Targeting Trehalose and Methylglucose Lipopolysaccharide Biosynthetic Pathways in M. tuberculosis - Structural and Functional Characterisation, and Early-Stage Drug Discovery of OtsA and Rv3030

Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is one of the most successful pathogens, with much of its virulence predominantly associated with its thick, lipid rich cellular envelope that modulates the host immune response. Tuberculosis, having a long history, has proved to be a challenging disease to eradicate. In mycobacteria, trehalose occurs as a free sugar in the cytoplasm, as well as a constituent of cellwall glycolipids, such as cord-factor, sulfolipid-1 and lipooligosaccharides. M. tuberculosis possesses two pathways for synthesis of trehalose, the OtsA-OtsB pathway and the TreY-TreZ pathway. Multiple biosynthetic pathways underline the critical role that trehalose plays in the bacteria. OtsA-OtsB is the dominant pathway, required for bacterial growth in laboratory culture and for virulence in a mouse model. OtsA, a retaining glucosyltransferase encoded by the otsA (Rv3490) gene, catalyzes the synthesis of trehalose-6-phosphate from ADP-glucose and glucose- 6-phosphate, which is subsequently dephosphorylated by a functional homologue of OtsB (encoded by otsB2/Rv3372 gene) to yield trehalose. Both otsA and otsB2 gene have been shown to be essential for the growth of M. tuberculosis. The present investigation focuses on this pathway, more specifically on OtsA. The recombinant orthologous protein from M. thermoresistibile was structurally and functionally characterised. The crystal structure revealed that there are two domains, with the catalytic site at their interface. The biochemical and structural data indicated that the enzyme had high preference for ADP-glucose as a glucose-donor. The protein activity was also shown to be regulated by feedback inhibition. Furthermore, a structure-guided, fragment-based drug-discovery exercise was carried out against the target, which led to identification of fragment hits that have an inhibitory effect on the activity of the enzyme. Polymethylated polysaccharides (PMPSs) are complex intracellular polysaccharides, which are exclusive to mycobacteria and closely related species. PMPSs include methylglucose lipopolysaccharides (MGLPs) and methylmannose polysaccharides (MMPs). MGLPs, which are composed of acylated glucose and 6-O-methylglucose units, are found in all mycobacterial species, including the slow growing pathogenic M. tuberculosis. On the other hand, MMPs are linear polysaccharides that are not found in M. tuberculosis, but are found in non-pathogenic M. smegmatis and other fast growing mycobacteria. Three gene clusters (Rv1208-Rv1212c, Rv2418c-Rv2419c and Rv3030-Rv3037c), initially thought to coordinate MGLPs biosynthesis, contain several genes that are considered to be essential for M. tuberculosis growth. One of the genes Rv3030, encoding for a SAM-dependent 6-O-methyltransferase, is essential for survival of the bacilli and plays a pivotal role in the biosynthesis of MGLPs. In the present study, an orthologue of Rv3030 from M. smegmatis has been structurally characterized. To overcome a myriad of issues with crystallization, low resolution and lack of reproducibility of the crystals, an alternative approach was taken to determine the structural features of the protein by NMR spectroscopy using 15N, 13C labelled protein. The protein has the β-sheet topology of the classic Rossmann fold, and exists as a monomer in solution. OtsA and Rv3030, essential for survival of M. tuberculosis, represent attractive anti-tuberculosis drug targets, and this study focuses on understanding structural, biophysical and biochemical properties of these targets. This knowledge may then be used to identify lead chemical entities that bind to these proteins and modulate their function, providing new chemical tools that may be of use in designing antimicrobials for the fight against tuberculosis

Pictorial Review of Congenital Anomalies of the Gallbladder and Biliary Ducts: Findings on Hepatobiliary Iminodiacetic Acid Scan

Learning Objectives: To become familiar with the imaging appearance on Hepatobiliary Iminodiaceetic Acid (HIDA) scan of congenital gallbladder and biliary duct anomalies, and to understand the diagnostic utility of functional imaging with HIDA when evaluating biliary tract anomalies

Association of biochemical markers with time of onset and severity of hypertensive disorder of pregnancy

Background: Hypertensive disorders of pregnancy (HDP) are a common cause of morbidity and mortality. Inflammation is considered as one of the etiologic factors along with a complex interplay of multiple genetic, nutritional and other environmental agents. This study was undertaken to find association of biochemical markers with severity and time of onset of HDP. Methods: Institutional review board and ethics committee approval was taken prior to the study. History and examination was done. Blood pressure was recorded at each antenatal visit. Blood samples were taken for the study of biochemical markers-hsCRP and interleukin 6 levels. Data was analyzed. P value <0.05 was taken as significant. Results: Of the 80 women, 27.5% had early onset HDP. 72.5% had severe HDP. Inflammatory indices were altered as compared to the range of normal pregnant women. Mean IL-6 and hsCRP levels were found to be raised but no statistically significant association was observed between IL-6 levels or hs-CRP levels and time of onset of HDP (p value = 1 and 0.5859 respectively and severity of onset of HDP (p value = 0.197 and 0.453 respectively). Conclusions: Biochemical indices, IL-6 and hs-CRP levels were elevated in women with HDP, indicative of increased inflammation

Mycobacterial OtsA Structures Unveil Substrate Preference Mechanism and Allosteric Regulation by 2-Oxoglutarate and 2-Phosphoglycerate.

Trehalose is an essential disaccharide for mycobacteria and a key constituent of several cell wall glycolipids with fundamental roles in pathogenesis. Mycobacteria possess two pathways for trehalose biosynthesis. However, only the OtsAB pathway was found to be essential in Mycobacterium tuberculosis, with marked growth and virulence defects of OtsA mutants and strict essentiality of OtsB2. Here, we report the first mycobacterial OtsA structures from Mycobacterium thermoresistibile in both apo and ligand-bound forms. Structural information reveals three key residues in the mechanism of substrate preference that were further confirmed by site-directed mutagenesis. Additionally, we identify 2-oxoglutarate and 2-phosphoglycerate as allosteric regulators of OtsA. The structural analysis in this work strongly contributed to define the mechanisms for feedback inhibition, show different conformational states of the enzyme, and map a new allosteric site.IMPORTANCE Mycobacterial infections are a significant source of mortality worldwide, causing millions of deaths annually. Trehalose is a multipurpose disaccharide that plays a fundamental structural role in these organisms as a component of mycolic acids, a molecular hallmark of the cell envelope of mycobacteria. Here, we describe the first mycobacterial OtsA structures. We show mechanisms of substrate preference and show that OtsA is regulated allosterically by 2-oxoglutarate and 2-phosphoglycerate at an interfacial site. These results identify a new allosteric site and provide insight on the regulation of trehalose synthesis through the OtsAB pathway in mycobacteria

Ovarian embedding of a transmigrated intrauterine device: a case report and literature review

To report an unusual location of a transmigrated IUD which become embedded in the right ovary causing chronic pelvic pain and dyspareunia. A 22-year-old woman who had an IUD (Copper T), inserted 7 years ago presented with complaint of lower abdominal pain. Pelvic ultrasonographic examination revealed ovarian embedding of the IUD. Laparoscopic IUD removal was successfully performed. De novo lower abdominal pain in a woman with an IUD in situ should alert the clinician to the possibility of total or partial transmigration of the device into the pelvis or abdomen. Ovarian penetration is very rare and this is the second case of IUD transmigration into the ovary reported in the medical literature

Benign Metastasizing Leiomyoma

Benign metastasizing leiomyoma is a rare diagnosis in women with history of uterine leiomyomata. Distant metastasis may occur anywhere but is most commonly seen to the lungs. The age of presentation is often young to mid age, with patients often having a history of hysterectomy or myomectomy for uterine leiomyomata. The clinical course of these patients is most often indolent. However, the size and number of nodules can result in pulmonary symptoms. Differentiating benign metastasizing leiomyoma from malignant metastatic disease, including from leiomyosarcoma, is important to avoid unnecessary procedures or delayed treatment. The treatment with hormonal suppression is often successful for decreasing the size of lesions and alleviating pulmonary symptoms

Do All Cases of Diagnosed Carcinoma Cervix Need HIV Screening?

Background: This observational study was aimed to determine the frequency of existence of sero-positivity for human immunodeficiency virus (HIV) infection among women of age between 25 to 75 years with invasive cervical carcinoma and to decide whether HIV testing should be included as part of the initial routine work-up of cervical cancer patients. Methods: Histologically proven 120 cases of invasive carcinoma cervix, who came for treatment between 2009-2013, in the department of radiotherapy, after counselling gave consent were investigated for HIV by immunochromatography based rapid test. Results: Out of 120 patients investigated, reports revealed that only two patients (1.67%) were HIV seropositive. Both of these patients were already HIV seropositive and on gynaecological screening were found to have malignancy of cervix. No patient of diagnosed carcinoma cervix was found seropositive for HIV. Most patients (106/120) were above 40 years of age, from rural background (92/120) and housewives (80/120). Only 15% (18/120) were smokers. 95.8% (115/120) were of stage II and III. None presented with metastasis. Most common pathology was moderately differentiated carcinoma, in 76 patients (63.3%). Conclusion: Screening for HIV, as part of the initial work up for cervical cancer is not necessary in countries with limited resources and low HIV prevalence

Pictorial Review of Congenital Anomalies of the Gallbladder and Biliary Ducts: Findings on Hepatobiliary Iminodiacetic Acid Scan

Poster presented at SNMMI 2016 Annual Meeting June 11 – June 15, 2016, San Diego, CALearning Objectives: To become familiar with the imaging appearance on Hepatobiliary Iminodiaceetic Acid (HIDA) scan of congenital gallbladder and biliary duct anomalies, and to understand the diagnostic utility of functional imaging with HIDA when evaluating biliary tract anomalies

A comparative study of liquid based cytology and visual inspection with acetic acid as screening method of premalignant lesions of cervix

The study was conducted to evaluate the prevalence of premalignant lesions of cervix and to compare the diagnostic efficacy of liquid based Cytology and Visual Inspection with Acetic Acid in predicting pre- malignant lesions of cervix. It was a facility based cross sectional study conducted on  sexually active women in the age group of 18 to 60 years. Detailed history was obtained from all the patients followed by thorough examination. Per Speculum Examination, LBC and VIA was performed in all cases using standard procedure. Women with either positive VIA or with positive Cervical Cytology (LBC) or both positive, were subjected to cervical biopsy. A total of 2195 women were enrolled for LBC and VIA both. Liquid based cytology revealed premalignant lesions in 4.2% cases whereas VIA was positive in 7% cases. Histopathology revealed CIN 1, 2 and 3 in 22.5%, 11.1% and 4.1% cases respectively. Sensitivity of VIA was 76% and that of LBC was 55% whereas specificity of LBC was higher (70.88%) as compared to VIA (46.5%). VIA is more sensitive than the LBC for the detection of pre malignant lesions of the cervix.&nbsp

Photoionization of Na-like Si IV using R-matrix method

Photoionization cross-section results for the ground state 2p63s (2S1/2) along with the lowest four excited states 2p63p (2Po1/2, 3/2) and 2p63d (2D5/2, 3/2) of Na-like Si IV are reported by employing the close-coupling Breit–Pauli R-matrix (BPRM) method. The target state wavefunctions of Si V ion have been obtained using the configuration interaction method (CIV3) in the LSJ-coupling scheme. Relativistic effects and all key physical effects such as short-range correlations, exchange and channel coupling are considered in the computations. Our evaluated target state energies of the Si V core ion show close agreement with the NIST database. Quigley and Berrington (QB) technique is applied to determine resonance positions (Er) and autoionization line widths (Γ) with quantum defects caused by the removal of the 2p electron from 2s22p63s2S1/2, the ground state of Si IV. To the best of our understanding, the study presented here details thorough relativistic photoionization calculations for this system for the first time. We hope that our results will be valuable to astrophysical and laboratory plasma modeling and diagnosis