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1.  Malassezia—Can it be Ignored? 
Indian Journal of Dermatology  2015;60(4):332-339.
Genus Malassezia comprises of 14 species of “yeast like fungi,” 13 of which are lipophilic and 1 is nonlipophilic. They are known commensals and in predisposed individuals they commonly cause a spectrum of chronic recurrent infections. They rarely also cause serious illnesses like catheter-related blood stream infections, CAPD associated peritonitis etc., Though these fungi have been known to man for over 150 years, their fastidious nature and cumbersome culture and speciation techniques have restricted research. Since the last taxonomic revision, seven new species have been added to this genus. Their ability to evade the host immune system and virulence has increased the spectrum of the diseases caused by them. These agents have been implicated as causal agents in common diseases like atopic dermatitis recently. Though culture-based research is difficult, the new molecular analysis techniques and facilities have increased research in this field such that we can devote more attention to this genus to study in detail, their characteristics and their growing implications implications in the clinical scenario.
PMCID: PMC4533528  PMID: 26288399
Malassezia; pityriasis versicolor; seborrhoeic dermatitis
2.  Palatal bone thickness measured by palatal index method using cone-beam computed tomography in nonorthodontic patients for placement of mini-implants 
Journal of Pharmacy & Bioallied Sciences  2015;7(Suppl 1):S107-S110.
The purpose of this study was to compare the bone thickness of the palatal areas in different palatal index (PI) groups
Materials and Methods:
Cone-beam computed tomography scans of 10 subjects were selected with ameanage group of 18 years. The measurements of palatal bone thickness were made at 36 sites using CareStream 3D Imaging software. The PIwas measured using Korkhaus ratio (palatal height/palatal width). One-way analysis of variance was used to analyze intergroup differences, as well as the PI difference.
Bone thickness was higher in the anterior region than in the middle and posterior regions P <0.001. Furthermore, significant differences were found among the midline, medial, and lateralareas of the palate.
These findings might be helpful for clinicians to enhance the successful useof temporary anchorage devices in the palate.
PMCID: PMC4439645  PMID: 26015685
Imaging; mid palatal area; palatal index
3.  Orthodontic instrument sterilization with microwave irradiation 
Journal of Pharmacy & Bioallied Sciences  2015;7(Suppl 1):S111-S115.
This study was designed to evaluate the efficiency of microwave sterilization of orthodontic instruments and molar bands immersed in plain distilled water with and without oral rinse, and to ascertain the minimum time of exposure required to sterilize.
Materials and Methods:
The orthodontic instruments (hinged and nonhinged), molar bands and mouth mirrorsused in the patient 's mouth were selected for the study. The instruments were divided into two groups – Group I with oral rinse-set A (0.01% chlorhexidine gluconate) and set B (0.025% betadine) and Group II (included sets C and D without oral rinse). The instruments of set A, B and C were microwaved at 2,450 MHz, 800 W for 5 min, whereas, set D was microwaved for 10 min at the same above mentioned specifications. The efficacy of sterilization was assessed by stab inoculation of the instruments onto trypticase soya agar plates. The plates were checked for bacterial growth following incubation at 37 °C for 24 h. For sterility control,Geobacillus stearothermophilus (MTCC 1518) was included.
No growth was observed in the plates that were inoculated with the microwaved orthodontic instruments of sets A, B and D, whereas scanty bacterial growth was observed in the plates inoculatedwith the microwaved set C instruments.
Effective sterilization was achieved when the orthodontic instruments and molar bands were immersed in distilled water without oral rinse and microwaved for 10 min as also for those that were immersed in distilled water with oral rinse and microwaved for 5 min.
PMCID: PMC4439646  PMID: 26015686
Biological indicator; microwave sterilization; molar bands; orthodontic pliers
4.  Smile: A review 
Journal of Pharmacy & Bioallied Sciences  2015;7(Suppl 1):S271-S275.
“Beauty is in the mind of the beholder, each mind perceives a different beauty” famously said by writer Margeret Wolfe Hungerford. A beautiful smile is a gateway to the world. The aim of this article was to identify the criteria for designing the perfect smile. It was determined, smile design is a multifactorial process and various steps are involved in designing a radiant smile.
PMCID: PMC4439690  PMID: 26015730
Arc; buccal corridor; smile
5.  Congenital Generalized Hypertrichosis, Gingival Hyperplasia, a Coarse Facies with Constriction Bands: A Rare Association 
Congenital generalized hypertrichosis terminalis is a rare primary hypertrichotic condition, of unknown etiology presenting in the pediatric population. Though benign in nature, there is considerable psychosocial trauma attached to this, owing to the cosmetic disfigurement it produces. The association of gingival fibromatosis and a coarse facies could further worsen the cosmesis. Thus, a multidisciplinary approach involving a psychologist, a dentist apart from the dermatologist would be mandatory. We present this rare syndrome with the purpose of getting a better insight regarding the inheritance, the clinical features and the best available treatment modalities, especially the modern and novel techniques of hair removal that could be utilized to manage such individuals.
PMCID: PMC4502477  PMID: 26180451
Coarse facies; constriction bands; gingival hyperplasia; hypertrichosis
6.  Bupropion for Interferon-Alpha-Induced Depression in Patients with Hepatitis C Viral Infection: An Open-Label Study 
Psychiatry Investigation  2015;12(1):142-145.
Interferon (IFN)-α therapy for chronic hepatitis C virus (HCV) infection is frequently associated with major depressive episodes. Bupropion, a commonly used antidepressant agent, has recently found to have strong anti-inflammatory effects in animal models. Despite of the theoretical relevancy, the antidepressant effect of bupropion in IFN-alpha-induced depression has never been studied. Ten HCV patients with IFN-α-induced depression were recruited to receive 8-week bupropion treatment and were assessed every 2 weeks for depressive symptoms by the Hamilton rating scale for depression (HAMD) and somatic symptoms by the Neurotoxicity Rating Scale (NRS). Four of the 10 patients met the criteria for remission (total HAMD scores≤7), and 5 patients met the criteria for response (at least 50% reduction in total HAMD scores). In addition, 5 patients had 50% decreases in NRS for neuropsychiatric symptoms. This preliminary open-label study suggests that bupropion is effective in treating IFN-alpha-induced depressive and somatic symptoms.
PMCID: PMC4310912  PMID: 25670957
Interferon-induced depression; Bupropion; Antidepressant; Hepatitis C viral infection
8.  Analysis of Skeletal Muscle Gene Expression Patterns and the Impact of Functional Capacity in Patients With Systolic Heart Failure 
Journal of cardiac failure  2014;20(6):422-430.
Declining physical function is common among systolic heart failure (HF) patients and heralds poor clinical outcomes. We hypothesized that coordinated shifts in expression of ubiquitin-mediated atrophy-promoting genes are associated with muscle atrophy and contribute to decreased physical function.
Systolic HF patients (left ventricular ejection fraction [LVEF] ≤40%) underwent skeletal muscle biopsies (nondominant vastus lateralis) and comprehensive physical assessments. Skeletal muscle gene expression was assessed with the use of real-time polymerase chain reaction. Aerobic function was assessed with the use of cardiopulmonary exercise and 6-minute walk tests. Strength capacity was assessed with the use of pneumatic leg press (maximum strength and power). Serologic inflammatory markers also were assessed.
54 male patients (66.6 ± 10.0 years) were studied: 24 systolic HF patients (mean LVEF 28.9 ± 7.8%) and 30 age-matched control subjects. Aerobic and strength parameters were diminished in HF versus control. FoxO1 and FoxO3 were increased in HF versus control (7.9 ± 6.2 vs 5.0 ± 3.5, 6.5 ± 4.3 vs 4.3 ± 2.8 relative units, respectively; P ≤.05 in both). However, atrogin-1 and MuRF-1 were similar in both groups. PGC-1α was also increased in HF (7.9 ± 5.4 vs. 5.3 ± 3.6 relative units; P < .05). Muscle levels of insulin-like growth factor (IGF) 1 as well as serum levels of tumor necrosis factor α, C-reactive protein, inter-leukin (IL) 1β, and IL-6 were similar in HF and control.
Expression of the atrophy-promoting genes FoxO1 and FoxO3 were increased in skeletal muscle in systolic HF compared with control, but other atrophy gene expression patterns (atrogin-1 and MuRF-1), as well as growth promoting patterns (IGF-1), were similar. PGC-1α, a gene critical in enhancing mitochondrial function and moderating FoxO activity, may play an important counterregulatory role to offset ubiquitin pathwayemediated functional decrements.
PMCID: PMC4278433  PMID: 24704539
Heart failure; skeletal muscle; gene expression
9.  Structure-activity relationships of 2-aminothiazoles effective against Mycobacterium tuberculosis 
Bioorganic & medicinal chemistry  2013;21(21):10.1016/j.bmc.2013.08.048.
A series of 2-aminothiazoles was synthesized based on a HTS scaffold from a whole-cell screen against Mycobacterium tuberculosis (Mtb). The SAR shows the central thiazole moiety and the 2-pyridyl moiety at C-4 of the thiazole are intolerant to modification. However, the N-2 position of the aminothiazole exhibits high flexibility and we successfully improved the antitubercular activity of the initial hit by more than 128-fold through introduction of substituted benzoyl groups at this position. N-(3-Chlorobenzoyl)-4-(2-pyridinyl)-1,3-thiazol-2-amine (55) emerged as one of the most promising analogues with a MIC of 0.024 μM or 0.008 μg/mL in 7H9 media and therapeutic index of nearly ~300. However, 55 is rapidly metabolized by human liver microsomes (t1/2 = 28 min) with metabolism occurring at the invariant aminothiazole moiety and Mtb develops spontaneous resistance with a high frequency of ~10−5.
PMCID: PMC3816974  PMID: 24075144
Mycobacterium tuberculosis; aminothiazole; high-throughput screening; antibacterial agent
10.  A Case of Lamellar Ichthyosis with Rickets and Carcinoma of the Hypopharynx 
Lamellar ichthyosis (LI) is an autosomal recessive disorder rarely associated with systemic organ involvement and development of carcinoma. Rickets has occasionally been described with LI owing to impaired vitamin D synthesis following altered keratinization. There has also been a high association of cutaneous cancers in patients of LI. We as Dermatologists should therefore be very meticulous while doing a full work up of these patients. We report here a case of LI associated with rickets and carcinoma of the hypopharynx.
PMCID: PMC4248537  PMID: 25484429
Lamellar ichthyosis; rickets; carcinoma of hypopharynx
11.  BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis 
The Journal of Cell Biology  2014;205(2):233-249.
The integral membrane glycoprotein BARP associates with voltage-gated calcium channel Cavβ subunits, disrupting their association with Cavα1 subunits and suppressing overall channel activity.
Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca2+-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca2+ overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC β-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavβ subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain–binding pocket in Cavβ and interferes with the association between Cavβ and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavβ via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca2+ channel activity at the plasma membrane, resulting in the inhibition of Ca2+-evoked exocytosis. Thus, BARP can modulate the localization of Cavβ and its association with the Cavα1 subunit to negatively regulate VGCC activity.
PMCID: PMC4003244  PMID: 24751537
12.  Comparison of screening for gestational diabetes mellitus by oral glucose tolerance tests done in the non-fasting (random) and fasting states 
Acta Diabetologica  2014;51(6):1007-1013.
The Diabetes in Pregnancy Study Group of India (DIPSI) guidelines recommend the non-fasting 75-g oral glucose tolerance test (OGTT) as a single-step screening and diagnostic test for gestational diabetes mellitus (GDM). The aim of this study was to compare the DIPSI criteria with the World Health Organization (WHO) 1999 and the International Association of Diabetes and Pregnancy Study Groups (IADPSG) criteria for GDM.
A total of 1,031 pregnant women attending antenatal clinics in urban and rural Tamil Nadu, India, underwent a 75-g OGTT in both non-fasting and fasting states, 2–3 days apart. Venous plasma glucose was measured using an autoanalyser, and GDM was diagnosed by DIPSI, WHO 1999 and IADPSG criteria.
Of the 83 women identified to have GDM by WHO 1999 criteria, only 23 were diagnosed by DIPSI criteria. Of the 106 women diagnosed to have GDM by the IADPSG criteria, only 24 were diagnosed by DIPSI. The DIPSI non-fasting OGTT 2-h VPG cut point of 140 mg/dl (7.8 mmol/l) had a very low sensitivity when compared to the WHO 1999 criteria (sensitivity 27.7 %, specificity 97.7 %) and IADPSG criteria (sensitivity 22.6 %, specificity 97.8 %).
The DIPSI non-fasting OGTT criteria cannot be recommended for diagnosis of GDM due to its low sensitivity. Thus, as a single-step diagnostic test for GDM, the fasting OGTT needs to be done. When this is not possible, the well-established two-step procedure using the 50-g glucose challenge test as an initial screening test, followed by the diagnostic fasting OGTT, can be continued.
PMCID: PMC4239809  PMID: 25315629
Gestational diabetes mellitus; Fasting OGTT; Non-fasting OGTT; Diabetes in Pregnancy Study Group of India (DIPSI); WHO 1999 criteria; IADPSG; Screening
13.  Differential Contribution of Tryptophans to the Folding and Stability of the Attachment Invasion Locus Transmembrane β-Barrel from Yersinia pestis 
Scientific Reports  2014;4:6508.
Attachment invasion locus (Ail) protein of Yersinia pestis is a crucial outer membrane protein for host invasion and determines bacterial survival within the host. Despite its importance in pathogenicity, surprisingly little is known on Ail biophysical properties. We investigate the contribution of micelle concentrations and interface tryptophans on the Ail β-barrel refolding and unfolding processes. Our results reveal that barrel folding is surprisingly independent of micelle amounts, but proceeds through an on-pathway intermediate that requires the interface W42 for cooperative barrel refolding. On the contrary, the unfolding event is strongly controlled by absolute micelle concentrations. We find that upon Trp→Phe substitution, protein stabilities follow the order W149F>WT>W42F for the refolding, and W42F>WT>W149F for unfolding. W42 confers cooperativity in barrel folding, and W149 clamps the post-folded barrel structure to its micelle environment. Our analyses reveal, for the first time, that interface tryptophan mutation can indeed render greater β-barrel stability. Furthermore, hysteresis in Ail stems from differential barrel-detergent interaction strengths in a micelle concentration-dependent manner, largely mediated by W149. The kinetically stabilized Ail β-barrel has strategically positioned tryptophans to balance efficient refolding and subsequent β-barrel stability, and may be evolutionarily chosen for optimal functioning of Ail during Yersinia pathogenesis.
PMCID: PMC4179465  PMID: 25266561
14.  Evaluation of initial stability and crestal bone loss in immediate implant placement: An in vivo study 
(1) To measure the crestal bone levels around implants immediately, and one month, three months, and six months after immediate implant placement, to evaluate the amount of bone level changes in six months. (2) To measure the initial stability in immediate implant placement.
Materials and Methods:
Ten patients were selected and a total of ten implants were placed in the immediate extraction sites. The change in the level of crestal bone was measured on standardized digital periapical radiographs taken at baseline, first month, third month, and sixth months for each patient, using the SOPRO imaging software. The initial stability of implants was measured with resonance frequency analysis (RFA) and an engine-driven torque. The measurements were statistically analyzed. The student's t-test was used, to identify the significance of the study parameters.
When mesial and distal bone losses were averaged, the radiographic evaluation with the SOPRO imaging software showed an average of 0.80 mm, with a standard deviation of ± 0.18 mm bone loss at the first month, followed by 1.03 mm with a standard deviation of ± 0.19 mm at the third month, and 1.23 mm with standard deviation of ± 0.6 mm at the sixth month. The initial stability with the RFA instrument showed a mean of 55 implant stability quotient (ISQ) values and the torque showed a value of 36.50 Nm.
The implant has to be placed 2 mm below the crestal bone level to compensate the crestal bone loss. The initial stability is achieved by apical preparation of the socket wall and use of straight screw implants. When the defect is more than 2 mm, autogenous grafts with membranes are the best choice.
PMCID: PMC4209610  PMID: 25374829
Crestal bone loss; immediate implants; initial stability in immediate implants
15.  Neutrophil Recruitment to the Lung in both C5a and CXCL1-Induced Alveolitis is Impaired in Vitamin D Binding Protein Deficient Mice 
Knowledge of how neutrophils respond to chemotactic signals in a complex inflammatory environment is not completely understood. Moreover, even less is known about factors in physiological fluids that regulate the activity of chemoattractants. The vitamin D binding protein (DBP) has been shown to significantly enhance chemotaxis to complement activation peptide C5a using purified proteins in vitro, and by ex vivo depletion of DBP in physiological fluids, but this function has not been determined in vivo. DBP null (-/-) mice were used to investigate how a systemic absence of this plasma protein affects leukocyte recruitment in alveolitis models of lung inflammation. DBP-/- mice had significantly reduced (~50%) neutrophil recruitment to the lungs compared to their wild-type DBP+/+ counterparts in three different alveolitis models, two acute and one chronic. The histology of DBP-/- mouse lungs also showed significantly less injury than wild-type animals. The chemotactic cofactor function of DBP appears to be selective for neutrophil recruitment, but in contrast to previous in vitro results, in vivo DBP can enhance the activity of other chemoattractants including CXCL1. The reduced neutrophil response in DBP-/- mice could be rescued to wild-type levels by administering exogenous DBP. Finally, in inflammatory fluids DBP binds to G-actin released from damaged cells and this complex may be the active chemotactic cofactor. Results show for the first time that DBP is a significant chemotactic cofactor in vivo and not specific for C5a, suggesting that this ubiquitous plasma protein may have a more significant role in neutrophil recruitment than previously recognized.
PMCID: PMC3702662  PMID: 23752613
16.  Evaluation of Rapid Blood Sample Collection in the Detection of Circulating Filarial Antigens for Epidemiological Survey by rWbSXP-1 Capture Assay 
PLoS ONE  2014;9(7):e102260.
Lymphatic filariasis is a neglected tropical disease leading to profound disfiguring causing socio economic burden in the tropics. Current diagnosis strategies available during field surveys and epidemics are based on traditional microscopic detections and a few antigen/antibody assays. We have compared different sampling methodologies and standardized the highly sensitive and reliable rWbSXP-1 antigen detection assay to our new sampling methodology.
Samples collected as serum, whole blood, whole blood on filter paper and whole blood on microscopic slides from patients belonging to various clinical groups of filariasis [endemic normal(EN), chronic pathology(CP), microfilaraemic(MF) and non-endemic normal(NEN)] were collected and standardized the rWbSXP-1 antigen detection assay using monoclonal antibody raised against rWbSXP-1 protein. The whole blood collected on microscopic slide based sampling method was employed in the field and the presence of circulating filarial antigen (CFA) was assessed using the rWbSXP-1 assay.
Principal Findings
The sampling methods were compared and no significant difference was observed for the detection of CFA (MF, P = 0.304, EN, P = 0.675, CP, P = 0.5698, NEN, P = 0.4494). Further the optimized sampling method was utilized to collect the 1106 samples from Polur, Tiruvannamalai. The rWbSXP-1 assay gave 98 antigen positive results whereas the microscopic method gave only 17.
Four sampling methodologies were analyzed and the new sampling methodology of whole blood collected on microscopic slide was found to be convenient for the detection of CFA using rWbSXP-1 antigen detection assay. The 1106 samples from Polur were collected using the new method. The rWbSXP-1 antigen assay perceived a 7.32% increased result which was read as false negatives on the conventional microscopic staining method. This new sampling methodology coupled with the rWbSXP-1 antigen assay can be used in epidemiological surveys for lymphatic filariasis and the same sampling methodology can be expanded to other antigen based high affinity assays.
PMCID: PMC4099071  PMID: 25025711
17.  An Unusual Content in a Congenital Hernia - Complete Spleno –gonadal Fusion Band 
The Indian Journal of Surgery  2012;75(Suppl 1):110-112.
Six years old boy underwent elective inguinal exploration for left congenital hernia. Per- operatively, an elongated, purplish-red, fleshy band of tissue was found inside the sac, adherent to the upper pole of testis. Biopsy was taken and the wound closed. An MRI done after 4 weeks proved the origin of the band from spleen. Laparotomy and excision of the band was done. The histo-pathology of the specimen was reported as normal splenic tissue. The above features are consistent with a diagnosis of spleno - gonadal fusion (SGF).
PMCID: PMC3693285  PMID: 24426532
Spleno-gonadal fusion; MRI for spleno-gonadal fusion; Splenic tissue in the testis; Splenic anomalies
20.  Sexually dimorphic neurons in the ventromedial hypothalamus govern mating in both sexes and aggression in males 
Cell  2013;153(4):896-909.
Sexual dimorphisms in the brain underlie behavioral sex differences, but the function of individual sexually dimorphic neuronal populations is poorly understood. Neuronal sexual dimorphisms typically represent quantitative differences in cell number, gene expression, or other features, and it is unknown if these dimorphisms control sex-typical behavior in one sex exclusively or in both sexes. The progesterone receptor (PR) controls female sexual behavior, and we find many sex differences in number, distribution, or projections of PR-expressing neurons in the adult mouse brain. We have ablated one such PR-expressing neuronal population located in the ventromedial hypothalamus (VMH) using a novel genetic strategy. Ablation of these neurons in females greatly diminishes sexual receptivity. Strikingly, the corresponding ablation in males reduces mating and aggression. Our findings reveal the functions of a molecularly-defined, sexually dimorphic neuronal population in the brain. Moreover we show that sexually dimorphic neurons can control distinct sex-typical behaviors in both sexes.
PMCID: PMC3767768  PMID: 23663785
21.  Clinical profile, outcomes, and progression to type 2 diabetes among Indian women with gestational diabetes mellitus seen at a diabetes center in south India 
To describe the clinical profile, maternal and fetal outcomes, and the conversion rates to diabetes in women with gestational diabetes mellitus (GDM) seen at a tertiary care diabetes center in urban south India.
Materials and Methods:
Clinical case records of 898 women with GDM seen between 1991 and 2011 were extracted from the Diabetes Electronic Medical Records (DEMR) of a tertiary care diabetes center in Chennai, south India and their clinical profile was analyzed. Follow-up data of 174 GDM women was available. To determine the conversion rates to diabetes, oral glucose tolerance test (OGTT) was done in these women. Glucose tolerance status postpartum was classified based on World Health Organization (WHO) 2006 criteria.
The mean maternal age of the women was 29 ± 4 years and mean age of gestation at first visit were 24 ± 8.4 weeks. Seventy percent of the women had a family history of diabetes. Seventy-eight percent of the women delivered full-term babies and 65% underwent a cesarean section. The average weight gain during pregnancy was 10.0 ± 4.2 kg. Macrosomia was present in 17.9% of the babies, hypoglycemia in 10.4%, congenital anomalies in 4.3%, and the neonatal mortality rate was 1.9%. Mean follow-up duration of the 174 women of whom outcome data was available was 4.5 years. Out of the 174, 101 women who were followed-up developed diabetes, of whom half developed diabetes within 5 years and over 90%, within 10 years of the delivery.
Progression to type 2 diabetes mellitus (T2DM) in Indian women with GDM is rapid. There is an urgent need to develop standardized protocols for GDM care in India that can improve the maternal and fetal outcomes and help prevent future diabetes in women with GDM.
PMCID: PMC4056142  PMID: 24944938
American Diabetes Association; Asian Indians; gestational diabetes; progression to type 2 diabetes; WHO
22.  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.
PMCID: PMC3967697  PMID: 24642864
23.  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.
PMCID: PMC3907894  PMID: 24494036
24.  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.
PMCID: PMC3876932  PMID: 24319267
25.  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.
PMCID: PMC3827151  PMID: 24265768

Results 1-25 (92)