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1.  Wdpcp, a PCP Protein Required for Ciliogenesis, Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton 
PLoS Biology  2013;11(11):e1001720.
Wdpcp, a protein required for both planar cell polarity and ciliogenesis, regulates cell polarity and alignment via direct modulation of the actin cytoskeleton.
Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet–Biedl/Meckel–Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration.
Author Summary
Cilia are microscopic cell surface hair-like protrusions that can act as antennae to mediate cell signaling. Mutations disrupting ciliogenesis can cause many developmental anomalies associated with syndromes known as “ciliopathies.” Some developmental defects, such as limb polydactyly, arise from disruption of cilia-transduced sonic hedgehog signaling, while other defects, such as aberrant patterning of hair cells in the inner ear, arise from disrupted Wnt signaling resulting in modulation of planar cell polarity (PCP)—a process whereby cells are polarized and aligned. While ciliopathy phenotypes would suggest that cilia are involved in modulating PCP, the mechanistic link between cilia and PCP has been elusive. Our study using a mouse model carrying a mutation in Wdpcp, a gene required for both ciliogenesis and PCP, suggest that Wdpcp modulation of PCP involves interactions with the actin cytoskeleton separate from its function in ciliogenesis. We observe Wdpcp localization in cilia, where it is required for recruitment of proteins essential for ciliogenesis. Wdpcp interacts with Sept2, and is also found in actin filaments, where it regulates actin dynamics essential for PCP. Together, these findings show that PCP regulation by Wdpcp is distinct from its function in ciliogenesis and involves direct modulation of the actin cytoskeleton.
doi:10.1371/journal.pbio.1001720
PMCID: PMC3841097  PMID: 24302887
2.  Nutritional Status of Adult Patients with Pulmonary Tuberculosis in Rural Central India and Its Association with Mortality 
PLoS ONE  2013;8(10):e77979.
Under-nutrition is a known risk factor for TB and can adversely affect treatment outcomes. However, data from India are sparse, despite the high burden of TB as well as malnutrition in India. We assessed the nutritional status at the time of diagnosis and completion of therapy, and its association with deaths during TB treatment, in a consecutive cohort of 1695 adult patients with pulmonary tuberculosis in rural India during 2004 - 2009.Multivariable logistic regression was used to obtain adjusted estimates of the association of nutritional status with deaths during treatment. At the time of diagnosis, median BMI and body weights were 16.0 kg/m2and 42.1 kg in men, and 15.0 kg/m2and 34.1 kg in women, indicating that 80% of women and 67% of men had moderate to severe under-nutrition (BMI<17.0 kg/m2). Fifty two percent of the patients (57% of men and 48% of women) had stunting indicating chronic under-nutrition. Half of women and one third of men remained moderately to severely underweight at the end of treatment. 60 deaths occurred in 1179 patients (5%) in whom treatment was initiated. Severe under-nutrition at diagnosis was associated with a 2 fold higher risk of death. Overall, a majority of patients had evidence of chronic severe under-nutrition at diagnosis, which persisted even after successful treatment in a significant proportion of them. These findings suggest the need for nutritional support during treatment of pulmonary TB in this rural population.
doi:10.1371/journal.pone.0077979
PMCID: PMC3812022  PMID: 24205052
3.  Tropical Pyomyositis 
Background:
Tropical pyomyositis is characterized by suppuration within skeletal muscles, manifesting as single or multiple abscesses. Though primarily a disease of tropics, it is increasingly being reported from temperate regions in immunosuppressed patients. However, India has only few sporadic case reports.
Aims:
The aim of this study is to evaluate the causative organisms, clinical presentations, diagnostic modalities, treatment protocols and outcome data in tropical pyomyositis patients.
Subjects and Methods:
The study was carried out in Nilratan Sircar Medical College and Hospital, Kolkata over 3 years (July 2010 to June 2013). A total of 12 patients were diagnosed with tropical pyomyositis (confirmed with aspiration and culture of pus from muscle). All the investigation and treatment data were recorded systematically.
Results:
The presenting feature was high fever and myalgia in all 12 patients. Quadriceps femoris was the most commonly involved muscle (50%); followed by iliopsoas (25%). Culture of the aspirate showed Staphylococcus aureus in nine patients (75%), Klebsiella pneumonia in one patient (8.33%) and no growth in two patients (16.67%) even after tubercular and fungal culture.
Conclusions:
Tropical pyomyositis can affect immune-competent individuals. S. aureus is the most commonly cultured organism. Immediate initiation of appropriate antibiotics and surgical debridement are required to avoid complications. The prognosis remains excellent if promptly treated.
doi:10.4103/1947-2714.120796
PMCID: PMC3842701  PMID: 24350072
Fever; Muscle pain; Staphylococcus aureus; Tropical pyomyositis
4.  Basal Cell Carcinoma, Oculo-Cutaneous Albinism and Actinic Keratosis in a Native Indian 
Indian Journal of Dermatology  2013;58(5):377-379.
The report highlights the occurrence of basal cell carcinoma in a native Indian with oculo-cutaneous albinism, an association not frequently encountered. The clinical and histopathological features, which assisted to form the diagnosis, are outlined. A high degree of suspicion and timely recognition of the potentially aggressive neoplasm, under this unusual circumstance, is the key to its diagnosis.
doi:10.4103/0019-5154.117308
PMCID: PMC3778778  PMID: 24082183
Actinic keratosis; basal Cell Carcinoma; oculo-cutaneous albinism
5.  Activation of protein kinase CK2 attenuates FOXO3a functioning in a PML-dependent manner: implications in human prostate cancer 
Cell Death & Disease  2013;4(3):e543-.
Protein kinase CK2 (also known as Caseine Kinase II) is an ubiquitous Ser/Thr protein kinase present in both the nucleus and cytoplasm of cells, targeting several key enzymes, growth factor receptors, transcription factors and cytoskeletal proteins. It is not only a key player in regulating cellular growth and proliferation, but also behaves as a potent suppressor of apoptosis. CK2 has been frequently found to be deregulated (mostly hyperactivated) in all cancers, prostate cancer being prominent of them. In the recent past, tumor suppressor PML (promyelocytic leukemia) has been shown to be a target of phosphorylation by CK2. This phosphorylation promotes the ubiquitin-mediated proteasomal degradation of PML thereby effectively curbing its role as a tumor suppressor. Among many others, PML has also been established to mediate its tumor suppressive role by mitigating the inactivation of active AKT (pAKT) inside the nucleus by assembling a dephosphorylating platform for nuclear pAKT. One of the immediate consequences, of this inactivation is the stabilization of FOXO3a, another well-established tumor suppressor, inside the nucleus and its downstream activities. Here, we propose a novel signaling axis apexed by deregulated CK2, dismantling the association of PML and PHLPP2 (we also report PHLPP2 to be a novel interacting partner of PML inside the nucleus), ultimately leading to the inactivation and nuclear exclusion of FOXO3a, thereby downregulating p21/p27/Bim in which degradation of PML and the concomitant stabilization of pAKT plays a cardinal part.
doi:10.1038/cddis.2013.63
PMCID: PMC3613841  PMID: 23492774
CK2; PML; FOXO3a
6.  Onychoheterotopia: A Unique Case 
Indian Journal of Dermatology  2013;58(2):150-151.
doi:10.4103/0019-5154.108064
PMCID: PMC3657221  PMID: 23716811
7.  Three-Dimensional Molecular Modeling of a Diverse Range of SC Clan Serine Proteases 
Serine proteases are involved in a variety of biological processes and are classified into clans sharing structural homology. Although various three-dimensional structures of SC clan proteases have been experimentally determined, they are mostly bacterial and animal proteases, with some from archaea, plants, and fungi, and as yet no structures have been determined for protozoa. To bridge this gap, we have used molecular modeling techniques to investigate the structural properties of different SC clan serine proteases from a diverse range of taxa. Either SWISS-MODEL was used for homology-based structure prediction or the LOOPP server was used for threading-based structure prediction. The predicted models were refined using Insight II and SCRWL and validated against experimental structures. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. The structural geometry of the catalytic core shows clear deviations between taxa, but the relative positions of the catalytic triad residues were conserved. Evolutionary divergence was also exhibited by large variation in secondary structure features outside the core, differences in overall amino acid distribution, and unique surface electrostatic potential patterns between species. Encompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on SC clan serine proteases.
doi:10.1155/2012/580965
PMCID: PMC3507156  PMID: 23213528
8.  Sporadic Intra-Abdominal Desmoid: A Rare Presentation as a Hepatic Mass 
Case Reports in Pathology  2012;2012:245671.
We report an unusual presentation of a sporadic intra-abdominal desmoid tumour, possibly arising from the diaphragm, masquerading as a hepatic mass in a young female without any history of surgery or trauma. Histopathology ruled out a hepatic origin of the tumour as was inferred from pre- and intraoperative evaluation. Immunohistochemistry showed positivity of lesional fibroblastic cells for β-catenin and negativity for CD34, CD117, EMA, SMA, desmin, vimentin, cytokeratin, and ALK1 thereby confirming the diagnosis of a desmoid tumour. There exist only a few reports in the literature on desmoids related to the diaphragm, but only one on a diaphragmatic desmoid that is possibly primary.
doi:10.1155/2012/245671
PMCID: PMC3471414  PMID: 23091764
9.  Evolutionary Link between the Mycobacterial Plasmid pAL5000 Replication Protein RepB and the Extracytoplasmic Function Family of σ Factors 
Journal of Bacteriology  2012;194(6):1331-1341.
Mycobacterial plasmid pAL5000 represents a family of plasmids found mostly in the Actinobacteria. It replicates using two plasmid-encoded proteins, RepA and RepB. While BLAST searches indicate that RepA is a replicase family protein, the evolutionary connection of RepB cannot be established, as no significant homologous partner (E < 10−3) outside the RepB family can be identified. To obtain insight into the structure-function and evolutionary connections of RepB, an investigation was undertaken using homology modeling, phylogenetic, and mutational analysis methods. The results indicate that although they are synthesized from the same operon, the phylogenetic affinities of RepA and RepB differ. Thus, the operon may have evolved through random breaking and joining events. Homology modeling predicted the presence of a three-helical helix-turn-helix domain characteristic of region 4 of extracytoplasmic function (ECF) σ factors in the C-terminal region of RepB. At the N-terminal region, there is a helical stretch, which may be distantly related to region 3 of σ factors. Mutational analysis identified two arginines indispensable for RepB activity, one each located within the C- and N-terminal conserved regions. Apart from analyzing the domain organization of the protein, the significance of the presence of a highly conserved A/T-rich element within the RepB binding site was investigated. Mutational analysis revealed that although this motif does not bind RepB, its integrity is important for efficient DNA-protein interactions and replication to occur. The present investigation unravels the possibility that RepB-like proteins and their binding sites represent ancient DNA-protein interaction modules.
doi:10.1128/JB.06218-11
PMCID: PMC3294839  PMID: 22247504
10.  Enteric fever in an HIV/AIDS patient: Atypical manifestations 
Iranian Journal of Microbiology  2012;4(3):150-152.
Bloodstream infections with Salmonella typhi, is uncommon in human immunodeficiency virus (HIV)-infected persons. The symptoms in such patients are often non-specific and have a rather insidious onset and progression. We report a patient with sepsis and lower limb gangrene due to Salmonella typhi infection in an HIV-infected patient.
PMCID: PMC3465542  PMID: 23066491
11.  Gallic Acid Enriched Fraction of Phyllanthus emblica Potentiates Indomethacin-Induced Gastric Ulcer Healing via e-NOS-Dependent Pathway 
The healing activity of gallic acid enriched ethanolic extract (GAE) of Phyllanthus emblica fruits (amla) against the indomethacin-induced gastric ulceration in mice was investigated. The activity was correlated with the ability of GAE to alter the cyclooxygenase- (COX-) dependent healing pathways. Histology of the stomach tissues revealed maximum ulceration on the 3rd day after indomethacin (18 mg/kg, single dose) administration that was associated with significant increase in inflammatory factors, namely, mucosal myeloperoxidase (MPO) activity and inducible nitric oxide synthase (i-NOS) expression. Proangiogenic parameters such as the levels of prostaglandin (PG) E2, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), von Willebrand Factor VIII, and endothelial NOS (e-NOS) were downregulated by indomethacin. Treatment with GAE (5 mg/kg/day) and omeprazole (3 mg/kg/day) for 3 days led to effective healing of the acute ulceration, while GAE could reverse the indomethacin-induced proinflammatory changes of the designated biochemical parameters. The ulcer healing activity of GAE was, however, compromised by coadministration of the nonspecific NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), but not the i-NOS-specific inhibitor, L-N6-(1-iminoethyl) lysine hydrochloride (L-NIL). Taken together, these results suggested that the GAE treatment accelerates ulcer healing by inducing PGE2 synthesis and augmenting e-NOS/i-NOS ratio.
doi:10.1155/2012/487380
PMCID: PMC3433150  PMID: 22966242
12.  A Comparative Study of the Management Decisions by IMNCI Algorithm and by Pediatricians of a Teaching Hospital for the Children Between 2 Months to 5 Years 
Background:
Integrated management of neonatal and childhood illness (IMNCI) is already operational in many states of India, but there are very few studies in Indian scenario comparing its validity and reliability with the pediatricians’ decisions.
Objectives:
1) To compare the IMNCI decision with the decision of pediatricians; 2) to assess the significance of multiple presenting symptoms in the IMNCI algorithm.
Materials and Methods:
The study was conducted among the sick children between 2 months to 5 years presented in pediatric department from January to March 2009. The IMNCI decision was compared with pediatrician's decisions by percent agreement, Kappa and weighted Kappa with the aids of SPSS version 10.
Results:
The overall diagnostic agreement between IMNCI algorithm and pediatrician's decisions was 36.64%, (Kappa 0.16 and weighted Kappa 0.29) with 51.15% over diagnosis and 12.21% under diagnosis. The importance given by IMNCI algorithm in cases of multiple presenting symptoms was also reflected as it was evident that 37.50% children presented with three symptoms were categorized as red, whereas it was 28.57% and 11.67% for those presented with two and one symptom, respectively, (P < 0.0001). Pediatricians also gave importance for presence of multiple symptoms by considering 50% as admissible in the group presented with three symptoms, 30.16% in the group presented with two symptoms, and 16.67% in the group presented with only one symptom. The association was also statistically significant (P = 0.018).
Conclusion:
Diagnostic discordance is seen mainly due to over diagnosis of all fever cases as malaria. Importance of presence of comorbidities was also reflected.
doi:10.4103/0970-0218.99923
PMCID: PMC3483511  PMID: 23112444
IMNCI; Kappa; validation study; weighted kappa
13.  Acute Intestinal Obstruction: A Rare Aetiology 
Case Reports in Surgery  2012;2012:501209.
Internal herniation of small intestine is a very rare entity, and it poses a real diagnostic challenge clinically. Recurrent entrapment of the bowel may lead to partial to complete intestinal obstruction and eventually strangulation of the small bowel. Of this rare clinical entity, left paraduodenal hernia is more common. High index of suspicion with prompt management may prevent bowel strangulation and gangrene. We present a case of acute intestinal obstruction due to left paraduodenal hernia with malrotation of midgut in a 55-year-old male patient.
doi:10.1155/2012/501209
PMCID: PMC3389660  PMID: 22792504
14.  Interaction of Virstatin with Human Serum Albumin: Spectroscopic Analysis and Molecular Modeling 
PLoS ONE  2012;7(5):e37468.
Virstatin is a small molecule that inhibits Vibrio cholerae virulence regulation, the causative agent for cholera. Here we report the interaction of virstatin with human serum albumin (HSA) using various biophysical methods. The drug binding was monitored using different isomeric forms of HSA (N form ∼pH 7.2, B form ∼pH 9.0 and F form ∼pH 3.5) by absorption and fluorescence spectroscopy. There is a considerable quenching of the intrinsic fluorescence of HSA on binding the drug. The distance (r) between donor (Trp214 in HSA) and acceptor (virstatin), obtained from Forster-type fluorescence resonance energy transfer (FRET), was found to be 3.05 nm. The ITC data revealed that the binding was an enthalpy-driven process and the binding constants Ka for N and B isomers were found to be 6.09×105 M−1 and 4.47×105 M−1, respectively. The conformational changes of HSA due to the interaction with the drug were investigated from circular dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. For 1∶1 molar ratio of the protein and the drug the far-UV CD spectra showed an increase in α- helicity for all the conformers of HSA, and the protein is stabilized against urea and thermal unfolding. Molecular docking studies revealed possible residues involved in the protein-drug interaction and indicated that virstatin binds to Site I (subdomain IIA), also known as the warfarin binding site.
doi:10.1371/journal.pone.0037468
PMCID: PMC3359307  PMID: 22649528
15.  Regenerative urethroplasty in reoperative hypospadias: Buried strip principle revisited 
Aim:
Reporting the feasibility of the Denis Browne (buried strip) principle along with tunica vaginalis (TV) pedicled wrapping of the strip in reoperative urethroplasty in hypospadias.
Materials and Methods:
Over a period of 5 years, 32 patients presented with failure of previous urethroplasty and the range of failure was between 2 and 6 times; mean 2.5 times. Mean age was 12.9 (range 2 to 26 years) years. “Buried strip” urethroplasty (i.e., without tubularization of urethral plate) and wrapping with TV were done along with supra pubic cystostomy (SPC) for diversion of urine. Mean follow-up was 29.8 (range 12 to 56 months) months.
Results:
One patient had fistula and vertical slit meatus was possible in 26 patients. The flow of urine was satisfactory in 31 patients and one patient developed pouch in penile urethra.
Conclusions:
The buried strip along with the additional coverage with TV was found to be simple and effective in salvaging the failed urethroplasty.
doi:10.4103/0971-9261.93965
PMCID: PMC3326824  PMID: 22529550
Denis Browne; hypospadias; regeneration; tubularized incised plate; tunica vaginalis; urethroplasty
16.  A study of histopathological spectrum and expression of Ki-67, TP53 in primary brain tumors of pediatric age group 
Objectives:
The primary brain tumors are the second most common cause of death due to malignancies in children. This study was done to analyze the histological spectrum of primary brain tumors in children and also to find out the expression of p53 and Ki67 in some of the common pediatric brain tumors.
Materials and Methods:
This study was done over a period of 2.5 years. The patients were followed up until 6 months to determine the outcome. We examined H and E sections from 61 pediatric brain tumors and also performed immunohistochemical stains with p53 and Ki67 on 52 of these samples.
Results:
Of the 61 cases of pediatric brain tumors the commonest were pilocytic astrocytomas and medulloblastomas both constituting 22.9% of total cases, followed by high grade gliomas, that is, anaplastic astrocytoma and glioblastoma taken together (14.7%), diffuse astrocytomas (11.4%), ependymomas (8.1%), and oligodendrogliomas (4.9%). Other cases comprised craniopharyngiomas, astroblastomas, and gangliocytoma. The mean age of presentation was 9.3 years, male children being more commonly affected. Ki67 labeling index (LI) and p53 expression in pilocytic astrocytomas and diffuse astrocytomas were significantly lower than that of high-grade astrocytomas. However, there was no significant difference of expression of these two antigens in pilocytic astrocytomas and diffuse astrocytomas. It was found that Ki67 LI was a better marker for distinguishing between grades of astrocytoma than p53 (P=0.000 and P=0.002, respectively). The survival in cases of pilocytic astrocytomas was far better than high-grade gliomas. However, there was no significant difference in survival between pilocytic astrocytoma and diffuse infiltrating astrocytoma. There was significant positive correlation between expression of p53 and Ki67 LI in cases of medulloblastomas. Both p53 (P=0.002) and Ki67 LI (P=0.000) taken individually correlated well with survival in these cases. Also, Ki67 LI is better predictor of outcome than p53.
Conclusion:
From this study, it can be concluded that Ki67 and p53 score correlated well with the grade of astrocytoma; however, Ki67 is a better marker for differentiating between the grades of astrocytoma than p53. Also, Ki67 LI is a better prognostic factor than p53 in case of medulloblastomas.
doi:10.4103/0971-5851.96965
PMCID: PMC3385275  PMID: 22754205
Pediatric brain tumors; p53; Ki67
17.  Health awareness and popularity of alternative medicines among people of Jamnagar town: A cross - sectional study 
Ayu  2012;33(1):33-37.
By 2020, it is predicted that non-communicable diseases will be causing seven out of every 10 deaths in developing countries. Indian traditional medicine system with the concept of personalized therapy in Ayurveda has the potential to offer remedies to these challenging health issues. Integration of Ayurveda, Siddha and Unani, the three Indian systems of medicine (ISM), along with homoeopathic and allopathic systems of medicine to ensure health for all citizens across the country is the new Mantra of the Union health ministry. To tap the potentials of our indigenous medicine systems and other popular systems of medicine it is important to assess the awareness among people and make efforts to popularize them. The present study was therefore carried out to assess the awareness among 200 respondents with the help of a multiple choice questionnaire by the interview method. Convenience sampling technique was employed. The awareness about lifestyle, diet, oil consumption needs more vigorous attention as observed in this study. The most popular choice was found to be groundnut oil. Around 4% of the participants used more than one medium of cooking. Forty-two percent of the participants observed fast regularly. Twenty-three percent of the participants did not include any form of exercise in their daily routine while walking was the most popular form of exercise performed by 43%. By using multiple comparisons it was observed that the difference between i) Allopathy- Homeopathy, ii) Allopathy – Ayurvedic and iii) Ayurvedic- Homeopathy as 15.5263, 7.1053 and 8.4211, respectively, are significant at α = 0.05. A larger sample size encompassing various economic strata could be a better index of popularity of various alternative medicine systems existing in different sections of our society.
doi:10.4103/0974-8520.100306
PMCID: PMC3456860  PMID: 23049181
Alternative and complementary medicine; health awareness; Indian system of medicine; popularity of Ayurveda
18.  Intramedullary tumors in children 
Journal of Pediatric Neurosciences  2011;6(Suppl1):S86-S90.
Intramedullary tumors of the spinal cord account for 35-40% of intraspinal tumors in children. The biological behavior of these tumors is of slow progression, and hence aggressive surgery has been advocated. Surgical adjuncts include use of intraoperative neurophysiological monitoring, preoperative ultrasound, microsurgical techniques and ultrasonic suction devices. Osteoplastic laminoplasty approaches avoid post-laminectomy deformities in younger children. Postoperative radiotherapy and more recently chemotherapy regimes have been proposed for incompletely resected tumors.
doi:10.4103/1817-1745.85718
PMCID: PMC3208914  PMID: 22069435
Intramedullary tumours; intra-operative monitoring; laminoplasty; astrocytomas; ependymomas
19.  A fadD mutant of Vibrio cholerae Is Impaired in the Production of Virulence Factors and Membrane Localization of the Virulence Regulatory Protein TcpP▿  
Infection and Immunity  2010;79(1):258-266.
In the enteric pathogen Vibrio cholerae, expression of the major virulence factors is controlled by the hierarchical expression of several regulatory proteins comprising the ToxR regulon. In this study, we demonstrate that disruption of the fadD gene encoding a long-chain fatty acyl coenzyme A ligase has marked effects on expression of the ToxR virulence regulon, motility, and in vivo lethality of V. cholerae. In the V. cholerae fadD mutant, expression of the major virulence genes ctxAB and tcpA, encoding cholera toxin (CT), and the major subunit of the toxin-coregulated pilus (TCP) was drastically repressed and a growth-phase-dependent reduction in the expression of toxT, encoding the transcriptional activator of ctxAB and tcpA, was observed. Expression of toxT from an inducible promoter completely restored CT to wild-type levels in the V. cholerae fadD mutant, suggesting that FadD probably acts upstream of toxT expression. Expression of toxT is activated by the synergistic effect of two transcriptional regulators, TcpP and ToxR. Reverse transcription-PCR and Western blot analysis indicated that although gene expression and production of both TcpP and ToxR are unaffected in the fadD mutant strain, membrane localization of TcpP, but not ToxR, is severely impaired in the fadD mutant strain from the mid-logarithmic phase of growth. Since the decrease in toxT expression occurred concomitantly with the reduction in membrane localization of TcpP, a direct correlation between the defect in membrane localization of TcpP and reduced toxT expression in the fadD mutant strain is suggested.
doi:10.1128/IAI.00663-10
PMCID: PMC3019873  PMID: 21041490
20.  Three dimensional modeling of C-terminal loop of CssA subunit in CS6 of Enterotoxigenic Escherichia coli and its interaction with the 70 KDa domain of Fibronectin 
Bioinformation  2011;6(8):307-310.
Colonization factor CS6 of enterotoxigenic Escherichia coli (ETEC) helps to establish the adherence of CS6-expressing ETEC in the intestinal wall. CS6 is composed of two structural subunits, known as CssA and CssB. During CS6-expressing ETEC adherence in intestinal wall, 15 amino acid residues containing Cterminal region of CssA subunit, help to bind with N-terminal 70kDa domain of fibronectin (Fn). In this study, we have predicted a theoretical structural model for C-terminal domain of CssA by homology modelling using protein data bank (PDB) file, 1NTY-A as template (66.67% sequence identity) in Discovery Studio. The structural model of N-terminal region of Fn was also determined by homology modelling using PDB files 1FBR and 1E88 as templates. The structure of the model was also validated by Ramachandran plot. The energy minimization for Fn was performed in standard dynamic cascade using Steepest Descent algorithm followed by Adopted Basis NR algorithm in Discovery studio. The docking model between C-terminal domain and fibronectin were generated by using ClusPro algorithm. This docking study would be help for better understanding how CS6 interacts with fibronectin of intestinal extracellular matrix in the host during infection, and would be of great help towards subunit vaccine generation.
PMCID: PMC3134779  PMID: 21769192
Colonization factor CS6; ETEC; fibronectin; homology modelling; Ramachandran plot; docking model
21.  Psorinum Therapy in Treating Stomach, Gall Bladder, Pancreatic, and Liver Cancers: A Prospective Clinical Study 
We prospectively studied the clinical efficacy of an alternative cancer treatment “Psorinum Therapy” in treating stomach, gall bladder, pancreatic and liver cancers. Our study was observational, open level and single arm. The participants' eligibility criteria included histopathology/cytopathology confirmation of malignancy, inoperable tumor, and no prior chemotherapy or radiation therapy. The primary outcome measures of the study were (i) to assess the radiological tumor response (ii) to find out how many participants survived at least 1 year, 2 years, 3 years, 4 years and finally 5 years after the beginning of the study considering each type of cancer. Psorinum-6x was administered orally to all the participants up to 0.02 ml/Kg body weight as a single dose in empty stomach per day for 2 years along with allopathic and homeopathic supportive cares. 158 participants (42 of stomach, 40 of gall bladder, 44 of pancreatic, 32 of liver) were included in the final analysis of the study. Complete tumor response occurred in 28 (17.72%) cases and partial tumor response occurred in 56 (35.44%) cases. Double-blind randomized controlled clinical trial should be conducted for further scientific exploration of this alternative cancer treatment.
doi:10.1155/2011/724743
PMCID: PMC3004411  PMID: 21197093
22.  Targeting Mitochondrial Cell Death Pathway to Overcome Drug Resistance with a Newly Developed Iron Chelate 
PLoS ONE  2010;5(6):e11253.
Background
Multi drug resistance (MDR) or cross-resistance to multiple classes of chemotherapeutic agents is a major obstacle to successful application of chemotherapy and a basic problem in cancer biology. The multidrug resistance gene, MDR1, and its gene product P-glycoprotein (P-gp) are an important determinant of MDR. Therefore, there is an urgent need for development of novel compounds that are not substrates of P-glycoprotein and are effective against drug-resistant cancer.
Methodology/Principal Findings
In this present study, we have synthesized a novel, redox active Fe (II) complex (chelate), iron N- (2-hydroxy acetophenone) glycinate (FeNG). The structure of the complex has been determined by spectroscopic means. To evaluate the cytotoxic effect of FeNG we used doxorubicin resistant and/or sensitive T lymphoblastic leukemia cells and show that FeNG kills both the cell types irrespective of their MDR phenotype. Moreover, FeNG induces apoptosis in doxorubicin resistance T lymphoblastic leukemia cell through mitochondrial pathway via generation reactive oxygen species (ROS). This is substantiated by the fact that the antioxidant N-acetyle-cysteine (NAC) could completely block ROS generation and, subsequently, abrogated FeNG induced apoptosis. Therefore, FeNG induces the doxorubicin resistant T lymphoblastic leukemia cells to undergo apoptosis and thus overcome MDR.
Conclusion/Significance
Our study provides evidence that FeNG, a redox active metal chelate may be a promising new therapeutic agent against drug resistance cancers.
doi:10.1371/journal.pone.0011253
PMCID: PMC2889820  PMID: 20582168
23.  RsmC of Erwinia carotovora subsp. carotovora Negatively Controls Motility, Extracellular Protein Production, and Virulence by Binding FlhD and Modulating Transcriptional Activity of the Master Regulator, FlhDC▿  
Journal of Bacteriology  2009;191(14):4582-4593.
RsmC and FlhDC are global regulators controlling extracellular proteins/enzymes, rsmB RNA, motility, and virulence of Erwinia carotovora subsp. carotovora. FlhDC, the master regulator of flagellar genes, controls these traits by positively regulating gacA, fliA, and rsmC and negatively regulating hexA. RsmC, on the other hand, is a negative regulator of extracellular proteins/enzymes, motility, and virulence since the deficiency of RsmC in FlhDC+ strain results in overproduction of extracellular proteins/enzymes, hypermotility, and hypervirulence. These phenotypes are abolished in an RsmC− FlhDC− double mutant. We show that RsmC interferes with FlhDC action. Indeed, the expression of all three targets (i.e., gacA, rsmC, and fliA) positively regulated in E. carotovora subsp. carotovora by FlhDC is inhibited by RsmC. RsmC also partly relieves the inhibition of hexA expression by FlhDC. The results of yeast two-hybrid analysis revealed that RsmC binds FlhD and FlhDC, but not FlhC. We propose that binding of RsmC with FlhD/FlhDC interferes with its regulatory functions and that RsmC acts as an anti-FlhD4FlhC2 factor. We document here for the first time that RsmC interferes with activation of fliA and motility in several members of the Enterobacteriaceae family. The extent of E. carotovora subsp. carotovora RsmC-mediated inhibition of FlhDC-dependent expression of fliA and motility varies depending upon enterobacterial species. The data presented here support the idea that differences in structural features in enterobacterial FlhD are responsible for differential susceptibility to E. carotovora subsp. carotovora RsmC action.
doi:10.1128/JB.00154-09
PMCID: PMC2704716  PMID: 19447906
24.  A Novel Copper Chelate Modulates Tumor Associated Macrophages to Promote Anti-Tumor Response of T Cells 
PLoS ONE  2009;4(9):e7048.
Background
At the early stages of carcinogenesis, the induction of tumor specific T cell mediated immunity seems to block the tumor growth and give protective anti-tumor immune response. However, tumor associated macrophages (TAMs) might play an immunosuppressive role and subvert this anti tumor immunity leading to tumor progression and metastasis.
Methodology/Principal Findings
The Cu (II) complex, (chelate), copper N-(2-hydroxy acetophenone) glycinate (CuNG), synthesized by us, has previously been shown to have a potential usefulness in immunotherapy of multiple drug resistant cancers. The current study demonstrates that CuNG treatment of TAMs modulates their status from immunosuppressive to proimmunogenic nature. Interestingly, these activated TAMs produced high levels of IL-12 along with low levels of IL-10 that not only allowed strong Th1 response marked by generation of high levels of IFN-γ but also reduced activation induced T cell death. Similarly, CuNG treatment of peripheral blood monocytes from chemotherapy and/or radiotherapy refractory cancer patients also modulated their cytokine status. Most intriguingly, CuNG treated TAMs could influence reprogramming of TGF-β producing CD4+CD25+ T cells toward IFN-γ producing T cells.
Conclusion/Significance
Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers through reprogramming of TAMs that in turn reprogram the T cells and reeducate the T helper function to elicit proper anti-tumorogenic Th1 response leading to effective reduction in tumor growth.
doi:10.1371/journal.pone.0007048
PMCID: PMC2737642  PMID: 19756150
25.  Regulatory Network Controlling Extracellular Proteins in Erwinia carotovora subsp. carotovora: FlhDC, the Master Regulator of Flagellar Genes, Activates rsmB Regulatory RNA Production by Affecting gacA and hexA (lrhA) Expression▿  
Journal of Bacteriology  2008;190(13):4610-4623.
Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC− mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (HarpinEcc) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC+ plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC− mutant are responsible for the inhibition of rsmB RNA production, a condition conducive to the accumulation of free RsmA. Indeed, studies with an RsmA− FlhDC− double mutant and multiple copies of rsmB+ DNA establish that the negative effect of FlhDC deficiency is exerted via RsmA. The FlhDC-mediated regulation of fliA has no bearing on exoprotein production in E. carotovora subsp. carotovora. Our observations for the first time establish a regulatory connection between FlhDC, HexA, GacA, and rsmB RNA in the context of the exoprotein production and virulence of E. carotovora subsp. carotovora.
doi:10.1128/JB.01828-07
PMCID: PMC2446818  PMID: 18441056

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