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1.  ADP-Ribosylation of NLRP3 by Mycoplasma pneumoniae CARDS Toxin Regulates Inflammasome Activity 
mBio  2014;5(6):e02186-14.
ABSTRACT
The inflammasome is a major regulator of inflammation through its activation of procaspase-1, which cleaves prointerleukin-1β (pro-IL-1β) into its mature form. IL-1β is a critical proinflammatory cytokine that dictates the severity of inflammation associated with a wide spectrum of inflammatory diseases. NLRP3 is a key component of the inflammasome complex, and multiple signals and stimuli trigger formation of the NLRP3 inflammasome complex. In the current study, we uncovered a yet unknown mechanism of NLRP3 inflammasome activation by a pathogen-derived factor. We show that the unique bacterial ADP-ribosylating and vacuolating toxin produced by Mycoplasma pneumoniae and designated community-acquired respiratory distress syndrome (CARDS) toxin activates the NLRP3 inflammasome by colocalizing with the NLRP3 inflammasome and catalyzing the ADP-ribosylation of NLRP3. Mutant full-length CARDS toxin lacking ADP-ribosyltransferase (ADPRT) activity and truncated CARDS toxins unable to bind to macrophages and be internalized failed to activate the NLRP3 inflammasome. These studies demonstrate that CARDS toxin-mediated ADP-ribosylation constitutes an important posttranslational modification of NLRP3, that ADPRT activity of CARDS toxin is essential for NLRP3 inflammasome activation, and that posttranslational ADPRT-mediated modification of the inflammasome is a newly discovered mechanism for inflammasome activation with subsequent release of IL-1β and associated pathologies.
IMPORTANCE
Inflammation is a fundamental innate immune response to environmental factors, including infections. The inflammasome represents a multiprotein complex that regulates inflammation via its ability to activate specific proinflammatory cytokines, resulting in an effective host protective response. However, excessive release of proinflammatory cytokines can occur following infection that skews the host response to “hyperinflammation” with exaggerated tissue damage. Mycoplasma pneumoniae, a common bacterial airway pathogen, possesses a unique protein toxin with ADP-ribosyltransferase and vacuolating properties capable of reproducing the robust inflammation and cytopathology associated with mycoplasma infection. Here, we show that the toxin uniquely activates the NLRP3 inflammasome by colocalizing with and ADP-ribosylating NLRP3, possibly leading to “hyperinflammation” and thus uncovering a novel target for therapeutic intervention.
doi:10.1128/mBio.02186-14
PMCID: PMC4278538  PMID: 25538194
2.  Annexin A2 Mediates Mycoplasma pneumoniae Community-Acquired Respiratory Distress Syndrome Toxin Binding to Eukaryotic Cells 
mBio  2014;5(4):e01497-14.
ABSTRACT
Mycoplasma pneumoniae synthesizes a novel human surfactant protein A (SP-A)-binding cytotoxin, designated community-acquired respiratory distress syndrome (CARDS) toxin, that exhibits ADP-ribosylating and vacuolating activities in mammalian cells and is directly linked to a range of acute and chronic airway diseases, including asthma. In our attempt to detect additional CARDS toxin-binding proteins, we subjected the membrane fraction of human A549 airway cells to affinity chromatography using recombinant CARDS toxin as bait. A 36-kDa A549 cell membrane protein bound to CARDS toxin and was identified by time of flight (TOF) mass spectroscopy as annexin A2 (AnxA2) and verified by immunoblotting with anti-AnxA2 monoclonal antibody. Dose-dependent binding of CARDS toxin to recombinant AnxA2 reinforced the specificity of the interaction, and further studies revealed that the carboxy terminus of CARDS toxin mediated binding to AnxA2. In addition, pretreatment of viable A549 cells with anti-AnxA2 monoclonal antibody or AnxA2 small interfering RNA (siRNA) reduced toxin binding and internalization. Immunofluorescence analysis of CARDS toxin-treated A549 cells demonstrated the colocalization of CARDS toxin with cell surface-associated AnxA2 upon initial binding and with intracellular AnxA2 following toxin internalization. HepG2 cells, which express low levels of AnxA2, were transfected with a plasmid expressing AnxA2 protein, resulting in enhanced binding of CARDS toxin and increased vacuolization. In addition, NCI-H441 cells, which express both AnxA2 and SP-A, upon AnxA2 siRNA transfection, showed decreased binding and subsequent vacuolization. These results indicate that CARDS toxin recognizes AnxA2 as a functional receptor, leading to CARDS toxin-induced changes in mammalian cells.
IMPORTANCE
Host cell susceptibility to bacterial toxins is usually determined by the presence and abundance of appropriate receptors, which provides a molecular basis for toxin target cell specificities. To perform its ADP-ribosylating and vacuolating activities, community-acquired respiratory distress syndrome (CARDS) toxin must bind to host cell surfaces via receptor-mediated events in order to be internalized and trafficked effectively. Earlier, we reported the binding of CARDS toxin to surfactant protein A (SP-A), and here we show how CARDS toxin uses an alternative receptor to execute its pathogenic properties. CARDS toxin binds selectively to annexin A2 (AnxA2), which exists both on the cell surface and intracellularly. Since AnxA2 regulates membrane dynamics at early stages of endocytosis and trafficking, it serves as a distinct receptor for CARDS toxin binding and internalization and enhances CARDS toxin-induced vacuolization in mammalian cells.
doi:10.1128/mBio.01497-14
PMCID: PMC4147866  PMID: 25139904
3.  Mycoplasma pneumoniae CARDS Toxin Exacerbates Ovalbumin-Induced Asthma-Like Inflammation in BALB/c Mice 
PLoS ONE  2014;9(7):e102613.
Mycoplasma pneumoniae causes a range of airway and extrapulmonary pathologies in humans. Clinically, M. pneumoniae is associated with acute exacerbations of human asthma and a worsening of experimentally induced asthma in mice. Recently, we demonstrated that Community Acquired Respiratory Distress Syndrome (CARDS) toxin, an ADP-ribosylating and vacuolating toxin synthesized by M. pneumoniae, is sufficient to induce an asthma-like disease in BALB/cJ mice. To test the potential of CARDS toxin to exacerbate preexisting asthma, we examined inflammatory responses to recombinant CARDS toxin in an ovalbumin (OVA) murine model of asthma. Differences in pulmonary inflammatory responses between treatment groups were analyzed by histology, cell differentials and changes in cytokine and chemokine concentrations. Additionally, assessments of airway hyperreactivity were evaluated through direct pulmonary function measurements. Analysis of histology revealed exaggerated cellular inflammation with a strong eosinophilic component in the CARDS toxin-treated group. Heightened T-helper type-2 inflammatory responses were evidenced by increased expression of IL-4, IL-13, CCL17 and CCL22 corresponding with increased airway hyperreactivity in the CARDS toxin-treated mice. These data demonstrate that CARDS toxin can be a causal factor in the worsening of experimental allergic asthma, highlighting the potential importance of CARDS toxin in the etiology and exacerbation of human asthma.
doi:10.1371/journal.pone.0102613
PMCID: PMC4109942  PMID: 25058417
4.  Anti-inflammatory and anti-oxidant activity of anionic dendrimer-N-acetyl cysteine conjugates in activated microglial cells 
Dendrimers are emerging as potential intracellular drug delivery vehicles. Understanding and improving the cellular efficacy of dendrimer-drug conjugates, can lead to significant in vivo benefits. This study explores efficacy of anionic polyamidoamine (PAMAM-COOH) dendrimer-N-acetyl cysteine (NAC) conjugates for applications in neuroinflammation. The anti-oxidative and anti-inflammatory effects of PAMAM-(COOH)46-(NAC)18 conjugate is evaluated on microglial cells in vitro. Cell entry and localization of PAMAM-(COOH)62-(FITC)2 conjugate in BV-2 microglial cells were assessed using flow cytometry and confocal microscopy. ELISA assays were used to evaluate markers of oxidative stress (ROS, NO) and inflammation (TNFα) after stimulation of microglial cells with lipopolysaccharides (LPS), following treatment with increasing doses of free N-acetyl--cysteine (NAC) or PAMAM-(COOH)46-(NAC)18 conjugate containing an equivalent molar concentration of NAC. Flow cytometry and confocal microscopy demonstrated the PAMAM-(COOH)62-(FITC)2 conjugate entered BV-2 rapidly with significant increase in fluorescence within 15 min and localized mostly in the cytoplasm. PAMAM-(COOH)46-(NAC)18 conjugate was non-toxic, and significantly reduced ROS, NO and TNFα release by activated microglial cells after 24 h and 72 h stimulation of LPS following 3 h pre-treatment when compared to the same concentration of free NAC (P<0.05 or P<0.01). Anionic PAMAM dendrimer-NAC conjugate was synthesized with a glutathione sensitive linker for intracellular release. The non-toxic conjugate is a more effective anti-oxidant and anti-inflammatory agent when compared to free NAC in vitro. The conjugate showed significant efficacy even at the lowest dose (0.5 mM NAC), where the activity was comparable or better than that of free drug at 8mM (16× higher dosage). The improved efficacy of the conjugate, when combined with the intrinsic neuroinflammation-targeting ability of the PAMAM dendrimer, may provide new opportunities for in vivo applications.
doi:10.1016/j.ijpharm.2009.04.050
PMCID: PMC3917717  PMID: 19463931
Dendrimers; PAMAM; drug delivery; neuroinflammation; N-acetyl cysteine
5.  Are intra-gastric adjustable balloon system safe? A case series☆ 
INTRODUCTION
Intra-gastric balloons have been in use as an aide to weight loss. Since its introduction, it has evolved from air filled to saline filled intra-gastric balloons. The SPATZ-ABS is a new adjustable saline filled balloon.
PRESENTATION OF CASE
Three patients have presented to our hospital as emergencies due to complications arising from this balloon. Two of these patients required emergency laparotomy and resection of small bowel due to pressure necrosis effects of the anchoring device. One patient had migration of the device into the duodenum that was removed endoscopically. Of the 2 patients who underwent a laparotomy, one patient did not have any symptoms or signs that correlated with the intra-operative findings.
DISCUSSION
The anchoring device meant to prevent the intra-gastric balloon from migrating distally has migrated in three patients. To our knowledge, there has been no reported incident of migration of this device. These serious complications pose a risk to patients having these balloons fitted.
CONCLUSION
There is a need to study our experience with a larger population of patients who have had this device inserted. Its safety needs to be questioned and its design may need to be addressed.
doi:10.1016/j.ijscr.2013.07.025
PMCID: PMC3785945  PMID: 24013094
Intra-gastric adjustable balloon system; Anchoring device; Migration; Infarction; Resection
6.  Mycoplasma pneumoniae CARDS Toxin Is Internalized via Clathrin-Mediated Endocytosis 
PLoS ONE  2013;8(5):e62706.
Bacterial toxins possess specific mechanisms of binding and uptake by mammalian cells. Mycoplasma pneumoniae CARDS (Community Acquired Respiratory Distress Syndrome) toxin is a 68 kDa protein, which demonstrates high binding affinity to human surfactant protein-A and exhibits specific biological activities including mono-ADP ribosylation and vacuolization. These properties lead to inflammatory processes in the airway and a range of cytopathologies including ciliostasis, loss of tissue integrity and injury, and cell death. However, the process by which CARDS toxin enters target cells is unknown. In this study, we show that CARDS toxin binds to mammalian cell surfaces and is internalized rapidly in a dose and time-dependent manner using a clathrin-mediated pathway, as indicated by inhibition of toxin internalization by monodansylcadaverine but not by methyl-β-cyclodextrin or filipin. Furthermore, the internalization of CARDS toxin was markedly inhibited in clathrin-depleted cells.
doi:10.1371/journal.pone.0062706
PMCID: PMC3647021  PMID: 23667510
7.  Fatal Outcomes in Family Transmission of Mycoplasma pneumoniae 
Mycoplasma pneumoniae continues to be a significant cause of community-acquired pneumonia and can manifest as fulminant disease leading to mortality in healthy individuals.
Background. Mycoplasma pneumoniae continues to be a significant cause of community-acquired pneumonia and, on rare occasions, manifests as fulminant disease that leads to mortality, even in healthy individuals.
Methods. We conducted a retrospective study on members of a family who were quarantined by the Centers for Disease Control and Prevention in 2002 for respiratory failure and death of a 15-year-old brother (sibling 1) and a 13-year-old sister (sibling 2). Collected airway, cerebrospinal fluid (CSF), and serum samples from both deceased siblings and serum samples from both parents and the remaining 3 ill siblings (sibling 3–5) were tested using a range of diagnostic assays. Autopsy lung tissue samples from sibling 2 were also assessed using immunohistochemical and immunoelectron microscopic methods.
Results. Autopsy evaluation of sibling 1 revealed cerebral edema consistent with hypoxic ischemic encepatholopathy and pulmonary findings of bronchiolitis obliterans with organizing pneumonia (BOOP). Postmortem lung examination of sibling 2 revealed lymphoplasmacytic bronchiolitis with intraluminal purulent exudate, BOOP, and pulmonary edema. Results of diagnostic assays implicated the household transmission of M. pneumoniae among all 5 siblings and both parents. Further analysis of lung tissue from sibling 2 demonstrated the presence of M. pneumoniae organisms and community-acquired respiratory distress syndrome toxin. M. pneumoniae was cultured directly from sibling 2 autopsy lung tissue.
Conclusion. Evidence is provided that M. pneumoniae was readily transmitted to all members of the household and that the resulting infections led to a spectrum of individual responses with variation in disease progression, including lymphoplasmacytic bronchiolitis, BOOP, and death.
doi:10.1093/cid/cir769
PMCID: PMC3245726  PMID: 22052890
8.  Synthesis and Distribution of CARDS Toxin During Mycoplasma pneumoniae Infection in a Murine Model 
The Journal of Infectious Diseases  2011;204(10):1596-1604.
Mice were infected with Mycoplasma pneumoniae and monitored for the synthesis and distribution of the unique adenosine diphosphate–ribosylating and vacuolating Community Acquired Respiratory Distress Syndrome (CARDS) toxin in bronchiolar lavage fluid (BALF) and lung. We noted direct relationships between the concentration of CARDS toxin and numbers of mycoplasma genomes in BALF and the degree of histologic pulmonary inflammation. Immunostaining of lungs revealed extensive colonization by mycoplasmas, including the detection of CARDS toxin in the corresponding inflamed airways. Lung lesion scores were higher during the early stages of infection, decreased gradually by day 14 postinfection, and reached substantially lower values at day 35. Infected mouse immunoglobulin (Ig) M and IgG titers were positive for CARDS toxin as well as for the major adhesin P1 of M. pneumoniae. These data reinforce the proposed pathogenic role of CARDS toxin in M. pneumoniae–mediated pathologies.
doi:10.1093/infdis/jir557
PMCID: PMC3222103  PMID: 21957154
9.  Mycoplasma pneumoniae Large DNA Repetitive Elements RepMP1 Show Type Specific Organization among Strains 
PLoS ONE  2012;7(10):e47625.
Mycoplasma pneumoniae is the smallest self-replicating bacterium with a streamlined genome of 0.81 Mb. Complete genome analysis revealed the presence of multiple copies of four large repetitive elements (designated RepMP1, RepMP2/3, RepMP4 and RepMP5) that are implicated in creating sequence variations among individual strains. Recently, we described RepMP1-associated sequence variations between reference strain M129 and clinical isolate S1 that involved three RepMP1-genes (i.e. mpn130, mpn137 and mpn138). Using PCR and sequencing we analyze 28 additional M. pneumoniae strains and demonstrate the existence of S1-like sequence variants in nine strains and M129-like variants in the remaining nineteen strains. We propose a series of recombination steps that facilitates transition from M129- to S1-like sequence variants. Next we examined the remaining RepMP1-genes and observed no other rearrangements related to the repeat element. The only other detected difference was varying numbers of the 21-nucleotide tandem repeats within mpn127, mpn137, mpn501 and mpn524. Furthermore, typing of strains through analysis of large RepMPs localized within the adhesin P1 operon revealed that sequence divergence involving RepMP1-genes mpn130, mpn137 and mpn138 is strictly type-specific. Once more our analysis confirmed existence of two highly conserved groups of M. pneumoniae strains.
doi:10.1371/journal.pone.0047625
PMCID: PMC3472980  PMID: 23091634
10.  Efficacy of increasing dosages of clarithromycin for treatment of experimental Mycoplasma pneumoniae pneumonia 
Journal of Antimicrobial Chemotherapy  2011;66(10):2323-2329.
Objectives
Mycoplasma pneumoniae respiratory infection is a common cause of acute respiratory infection in children and adults. We evaluated the efficacy of increasing dosages of clarithromycin for the optimized therapy of M. pneumoniae respiratory infection in a mouse model.
Methods
BALB/c mice were intranasally inoculated once with M. pneumoniae or SP4 broth (control). Groups of mice were treated with increasing dosages of clarithromycin (10, 25 or 75 mg/kg/day) or placebo subcutaneously daily. Groups of mice were evaluated after 1, 2, 3, 6 and 12 days of therapy. Outcome variables included quantitative M. pneumoniae culture, histopathological score of the lungs, bronchoalveolar lavage (BAL) cytokine/chemokine/growth factor concentrations and plethysmography after aerosolized methacholine to assess airway hyperresponsiveness.
Results
Elevated dosages of clarithromycin resulted in greater antimicrobial efficacy with significantly reduced M. pneumoniae quantitative cultures (P < 0.05), as well as greater improvement in markers of disease severity with significantly reduced lung histopathology scores, BAL cytokine concentrations and airway hyperresponsiveness (P < 0.05).
Conclusions
Escalated dosing of clarithromycin resulted in significantly greater therapeutic efficacy in the treatment of experimental M. pneumoniae respiratory infection.
doi:10.1093/jac/dkr306
PMCID: PMC3172041  PMID: 21791441
macrolides; cytokines; asthma; pharmacokinetics; pharmacodynamics
11.  Regional anesthesia in faciomaxillary and oral surgery 
Journal of Pharmacy & Bioallied Sciences  2012;4(Suppl 2):S264-S269.
Faciomaxillary and oral surgical procedures are frequently done under local anesthesia. Only few techniques are used widely in these areas in spite of the numerous blocks available. Knowledge about these techniques could encourage use of these techniques for the benefit of patients and operators’ comfort. Leaving aside the commonly used intraoral anesthetic technique by faciomaxillary and dental surgeons, focus is given on regional blocks of extraoral route, like maxillary block, mandibular block, superficial cervical plexus block, forehead and scalp block, trigeminal nerve block, sphenopalatine nerve block, and they are discussed with their indications and technical details involved in administering them. Advantages of using the regional blocks over general anesthesia and multiple pricks include reduced dosage and number of needle pricks. Pediatric considerations like prolonged duration of anesthesia and wider area of action for regional blocks warrant that they should be used with caution.
doi:10.4103/0975-7406.100247
PMCID: PMC3467933  PMID: 23066267
Faciomaxillary; nerve blocks; regional blocks
12.  Significance of localization of mandibular foramen in an inferior alveolar nerve block 
Background:
The mandibular foramen (MF) is an opening on the internal surface of the ramus for divisions of the mandibular vessels and nerve to pass. The aim of this study is to determine the position of the MF from various anatomical landmarks in several dry adult mandibles.
Materials and Methods:
A total of 102 human dry mandibles were examined, of which 93 were of dentulous and 9 were of edentulous. The measurements were taken from the anterior border of the ramus (coronoid notch) to the midportion of the MF and then from the midportion of the MF to the other landmarks such as internal oblique ridge, inferior border, sigmoid notch, and condyle were measured and recorded.
Results:
The data were compared using Student's t-test. The MF is positioned at a mean distance of 19 mm (with SD 2.34) from coronoid notch of the anterior border of the ramus. Superio-inferiorly from the condyle to the inferior border MF is situated 5 mm inferior to the midpoint of condyle to the inferior border distance (ramus height).
Conclusion:
We conclude that failures in the anesthesia of the inferior alveolar nerve are due to the operator error and not due to the anatomical variation.
doi:10.4103/0976-9668.101896
PMCID: PMC3510910  PMID: 23225978
Failure of anesthesia; inferior alveolar nerve; mandible foramen
13.  Identity and pharmacognosy of Ruta graveolens Linn 
Ancient Science of Life  2012;32(1):16-19.
Ruta graveolens L., is a odoriferous herb belonging to the family Rutaceae. It is the source of Rue or Rue oil, called as Sadab or Satab in Hindi. It is distributed throughout the world and cultivated as a medicinal and ornamental herb. The ancient Greeks and Romans, held the plant in high esteem. It is used in Ayurveda, Homoeopathy and Unani. Phytochemical constituents and pharmacological properties were studied in depth. In 14 species of genus Ruta, R. graveolens and R. chalepensis are available in India and also cultivated in gardens. Taxonomical characters to identify the Indian plants are very clear with fringed and or non-fringed petals. However, references to it are confused in the traditional literature. Due to sharing of regional language name, its identity is confused with Euphorbia dracunculoides. Morphological and anatomical characters were described. Pharmacognostic studies with microscopic characters were also published. Upon reviewing the anatomical characters and pharmacognostic characters one finds that it is highly confused and conflicting. The characters described are opposite of each other and authenticity of the market sample of R. graveolens cannot be guaranteed and able to be differentiated from R. chalepensis. Present work is to describe the pharmacognostic characters of R. graveolens to differentiate it from R. chalepensis. It is concluded that morphologically, R. graveolens can be identified with its non-fringed petals and blunted apices of fruit lobes. Whereas, in R. chalepensis petals are fringed or ciliated and apices of the fruit lobes are sharp and projected. Microscopically, in stem of R. graveolens pericyclic fibers have wide lumen. Whereas, in R. chalepensis, it is narrow. The published pharmacognosy reports do not pertain to authentic plant or some of the characteristic features like glandular trichomes are not observed in our samples.
doi:10.4103/0257-7941.113792
PMCID: PMC3733200  PMID: 23929988
Pharmacognosy; ruta chalepensis; ruta graveolens; rutaceae
14.  Botanical pharmacognosy of stem of Gmelina asiatica Linn 
Ancient Science of Life  2012;31(4):190-193.
Gmelina asiatica Linn (G. parvifolia Roxb.) is a large shrub or a small tree. Roots and aerial parts are used in Ayurvedic medicine and also have ethno-medical uses. Root is reported as adulterant to G. arborea roxb roots. Pharmacognostical characters of root were reported. Owing to the shortage of genuine drug and ever-increasing demands in market, it becomes necessary to search an alternative with equal efficacy without compromising the therapeutic value. Nowadays, it becomes a common practice of using stem. In case of roots phytochemical and pharmacological analysis of stem was reported. However, there is no report on the pharmacognostical characters of stem and to differentiate it from roots. The present report describes the botanical pharmacognostical characters of stem and a note to differentiate it from root. Hollow pith, faint annual rings in cut ends, alternatively arranged macrosclereids and bundle cap fibers, and presence of abundant starch grains and calcium oxalates in pith and in ray cells are the diagnostic microscopic characters of stem. Stem pieces can be differentiated from roots by absence of tylosis.
doi:10.4103/0257-7941.107347
PMCID: PMC3644757  PMID: 23661867
Botanical pharmacognosy; ethnobotany; Gmelina arborea; Gmelina asiatica; pharmacognosy; root; stem
15.  Crystallization of community-acquired respiratory distress syndrome toxin from Mycoplasma pneumoniae  
Community-acquired respiratory distress syndrome toxin (CARDS TX) from M. pneumoniae was cloned, expressed, purified and crystallized. A diffraction data set from a native CARDS TX crystal was obtained at 2.2 Å resolution.
Community-acquired respiratory distress syndrome toxin (CARDS TX) is a 591-amino-acid protein with ADP-ribosyltransferase and vacuolating activities that damages the cells lining the respiratory tracts of patients infected with the bacterial pathogen Mycoplasma pneumoniae. Crystals of CARDS TX were grown in space group C2, with unit-cell parameters a = 191.4, b = 107.4, c = 222.1 Å, β = 90.6°. A complete 2.2 Å data set was obtained from a single CARDS TX crystal.
doi:10.1107/S1744309110000114
PMCID: PMC2833040  PMID: 20208164
CARDS TX; community-acquired respiratory distress syndrome; Mycoplasma pneumoniae; ADP-ribosyltransferases; ADP-ribosylation
16.  Reconstruction of Ala of Nose with Bilobed Flap: A 2 Year Follow-up 
Basal cell carcinoma accounts for 70–80% of all cutaneous malignancies in the head, face and neck region [2]. This article reports a case of basal cell carcinoma involving the ala of the nose with the excision of the lesion and reconstruction.
doi:10.1007/s12663-010-0102-z
PMCID: PMC3177493  PMID: 22379322
Bilobed flap; Ala reconstruction; Nasolabial fold
17.  Probing the Reaction Coordinate of the p300/CBP Histone Acetyltranferase with Bisubstrate Analogs 
Bioorganic chemistry  2010;39(1):42-47.
Histone and protein acetylation catalyzed by p300/CBP transcriptional coactivator regulates a variety of key biological pathways. This study investigates the proposed Theorell-Chance or “hit-and-run” catalytic mechanism of p300/CBP histone acetyltransferase (HAT) using bisubstrate analogs. A range of histone peptide tail peptide-CoA conjugates with different length linkers were synthesized and evaluated as inhibitors of p300 HAT. We show that longer linkers between the histone tail peptide and the CoA substrate moieties appear to allow for dual engagement of the two binding surfaces. Results with D1625R/D1628R double mutant p300 HAT further confirm the requirement for a negatively charged surface on the enzyme to interact with the histone tail.
doi:10.1016/j.bioorg.2010.10.004
PMCID: PMC3076313  PMID: 21111442
18.  Isolated cleft of alar rim 
Alar rim defects are most commonly acquired as a result of trauma, burns, tumor excision or sometimes accompanying craniofacial clefts. However, isolated congenital alar defects are extremely rare occurring in about 1 in 20,000 to 40,000 live births. We are presenting a case report of an isolated congenital cleft of the alar rim. The defect was closed by the use of a rotation advancement full-thickness flap. With this technique, both symmetry and desired thickness of the nostrils were achieved. The skin color and texture of the alar rim were good with minimal scars.
doi:10.4103/2231-0746.95323
PMCID: PMC3591078  PMID: 23482726
Advancement rotation flap; alar cleft; congenital; nasal deformities
19.  Inferior alveolar nerve block: Alternative technique 
Background:
Inferior alveolar nerve block (IANB) is a technique of dental anesthesia, used to produce anesthesia of the mandibular teeth, gingivae of the mandible and lower lip. The conventional IANB is the most commonly used the nerve block technique for achieving local anesthesia for mandibular surgical procedures. In certain cases, however, this nerve block fails, even when performed by the most experienced clinician. Therefore, it would be advantageous to find an alternative simple technique.
Aim and Objective:
The objective of this study is to find an alternative inferior alveolar nerve block that has a higher success rate than other routine techniques. To this purpose, a simple painless inferior alveolar nerve block was designed to anesthetize the inferior alveolar nerve.
Materials and Methods:
This study was conducted in Oral surgery department of Vinayaka Mission's dental college Salem from May 2009 to May 2011. Five hundred patients between the age of 20 years and 65 years who required extraction of teeth in mandible were included in the study. Out of 500 patients 270 were males and 230 were females. The effectiveness of the IANB was evaluated by using a sharp dental explorer in the regions innervated by the inferior alveolar, lingual, and buccal nerves after 3, 5, and 7 min, respectively.
Conclusion:
This study concludes that inferior alveolar nerve block is an appropriate alternative nerve block to anesthetize inferior alveolar nerve due to its several advantages.
doi:10.4103/0259-1162.103375
PMCID: PMC4173425
Aanesthesia; inferior alveolar nerve block; mandible
20.  Simple and safe posterior superior alveolar nerve block 
Background:
The posterior superior alveolar nerve (PSAN) block is a dental nerve block used for profound anesthesia of the maxillary molars. Although it is being written in texts as a commonly used technique, but in dentistry it is rarely followed due to its nonreliable landmarks, variation in depth of insertion and frequent complications. The aim and objective are to find a technically simple method of the PSAN block without any complications.
Study and Design:
This study was based on the experience gained from 200 patients of 125 males and 75 female in age group of 20 to 65 years in University of Vinayaka and department of oral and maxillofacial surgery of VMS Dental College and hospital, Salem, Tamil Nadu.
Results:
In 200 patients’ positive anesthesia obtained within a period of 5 to 10 min. No visual complications reported in this study. There was no pain during and after extraction.
Conclusion:
This study shows this PSA nerve block using curved needle would avoid all complications reported in the literature. Therefore, the technique described in this study is an ideal option to anesthetize PSA nerve.
doi:10.4103/0259-1162.103379
PMCID: PMC4173426
Complications; curved needle; nerve block; posterior superior alveolar nerve
21.  Maxillary nerve block in management of maxillary bone fractures: Our experience 
Background and Objectives:
The objective of this study is to evaluate the intraoral high tuberocity maxillary nerve block technique in zygoma and arch fracture reduction and fixation.
Study and Design:
This study was carried out at Arvind Multi-Specialty Hospital, Namakkal, Tamil Nadu on seven male patients with zygomatic bone and arch fracture.
Materials and Methods:
Intraoral high tuberocity maxillary nerve block administered in seven patients for management of isolated zygomatic bone and arch fracture. Lidocaine 2% measuring 4 mL with 1:80000 adrenaline anesthetic solutions was used to anesthetize maxillary nerve through a 3.2 cm length and 24G, needle. The following parameters were evaluated namely onset of anesthesia, nerve block duration, outcome of treatment and Patient's comfort.
Results:
The blocks were effective and patients were comfortable without pain during initial stage of surgery, but in latter stages two patients had mild to moderate pain. Duration of block varied from 60 to 90 min while onset varied from 3 to 10 min. There were vascular punctures in three patients, however, without hematoma.
Conclusions:
The maxillary nerve block is a good alternative option in selective cases of zygomatic bone fracture reduction.
doi:10.4103/0259-1162.103376
PMCID: PMC4173432
Local anesthesia; maxilla fracture; maxillary nerve block; zygoma
22.  Mycoplasma pneumoniae Mpn133 is a cytotoxic nuclease with a glutamic acid, lysine and serine rich region essential for binding and internalization but not enzymatic activity 
Cellular microbiology  2010;12(12):1821-1831.
Summary
We identified Mpn133 as a Ca2+-dependent cytotoxic nuclease of Mycoplasma pneumoniae. Flow cytometry analysis and immunofluorescence studies revealed the binding and internalization of recombinant Mpn133 (rMpn133) in human airway A549 cells. Amino acid sequence comparisons of Mpn133 with other mycoplasma nucleases demonstrated the presence of a unique glutamic acid, lysine and serine rich region (EKS region; amino acids 72–110). Deletion of this EKS peptide (rMpn133Δ72–110) abrogated its binding and internalization but not its nuclease activity. The function of the EKS region in host cell trafficking and nuclear localization was reinforced by the successful delivery of EKS-conjugated mCherry protein into A549 cells. rMpn133, but not rMpn133Δ72–110, induced apoptosis-like death in A549 cells. This observation suggested a unique role of Mpn133 as an important contributor to M. pneumoniae-associated life cycle events and as a virulence factor in host-associated cytopathologies. In addition, the distinct property of the EKS peptide in delivery of proteins, like mCherry, into target cells opens new avenues to the establishment of novel concepts of drug delivery and therapy.
doi:10.1111/j.1462-5822.2010.01513.x
PMCID: PMC3013625  PMID: 20690923
Mycoplasma pneumoniae; Mpn133; nuclease; apoptosis; lysine rich region
23.  Cellular Vacuoles Induced by Mycoplasma pneumoniae CARDS Toxin Originate from Rab9-Associated Compartments 
PLoS ONE  2011;6(7):e22877.
Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS) toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN) Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.
doi:10.1371/journal.pone.0022877
PMCID: PMC3146493  PMID: 21829543
24.  Pharmacognostical Studies on Balanophora fungosa - a Negative Listed Plant 
Ancient Science of Life  2011;31(1):22-25.
Balanophora fungosa Forster & Forster ssp. indica (Arn.) B. Hansen var. indica, (Balanophoraceae) syn. B. indica, is a root parasite found in hills of south India. This plant is included in the list of negative list, which are restricted and prohibited for export. Though it is not an official drug in any of the indigenous systems of medicine in India, it is used in tribal medicines in south India. However, it is found in crude drug markets as substitute/adulterant for the Ayurvedic drug Gajapippali (Scindapsus officinalis). Few phytochemical constituents were reported on this plant. However, there is no pharmacognostical report to authenticate the commercial samples of B. fungosa and to differentiate them from Scindapsus officinalis. This article describes the pharmacognostical characteristics of Balanophora fungosa and diagnostic features to differentiate it from Scindapsus officinalis.
PMCID: PMC3377038  PMID: 22736886
Balanophora fungosa Forster & Forster ssp. Indica (Arn.) B. Hansen var. Indica; B. indica (Arn.) Wallich ex Griffith; Langsdorffia indica Arn.; Balanophoraceae; root parasite; pharmacognosy; ethnobotany; indigenous systems of medicine; Gajapippali; Scindapsus officinalis; adulterant; substitute; Negative list; prohibited plants
25.  Molecular Cloning, Expression, and Characterization of a Ca2+-Dependent, Membrane-Associated Nuclease of Mycoplasma genitalium▿  
Journal of Bacteriology  2010;192(19):4876-4884.
In this study, we identified and characterized the enzymatic properties of MG_186, a calcium-dependent Mycoplasma genitalium nuclease. MG_186 displays the hallmarks of nucleases, as indicated by its amino acid sequence similarity to other nucleases. We cloned, UGA corrected, expressed, purified, and demonstrated that recombinant MG_186 (rMG_186) exhibits nuclease activity similar to that of typical sugar-nonspecific endonucleases and exonucleases. Biochemical characterization indicated that Ca2+ alone enhances its activity, which was inhibited by divalent cations, such as Zn2+ and Mn2+. Chelating agents EGTA and EDTA also inhibited nuclease activity. Mycoplasma membrane fractionation and Triton X-114 phase separation showed that MG_186 was a membrane-associated lipoprotein, and electron microscopy revealed its surface membrane location. Incubation of purified human endometrial cell nuclei with rMG_186 resulted in DNA degradation and morphological changes typical of apoptosis. Further, immunofluorescence analysis of rMG_186-treated nuclei indicated that morphological changes were linked to the disintegration of lamin and the internalization of rMG_186. Since M. genitalium has the capacity to invade eukaryotic cells and localize to the perinuclear and nuclear region of parasitized target cells, MG_186 has the potential to provide M. genitalium, which possesses the smallest genome of any self-replicating cell, with the ability to degrade host nucleic acids both as a source of nucleotide precursors for growth and for pathogenic purposes.
doi:10.1128/JB.00401-10
PMCID: PMC2944508  PMID: 20639320

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