Interaction Dynamics of the Human Mitochondrial β-Barrel Anion Channel hVDAC-2 
PLoS ONE  2014;9(3):e92183.
The anti-apoptotic 19-stranded transmembrane human voltage dependent anion channel isoform 2 (hVDAC-2) β-barrel stability is crucial for anion transport in mitochondria. The role of the unusually high number of cysteine residues in this isoform is poorly understood. Using a Cys-less construct of hVDAC-2, we haveinvestigated the contribution of cysteines to channel function, barrel stability and its influence on the strength of protein-micelle interactions. We observe that despite the overall preservation in barrel structure upon cysteine mutation, subtle local variations in the mode of interaction of the barrel with its refolded micellar environment arise, which may manifest itself in the channel activity of both the proteins.Fluorescence measurements of the Trp residues in hVDAC-2 point to possible differences in the association of the barrel with lauryldimethylamine oxide (LDAO) micelles. Upon replacement of cysteines in hVDAC-2, our data suggests greater barrel rigidity by way of intra-protein interactions. This, in turn, lowers the equilibrium barrel thermodynamic parameters in LDAOby perturbingthe stability of the protein-micelle complex. In addition to this, we also find a difference in the cooperativity of unfolding upon increasing the LDAO concentration, implying the importance of micelle concentration and micelle-protein ratios on the stability of this barrel. Our results indicate that the nine cysteine residues of hVDAC-2 are the key in establishing strong(er) barrel interactions with its environment and also impart additional malleability to the barrel scaffold.
doi:10.1371/journal.pone.0092183
PMCID: PMC3967697  PMID: 24642864
2.  Influence of Protein – Micelle Ratios and Cysteine Residues on the Kinetic Stability and Unfolding Rates of Human Mitochondrial VDAC-2 
PLoS ONE  2014;9(1):e87701.
Delineating the kinetic and thermodynamic factors which contribute to the stability of transmembrane β-barrels is critical to gain an in-depth understanding of membrane protein behavior. Human mitochondrial voltage-dependent anion channel isoform 2 (hVDAC-2), one of the key anti-apoptotic eukaryotic β-barrel proteins, is of paramount importance, owing to its indispensable role in cell survival. We demonstrate here that the stability of hVDAC-2 bears a strong kinetic contribution that is dependent on the absolute micellar concentration used for barrel folding. The refolding efficiency and ensuing stability is sensitive to the lipid-to-protein (LPR) ratio, and displays a non-linear relationship, with both low and high micellar amounts being detrimental to hVDAC-2 structure. Unfolding and aggregation process are sequential events and show strong temperature dependence. We demonstrate that an optimal lipid-to-protein ratio of 2600∶1 – 13000∶1 offers the highest protection against thermal denaturation. Activation energies derived only for lower LPRs are ∼17 kcal mol−1 for full-length hVDAC-2 and ∼23 kcal mol−1 for the Cys-less mutant, suggesting that the nine cysteine residues of hVDAC-2 impart additional malleability to the barrel scaffold. Our studies reveal that cysteine residues play a key role in the kinetic stability of the protein, determine barrel rigidity and thereby give rise to strong micellar association of hVDAC-2. Non-linearity of the Arrhenius plot at high LPRs coupled with observation of protein aggregation upon thermal denaturation indicates that contributions from both kinetic and thermodynamic components stabilize the 19-stranded β-barrel. Lipid-protein interaction and the linked kinetic contribution to free energy of the folded protein are together expected to play a key role in hVDAC-2 recycling and the functional switch at the onset of apoptosis.
doi:10.1371/journal.pone.0087701
PMCID: PMC3907894  PMID: 24494036
3.  Soluble gC1qR Is an Autocrine Signal That Induces B1R Expression on Endothelial Cells 
Bradykinin (BK) is one of the most potent vasodilator agonists known and belongs to the kinin family of proinflammatory peptides. BK induces its activity via two G protein–coupled receptors: BK receptor 1 (B1R) and BK receptor 2. Although BK receptor 2 is constitutively expressed on endothelial cells (ECs), B1R is induced by IL-1β. The C1q receptor, receptor for the globular heads of C1q (gC1qR), which plays a role in BK generation, is expressed on activated ECs and is also secreted as soluble gC1qR (sgC1qR). Because sgC1qR can bind to ECs, we hypothesized that it may also serve as an autocrine/paracrine signal for the induction of B1R expression. In this study, we show that gC1qR binds to ECs via a highly conserved domain consisting of residues 174–180, as assessed by solid-phase binding assay and deconvolution fluorescence microscopy. Incubation of ECs (24 h, 37°C) with sgC1qR resulted in enhancement of B1R expression, whereas incubation with gC1qR lacking aa 174–180 and 154–162 had a diminished effect. Binding of sgC1qR to ECs was through surface-bound fibrinogen and was inhibited by anti-fibrinogen. In summary, our data suggest that, at sites of inflammation, sgC1qR can enhance vascular permeability by upregulation of B1R expression through de novo synthesis, as well as rapid translocation of preformed B1R.
doi:10.4049/jimmunol.1302031
PMCID: PMC3876932  PMID: 24319267
4.  Methionine Mutations of Outer Membrane Protein X Influence Structural Stability and Beta-Barrel Unfolding 
PLoS ONE  2013;8(11):e79351.
We report the biochemical and biophysical characterization of outer membrane protein X (OmpX), an eight-stranded transmembrane β-barrel from E. coli, and compare the barrel behavior with a mutant devoid of methionine residues. Transmembrane outer membrane proteins of bacterial origin are known to display high tolerance to sequence rearrangements and mutations. Our studies with the triple mutant of OmpX that is devoid of all internal methionine residues (M18L; M21L; M118L) indicate that Met replacement has no influence on the refolding efficiency and structural characteristics of the protein. Surprisingly, the conserved substitution of Met→Leu leads to barrel destabilization and causes a lowering of the unfolding free energy by a factor of ∼8.5 kJ/mol, despite the mutations occurring at the loop regions. We report that the barrel destabilization is accompanied by a loss in cooperativity of unfolding in the presence of chemical denaturants. Furthermore, we are able to detect an unfolding intermediate in the Met-less barrel, whereas the parent protein exhibits a classic two-state unfolding. Thermal denaturation measurements also suggest a greater susceptibility of the OmpX barrel to heat, in the Met-less construct. Our studies reveal that even subtle variations in the extra-membrane region of rigid barrel structures such as OmpX, may bear severe implications on barrel stability. We propose that methionines contribute to efficient barrel structuring and protein-lipid interactions, and are therefore important elements of OmpX stability.
doi:10.1371/journal.pone.0079351
PMCID: PMC3827151  PMID: 24265768
5.  Semen preparation techniques in intrauterine insemination: A comparison of non-temperature and temperature controlled centrifugation in cases of unexplained infertility 
AIM:
The aim of the following study is to compare pregnancy rates between the use of non-temperature and temperature controlled centrifugation on semen preparation technique in intrauterine insemination.
MATERIALS AND METHODS:
The retrospective study was conducted on 671 patients of idiopathic infertility who underwent homologous artificial insemination at Fertility Research Center from the period of January 2007 to September 2012. The couples were randomized into two groups namely, Group A-patients (n = 303) being treated with sperm prepared by using non-temperature controlled centrifuge and Group B-Patients (n = 368) being treated with sperm prepared by temperature (37°C) controlled centrifuge.
RESULTS AND CONCLUSION:
The clinical pregnancy rate Group A was 13.86% and Group B was 12.77%. The clinical pregnancy fetal loss rate between the two groups was 38% and 42% respectively. The delivery rate per transfer was 62% and 58% respectively. No statistically significant difference was found between these two groups.
doi:10.4103/0974-1208.126289
PMCID: PMC3963306  PMID: 24672162
Intrauterine insemination; morphology; pregnancy rate; sperm concentration; sperm motility
6.  Antibacterial efficacy of Mangifera indica L. kernel and Ocimum sanctum L. leaves against Enterococcus faecalis dentinal biofilm 
Introduction:
The Enterococcus faecalis biofilm in the root canal makes it difficult to be eradicated by the conventional irrigants with no toxicity to the tissues. Hence, plant products with least side effects are explored for their use as irrigants in the root canal therapy.
Aim:
To evaluate and compare the antibacterial efficacy of Mangifera indica L. kernel (mango kernel) and Ocimum sanctum L. leaves (tulsi) extracts with conventional irrigants (5% sodium hypochlorite (NaOCl) and 2% chlorhexidine) against E. faecalis dentinal biofilm.
Materials and Methods:
Agar diffusion and broth microdilution assay was performed with the herbal extracts and conventional irrigants (2% chlorhexidine and 5% NaOCl) against E. faecalis planktonic cells. The assay was extended onto 3 week E. faecalis dentinal biofilm.
Results:
Significant reduction of colony forming units (CFU)/mL was observed for the herbal groups and the antibacterial activity of the herbal groups was at par with 5% NaOCl.
Conclusions:
The antibacterial activity of these herbal extracts is found to be comparable with that of conventional irrigants both on the biofilm and planktonic counterparts.
doi:10.4103/0972-0707.117507
PMCID: PMC3778630  PMID: 24082577
Antibacterial activity; Enterococcus faecalis biofilm; mango kernel; root canal; tulsi leaves
7.  Comparison of the world health organization and the International association of diabetes and pregnancy study groups criteria in diagnosing gestational diabetes mellitus in South Indians 
Aim:
We aimed to compare the International Association of Diabetes and Pregnancy Study Groups (IADPSG) and the World Health Organization (WHO) criteria to diagnose gestational diabetes mellitus (GDM) in Chennai, India.
Materials and Methods:
We reviewed the retrospective data of 1351 pregnant women who underwent screening for GDM at four selected diabetes centers at Chennai (three private and one government). All women underwent an oral glucose tolerance test using 75g glucose load and fasting, 1-h, and 2-h samples were collected. The IADPSG and WHO criteria were compared for diagnosis of GDM.
Results:
A total of 839 women had GDM by either the IADPSG or the WHO criteria, of whom the IADPSG criteria identified 699 and the WHO criteria also identified 699 women as having GDM. However, only 599/839 women (66.6%) were identified by both criteria. Thus, 140/839 women (16.7%) were missed by both the IADPSG and the WHO criteria. 687/699 (98.2%) of the women with GDM were identified by the WHO criteria. In contrast, each value of IADPSG criteria i.e., fasting, 1 h, and 2 h identified only 12.5%, 14%, and 22%, respectively.
Conclusions:
A single WHO cut-point of 2 h > 140 mg/dl appears to be suitable for large-scale screening for GDM in India and other developing countries.
doi:10.4103/2230-8210.117241
PMCID: PMC3784877  PMID: 24083175
Asian Indians; gestational diabetes; international association of diabetes and pregnancy study groups; south indians; world health organization
8.  (2E,4E)-Ethyl 5-(2,4-di­chloro­phenyl­sulfon­yl)penta-2,4-dienoate 
In the title compound, C13H12Cl2O4S, both C=C double bonds adopt an E conformation. The S atom has a distorted tetra­hedral geometry with bond angles ranging from 103.03 (12) to 118.12 (13)°. The eth­oxy­carbonyl group is disordered over two sets of sites, with site-occupancy factors of 0.739 (11) and 0.261 (11). In the crystal, C—H⋯O inter­actions link the mol­ecules into chains mol­ecules running parallel to the a axis.
doi:10.1107/S1600536813021429
PMCID: PMC3884450  PMID: 24427029
9.  Salmonella paratyphi B meningitis in an infant 
The Australasian Medical Journal  2013;6(7):350-353.
We report a case of Salmonella paratyphi B meningitis in a 90 day-old male infant who was admitted with complaints of fever, vomiting and one episode of vacant stare. Clinically, the infant was found to be toxic and dull with a bulging anterior fontanelle. Subsequently, blood and cerebrospinal fluid cultures demonstrated the presence of Salmonella Paratyphi B organism.
doi:10.4066/AMJ.2013.1667
PMCID: PMC3737760  PMID: 23940494
Salmonella Paratyphi B; meningitis; infant
10.  FEV1 over time in patients with connective tissue disease-related bronchiolitis 
Respiratory medicine  2013;107(6):883-889.
Summary
Background
Fibrosis or inflammation of the bronchioles is a well-known manifestation of connective tissue disease (CTD). However, the natural history of CTD-related bronchiolitis is largely unknown.
Methods
We analyzed consecutive patients evaluated at National Jewish Health (Denver, CO) from 1998 to 2008 with CTD and surgical lung biopsy-confirmed bronchiolitis. Linear mixed effects models were used to estimate the longitudinal postbronchodilator FEV1 %predicted (%pred) course and differences between subjects with or without constrictive bronchiolitis (CB).
Results
Of 28 subjects with a mean age of 53 ± 9 years, fourteen (50%) had CB. The most common CTD diagnosis was rheumatoid arthritis (n = 14; 50%). There were no significant differences in demographics, smoking status, underlying CTD diagnoses, 6-min walk distance, dyspnea score or drug therapy between subjects with CB and those with cellular bronchiolitis. Three subjects with CB (11%) and four with cellular bronchiolitis (14%) died. Compared with subjects with CB, those with cellular bronchiolitis had higher mean FEV1 %pred at all times. There were no significant differences in FEV1 %pred slope within- or between-groups (CB vs. cellular bronchiolitis) preceding surgical lung biopsy or afterward.
Conclusion
Subjects with CTD-related CB had lower FEV1 %pred values than those with CTD-related cellular bronchiolitis at all time points, but FEV1 %pred remained stable over time in both groups regardless of therapy received.
doi:10.1016/j.rmed.2013.02.019
PMCID: PMC3697096  PMID: 23582575
Pulmonary function testing; Autoimmune disease; Obliterative bronchiolitis
11.  Modulating lipid dynamics and membrane fluidity to drive rapid folding of a transmembrane barrel 
Scientific Reports  2013;3:1989.
Lipid-protein interactions, critical for the folding, stability and function of membrane proteins, can be both of mechanical and chemical nature. Mechanical properties of lipid systems can be suitably influenced by physical factors so as to facilitate membrane protein folding. We demonstrate here that by modulating lipid dynamics transiently using heat, rapid folding of two 8-stranded transmembrane β-barrel proteins OmpX and OmpA1–171, in micelles and vesicles, can be achieved within seconds. Folding kinetics using this ‘heat shock’ method shows a dramatic ten to several hundred folds increase in refolding rate along with ~100% folding efficiency. We establish that OmpX thus folded is highly thermostable even in detergent micelles, and retains structural characteristics comparable to the protein in bilayers.
doi:10.1038/srep01989
PMCID: PMC3683699  PMID: 23771099
12.  Mycophenolate Mofetil Improves Lung Function in Connective Tissue Disease-associated Interstitial Lung Disease 
The Journal of rheumatology  2013;40(5):640-646.
Objective
Small series suggest mycophenolate mofetil (MMF) is well tolerated and may be an effective therapy for connective tissue disease-associated interstitial lung disease (CTD-ILD). We examined the tolerability and longitudinal changes in pulmonary physiology in a large and diverse cohort of patients with CTD-ILD treated with MMF.
Methods
We identified consecutive patients evaluated at our center between January 2008 and January 2011 and prescribed MMF for CTD-ILD. We assessed safety and tolerability of MMF and used longitudinal data analyses to examine changes in pulmonary physiology over time, before and after initiation of MMF.
Results
We identified 125 subjects treated with MMF for a median 897 days. MMF was discontinued in 13 subjects. MMF was associated with significant improvements in estimated percentage of predicted forced vital capacity (FVC%) from MMF initiation to 52, 104, and 156 weeks (4.9% ± 1.9%, p = 0.01; 6.1% ± 1.8%, p = 0.0008; and 7.3% ± 2.6%, p = 0.004, respectively); and in estimated percentage predicted diffusing capacity (DLCO%) from MMF initiation to 52 and 104 weeks (6.3% ± 2.8%, p = 0.02; 7.1% ± 2.8%, p = 0.01). In the subgroup without usual interstitial pneumonia (UIP)-pattern injury, MMF significantly improved FVC% and DLCO%, and in the subgroup with UIP-pattern injury, MMF was associated with stability in FVC% and DLCO%.
Conclusion
In a large diverse cohort of CTD-ILD, MMF was well tolerated and had a low rate of discontinuation. Treatment with MMF was associated with either stable or improved pulmonary physiology over a median 2.5 years of followup. MMF appears to be a promising therapy for the spectrum of CTD-ILD.
doi:10.3899/jrheum.121043
PMCID: PMC3676865  PMID: 23457378
INTERSTITIAL LUNG DISEASE; CONNECTIVE TISSUE DISEASE; MYCOPHENOLATE MOFETIL
13.  Synovial Sarcoma of the Buccal Mucosa: A Rare Case Report 
Case Reports in Dentistry  2013;2013:938291.
Synovial sarcoma (SS) is a rare malignant neoplasm that arises most commonly in joint capsules and articular tendons, but its relationship to the synovium is not always obvious. Synovial sarcoma is a malignant soft tissue tumor representing 5.6% to 10% of all soft tissue sarcomas. They are termed SS because of their histologic resemblance to the synovium, but they rarely involve a synovial structure and are thought to arise from pluripotential mesenchymal cells. The tumor usually occurs in close association with tendon sheaths, bursae, and joint capsules, primarily in the para-articular regions of the extremities, with approximately 9% occurring in the head and neck region. Synovial sarcoma has been reported rarely in the oral cavity. We report a very rare case of Synovial sarcoma of the buccal mucosa in a 24-year-old male patient.
doi:10.1155/2013/938291
PMCID: PMC3670524  PMID: 23762651
14.  Cofactor Regulation of C5a Chemotactic Activity in Physiological Fluids. Requirement for the Vitamin D Binding Protein, Thrombospondin-1 and its Receptors 
Molecular immunology  2011;49(3):495-503.
Factors in physiological fluids that regulate the chemotactic activity of complement activation peptides C5a and C5a des Arg are not well understood. The vitamin D binding protein (DBP) has been shown to significantly enhance chemotaxis to C5a/C5a des Arg. More recently, platelet-derived thrombospondin-1 (TSP-1) has been shown to facilitate the augmentation of C5a-induced chemotaxis by DBP. The objective of this study was to better characterize these chemotactic cofactors and investigate the role that cell surface TSP-1 receptors CD36 and CD47 may play in this process. The chemotactic activity in C-activated normal serum, citrated plasma, DBP-depleted serum or C5 depleted serum was determined for both normal human neutrophils and U937 cell line transfected with the C5a receptor (U937-C5aR). In addition, levels of C5a des Arg, DBP and TSP-1 in these fluids were measured by RIA or ELISA. Results show that there is a clear hierarchy with C5a being the essential primary signal (DBP or TSP-1 will not function in the absence of C5a), DBP the necessary cofactor and TSP-1 a dependent tertiary factor, since it cannot function to enhance chemotaxis to C5a without DBP. Measurement of the C5a-induced intracellular calcium flux confirmed the same hierarchy observed with chemotaxis. Moreover, analysis of bronchoalveolar lavage fluid (BALF) from patients with the adult respiratory distress syndrome (ARDS) demonstrated that C5a-dependent chemotactic activity is significantly decreased after anti-DBP treatment. Finally, results show that TSP-1 utilizes cell surface receptors CD36 and CD47 to augment chemotaxis, but DBP does not bind to TSP-1, CD36 or CD47. The results clearly demonstrate that C5a/C5a des Arg needs both DBP and TSP-1 for maximal chemotactic activity and suggest that the regulation of C5a chemotactic activity in physiological fluids is more complex than previously thought.
doi:10.1016/j.molimm.2011.09.024
PMCID: PMC3224156  PMID: 22014686
Complement; C5a; Chemotaxis; Inflammation; Neutrophils
15.  Visualization and Analysis of Microtubule Dynamics Using Dual Color-Coded Display of Plus-End Labels 
PLoS ONE  2012;7(11):e50421.
Investigating spatial and temporal control of microtubule dynamics in live cells is critical to understanding cell morphogenesis in development and disease. Tracking fluorescently labeled plus-end-tracking proteins over time has become a widely used method to study microtubule assembly. Here, we report a complementary approach that uses only two images of these labels to visualize and analyze microtubule dynamics at any given time. Using a simple color-coding scheme, labeled plus-ends from two sequential images are pseudocolored with different colors and then merged to display color-coded ends. Based on object recognition algorithms, these colored ends can be identified and segregated into dynamic groups corresponding to four events, including growth, rescue, catastrophe, and pause. Further analysis yields not only their spatial distribution throughout the cell but also provides measurements such as growth rate and direction for each labeled end. We have validated the method by comparing our results with ground-truth data derived from manual analysis as well as with data obtained using the tracking method. In addition, we have confirmed color-coded representation of different dynamic events by analyzing their history and fate. Finally, we have demonstrated the use of the method to investigate microtubule assembly in cells and provided guidance in selecting optimal image acquisition conditions. Thus, this simple computer vision method offers a unique and quantitative approach to study spatial regulation of microtubule dynamics in cells.
doi:10.1371/journal.pone.0050421
PMCID: PMC3511552  PMID: 23226282
16.  Mutations in the Transcription Elongation Factor SPT5 Disrupt a Reporter for Dosage Compensation in Drosophila 
PLoS Genetics  2012;8(11):e1003073.
In Drosophila, the MSL (Male Specific Lethal) complex up regulates transcription of active genes on the single male X-chromosome to equalize gene expression between sexes. One model argues that the MSL complex acts upon the elongation step of transcription rather than initiation. In an unbiased forward genetic screen for new factors required for dosage compensation, we found that mutations in the universally conserved transcription elongation factor Spt5 lower MSL complex dependent expression from the miniwhite reporter gene in vivo. We show that SPT5 interacts directly with MSL1 in vitro and is required downstream of MSL complex recruitment, providing the first mechanistic data corroborating the elongation model of dosage compensation.
Author Summary
Drosophila males hypertranscribe most of the genes along their single X chromosome to match the output of females with two X chromosomes. It had been difficult to imagine how the MSL dosage compensation complex could impose a modest, but essential, ∼two-fold increase by interacting with hundreds of different factors that control transcription initiation for such a diverse collection of genes. An alternative model proposed that dosage compensation instead acted at some step of transcription elongation common to all genes. We performed a genetic screen for mutations that subtly reduce dosage compensation and recovered mutations in the Spt5 gene that encodes a universally conserved elongation factor. SPT5 closes the RNA polymerase II clamp around the DNA template to prevent pausing or premature termination. We find that the dosage compensation complex genetically and physically interacts with SPT5 on actively transcribed genes providing direct molecular support for the elongation model of dosage compensation.
doi:10.1371/journal.pgen.1003073
PMCID: PMC3510053  PMID: 23209435
17.  Acute stress to excised vocal fold epithelium from reactive oxygen species 
The Laryngoscope  2011;121(10):2180-2184.
Objectives
Vocal fold epithelium is exposed to reactive oxygen species from the inhaled environment and from tissue inflammation. The objective of this study was to explore the functional and structural consequences of reactive oxygen species exposure on vocal fold epithelium.
Study Design
In vitro, prospective study design.
Methods
Hydrogen peroxide (H2O2), a common reactive oxygen species, was utilized in this study. Freshly excised, viable porcine vocal fold epithelia (N = 32) were exposed to H2O2 or sham challenge for 2 hours. Electrophysiology, western blotting, and light microscopy were used to quantify the functional and structural effects of reactive oxygen species on vocal fold epithelia.
Results
Exposure to reactive oxygen species did not significantly alter transepithelial resistance. There was a small, non-significant trend for decreased concentration of epithelial junctional complex protein with reactive oxygen species challenge. Minimal changes to the gross structural appearance of vocal fold epithelia were also noted.
Conclusions
The stratified squamous epithelia of the vocal folds effectively defend against an acute reactive oxygen species challenge. The current study lays the groundwork for future investigations on the effects of reactive oxygen species on vocal fold epithelia that are compromised from phonotrauma.
doi:10.1002/lary.22157
PMCID: PMC3183277  PMID: 21898441
H2O2; vocal fold epithelia; reactive oxygen species
18.  The Effect of Long Term Calorie Restriction on in Vivo Hepatic Proteostatis: A Novel Combination of Dynamic and Quantitative Proteomics 
Molecular & Cellular Proteomics : MCP  2012;11(12):1801-1814.
Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden.
doi:10.1074/mcp.M112.021204
PMCID: PMC3518108  PMID: 22984287
19.  Glucocorticoids regulate extracellular matrix metabolism in human vocal fold fibroblasts 
The Laryngoscope  2011;121(9):1915-1919.
Objectives/Hypothesis
Given the recent emergence of encouraging efficacy data regarding the utility of intralesional glucocorticoid (GC) injection for a variety of vocal fold pathologies, we sought to describe the location and expression pattern of the GC receptors within the vocal folds and quantify the effects of GCs on vocal fold fibroblasts.
Study Design
In vitro, in vivo
Methods
Immunolocalization of the GC receptor (GCr) was performed on normal rat vocal fold tissue. Receptor expression was also assayed in our human vocal fold fibroblast cell line. These cells were then treated with exogenous dexamethasone (DM) to quantify the effects of GCs on receptor expression, proliferation, TGF-β-induced collagen secretion, and matrix protease synthesis.
Results
Positive immunostaining for the GC receptor was found throughout the vocal fold with particularly strong staining in the epithelium and capillaries. Human vocal fold fibroblasts constitutively express the GC receptor, but this expression decreased in response to exogenous DM. DM also decreased fibroblast proliferation and TGF-β-induced collagen synthesis. DM also abrogated TGF-β-mediated effects on enzymes related extracellular matrix turnover.
Conclusions
Our data are the first to provide mechanistic insight regarding the recently-published favorable data regarding the utility of GCs in patients with vocal fold scar. Although further investigation is warranted, both the accessibility of this class of agents and the amenability to office-based procedures are likely to direct patient care models.
Level of Evidence
N/A
doi:10.1002/lary.21920
PMCID: PMC3205428  PMID: 22024844
vocal fold; voice; steroids; scar; dexamethasone; glucocorticoids
20.  MGEx-Udb: A Mammalian Uterus Database for Expression-Based Cataloguing of Genes across Conditions, Including Endometriosis and Cervical Cancer 
PLoS ONE  2012;7(5):e36776.
Background
Gene expression profiling of uterus tissue has been performed in various contexts, but a significant amount of the data remains underutilized as it is not covered by the existing general resources.
Methodology/Principal Findings
We curated 2254 datasets from 325 uterus related mass scale gene expression studies on human, mouse, rat, cow and pig species. We then computationally derived a ‘reliability score’ for each gene's expression status (transcribed/dormant), for each possible combination of conditions and locations, based on the extent of agreement or disagreement across datasets. The data and derived information has been compiled into the Mammalian Gene Expression Uterus database (MGEx-Udb, http://resource.ibab.ac.in/MGEx-Udb/). The database can be queried with gene names/IDs, sub-tissue locations, as well as various conditions such as the cervical cancer, endometrial cycles and disorders, and experimental treatments. Accordingly, the output would be a) transcribed and dormant genes listed for the queried condition/location, or b) expression profile of the gene of interest in various uterine conditions. The results also include the reliability score for the expression status of each gene. MGEx-Udb also provides information related to Gene Ontology annotations, protein-protein interactions, transcripts, promoters, and expression status by other sequencing techniques, and facilitates various other types of analysis of the individual genes or co-expressed gene clusters.
Conclusions/Significance
In brief, MGEx-Udb enables easy cataloguing of co-expressed genes and also facilitates bio-marker discovery for various uterine conditions.
doi:10.1371/journal.pone.0036776
PMCID: PMC3350469  PMID: 22606288
23.  Orbitotemporal Neurofibromatosis: Case Report 
Plexiform neurofibromas occur in about 60% of neurofibromatosis type 1(NF-1) patients and can lead to severe morbidity by disfigurement or compression of vital structures. Moreover, these tumors can undergo malignant transformation. Both focal and localized bone abnormalities are part of the phenotypic expression of NF-1. We report a rare case of severe cranioorbital plexiform neurofibromatosis in a young male and discuss the classification, clinical features, and various treatment options of orbit-temporal neurofibromatosis type 1.
doi:10.1155/2012/498186
PMCID: PMC3350063  PMID: 22606495
24.  Morphological properties of collagen fibers in porcine lamina propria 
Objectives
Collagen influences the biomechanical properties of vocal folds. Altered collagen morphology has been implicated in dysphonia associated with aging and scarring. Documenting the morphological properties of native collagen in healthy vocal folds is essential to understand the structural and functional alterations to collagen with aging and disease. Our primary objective was to quantify the morphological properties of collagen in the vocal fold lamina propria. Our secondary exploratory objective was to investigate the effects of pepsin exposure on the morphological properties of collagen in the lamina propria.
Design
Experimental, in vitro study with porcine model.
Methods
Lamina propria was dissected from 26 vocal folds and imaged with Atomic Force Microscopy (AFM). Morphological data on d-periodicity, diameter, and roughness of collagen fibers were obtained. To investigate the effects of pepsin exposure on collagen morphology, vocal fold surface was exposed to pepsin or sham challenge prior to lamina propria dissection and AFM imaging.
Results
The d-periodicity, diameter, and roughness values for native vocal fold collagen are consistent with literature reports for collagen fibers in other body tissue. Pepsin exposure on vocal fold surface did not appear to change the morphological properties of collagen fibers in the lamina propria.
Conclusions
Quantitative data on collagen morphology were obtained at nanoscale resolution. Documenting collagen morphology in healthy vocal folds is critical for understanding the physiological changes to collagen with aging and scarring, and for designing biomaterials that match the native topography of lamina propria.
doi:10.1016/j.jvoice.2009.09.006
PMCID: PMC2889026  PMID: 20171830
vocal fold collagen; pepsin; atomic force microscopy; bioimplants
25.  Pseudomonas sp. as a Source of Medium Chain Length Polyhydroxyalkanoates for Controlled Drug Delivery: Perspective 
Controlled drug delivery technology represents one of the most rapidly advancing areas of science. They offer numerous advantages compared to conventional dosage forms including improved efficacy, reduced toxicity, improved patient compliance and convenience. Over the past several decades, many delivery tools or methods were developed such as viral vector, liposome-based delivery system, polymer-based delivery system, and intelligent delivery system. Recently, nonviral vectors, especially those based on biodegradable polymers, have been widely investigated as vectors. Unlike the other polymers tested, polyhydroxyalkanoates (PHAs) have been intensively investigated as a family of biodegradable and biocompatible materials for in vivo applications as implantable tissue engineering material as well as release vectors for various drugs. On the other hand, the direct use of these polyesters has been hampered by their hydrophobic character and some physical shortcomings, while its random copolymers fulfilled the expectation of biomedical researchers by exhibiting significant mechanical and thermal properties. This paper reviews the strategies adapted to make functional polymer to be utilized as delivery system.
doi:10.1155/2012/317828
PMCID: PMC3299479  PMID: 22518140

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