Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein.

Lipoic acid is a sulfur-containing cofactor indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential actions of lipoate protein ligase B (LipB) and lipoyl synthase (LipA). LipB takes up the octanoyl chain from C8-acyl carrier protein (C8-ACP), a byproduct of the type II fatty acid synthesis pathway, and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. However, the molecular basis of its substrate recognition is still not fully understood. Using Escherichia coli LipB as a model enzyme, we show here that the octanoyl-transferase mainly recognizes the 4'-phosphopantetheine-tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. We demonstrate LipB can accept octanoate from its own ACP and noncognate ACPs, as well as C8-CoA. Furthermore, our 1H saturation transfer difference and 31P NMR studies demonstrate the binding of adenosine, as well as the phosphopantetheine arm of CoA to LipB, akin to binding to LplA. Finally, we show a conserved 71RGG73 loop, analogous to the lipoate-binding loop of LplA, is required for full LipB activity. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge could be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.

Refined premature chromosome condensation (G0-PCC) with cryo-preserved mitotic cells for rapid radiation biodosimetry.

Mitotic cell fusion induced Premature Chromosome Condensation (G0-PCC) assay in human lymphocytes allows rapid detection of cytogenetic damage in interphase stage, within few hours after blood collection. Hence, it is the most suitable method for rapid and high dose biodosimetry. Mitotic cells, used for G0-PCC could be either freshly isolated or previously cryo-preserved. However, under emergency scenarios, only cryo-preserved cells can be relied upon, fresh isolation will only delay the process by 18-24 h. Impact of cryopreservation on mitotic cells and their efficacy to induce PCC are not reported. In the present study, we investigated effect of cryopreservation on mitotic cells and refined the parameters for G0-PCC. More than 95% of the cells were recoverable after 4 months of cryopreservation, within 20 min recovery at 37 °C, without significant change in the mitotic index or viability. Recovered mitotic cells have shown mitotic index of 89 ± 4% and viability of 90 ± 4%, similar to that of freshly isolated cells. Decrease in metaphases was observed within 40 min after recovery as the mitotic cells progressed through cell cycle and reduced to 21% at 1.5 h. Nevertheless, in presence of Colcemid, the cells progressed slowly and considerably high metaphase index (60%) persisted up to ~ 2 h. The recovered cells efficiently fused with lymphocytes and induced PCC. Average PCC index varied from 10 to 20%, which did not change with cryopreservation duration. Post fusion incubation duration of 2 h was found to be optimum for proper chromosome condensation. In conclusion, use of cryo-preserved mitotic cells is the most practical approach for rapid biodosimetry. The cells can be recovered quickly and efficiently without alteration in viability or mitotic index. Recovered cells are fully competent to induce G0-PCC.

Development of a Sensitive and Specific Novel qPCR Assay for Simultaneous Detection and Differentiation of Mucormycosis and Aspergillosis by Melting Curve Analysis.

Molecular diagnostic assays can expedite the diagnosis of fungal infections, and subsequently help in early interventions and appropriate management of patients. The aim of this study was to develop a single set of primers for a real-time quantitative polymerase chain reaction (qPCR) assay to detect and identify commonly reported, clinically relevant molds i.e., Aspergillus spp, Mucorales and Fusarium spp., up to genus level by melting curve analysis. This assay was evaluated in whole blood from patients with suspected invasive aspergillosis (IA), and in tissue biopsy, bronchoalveolar lavage (BAL) fluid and other site-specific samples from patients with suspected invasive mucormycosis (IM). The limit of detection (LoD) was determined as 10 copies/µl for all three molds. The mean coefficient of variation (CV) across all sets of intra- and inter-assay data was 0.63% (ranging from 0.42 to 1.56%), showing high reproducibility of the assay. Sensitivity and specificity of the assay were 93.3 and 97.1% respectively for diagnosis of IA, and 99.29 and 83.84% respectively for diagnosis of IM. Fusarium was not detected in any of the clinical samples included and the few laboratory confirmed cases of fusariosis did not meet the inclusion criteria of the study. Hence no ROC curve or cutoff value could be generated for the same. This newly developed qPCR assay therefore appears to be a promising tool in detection of IA and IM.

Multifaceted applications of pre-mature chromosome condensation in radiation biodosimetry.

BACKGROUND: Biodosimetry with persistent cytogenetic indicators in peripheral blood lymphocytes (PBLs) plays crucial role in regulatory/medical management of individuals overexposed to radiation. Conventional methods require ∼48 h culture and have limited dose range (0.1-5Gy) applications due to checkpoint arrest/poor stimulation. G0-Phase Premature chromosome condensation (G0-PCC) allows chromosome aberration analysis within hours after blood collection. Due to high skill demand, applications of G0-PCC were not very well explored and being re-visited worldwide. Among all aberrations, analysis of excess chromosomal fragments is quickest. Radiation dose response curve for the fragments has been reported. PURPOSE: In present study, excess fragment analysis has been addressed in detail, in addition to validation of radiation dose response curve, gender variation in the response, dose dependent repair kinetics, minimum detection limit (MDL), duration and accuracy of final dose estimation with 5blindfolded, ex vivo irradiated samples have been studied. In extension, feasibility of multiparametric dosimetry with Fluorescent in situ hybridization (FISH) based endpoints were qualitatively explored. MATERIAL AND METHODS: PBLs were exposed to Gamma-Radiation and G0-PCC was performed at different time points. Decay kinetics and dose response curve were established. Gender Variation of the frequency of the fragments was assessed at 0, 2 and 4 Gy. FISH was performed with G0-PCC applying centromere probe, whole chromosome paints, multi-color FISH and multi-color banding probes. RESULTS: Radiation response curve for fragments was found to be linear (Slope 1.09 ± 0.031 Gy-1). Background frequency as well as dose response did not show significant gender bias. Based on variation in background frequency of fragments MDL was calculated to be ∼0.3 Gy. Kinetics of fragment tested at 0, 4, 8, 16 and 24 h showed exponential decay pattern from 0 to 8 h and without further decay. Final dose estimation of five samples was completed within 13 man-hours. Dicentric chromosomes, translocations, insertions and breaks were identifiable in combination with centromere FISH and WCP. Advanced methods employing multicolor FISH and multi-color banding were also demonstrated with PCC spreads. CONCLUSION: G0-PCC, can be useful tool for high dose biodosimetry with quick assessment of fragment frequency. Further, it holds potential for multi-parametric dosimetry in combination with FISH.

Modulation of azole sensitivity and filamentation by GPI15, encoding a subunit of the first GPI biosynthetic enzyme, in Candida albicans.

Glycosylphosphatidylinositol (GPI)-anchored proteins are important for virulence of many pathogenic organisms including the human fungal pathogen, Candida albicans. GPI biosynthesis is initiated by a multi-subunit enzyme, GPI-N-acetylglucosaminyltransferase (GPI-GnT). We showed previously that two GPI-GnT subunits, encoded by CaGPI2 and CaGPI19, are mutually repressive. CaGPI19 also co-regulates CaERG11, the target of azoles while CaGPI2 controls Ras signaling and hyphal morphogenesis. Here, we investigated the role of a third subunit. We show that CaGpi15 is functionally homologous to Saccharomyces cerevisiae Gpi15. CaGPI15 is a master activator of CaGPI2 and CaGPI19. Hence, CaGPI15 mutants are azole-sensitive and hypofilamentous. Altering CaGPI19 or CaGPI2 expression in CaGPI15 mutant can elicit alterations in azole sensitivity via CaERG11 expression or hyphal morphogenesis, respectively. Thus, CaGPI2 and CaGPI19 function downstream of CaGPI15. One mode of regulation is via H3 acetylation of the respective GPI-GnT gene promoters by Rtt109. Azole sensitivity of GPI-GnT mutants is also due to decreased H3 acetylation at the CaERG11 promoter by Rtt109. Using double heterozygous mutants, we also show that CaGPI2 and CaGPI19 can independently activate CaGPI15. CaGPI15 mutant is more susceptible to killing by macrophages and epithelial cells and has reduced ability to damage either of these cell lines relative to the wild type strain, suggesting that it is attenuated in virulence.

Differential killing and radio-modifying effects of iodoacetate in mammalian normal and cancer cells.

To explore possible applications of iodoacetate (IA), a glycolytic inhibitor, in cancer treatment, we screened its cytotoxicity and radioprotective/sensitizing efficacy in three different mammalian cell lines; A549 (human lung carcinoma), MCF7 (human mammary cancer), a non-cancerous CHO (Chinese hamster ovary) cells and human lymphocytes. Experiments were carried out using IA concentrations ranging from 0.01 to 2.5 µg/ml, with or without 60Coγ-radiation. In the outcomes, IA was found to exhibit higher toxicity in the cancer cells, whereas it was non-toxic/marginally toxic to the non-cancerous cells. Considerably higher glucose uptake in both cancer cells lines was observed indicating higher rates of glycolysis. IA significantly inhibited glycolysis as reflected by GAPDH activity inhibition. Radiomodifying effects of IA were found to be concentration dependent in both cancerous and non-cancerous cells. The response in non-cancerous was found to be biphasic: at lower concentrations, it offered significant radioprotection; however, the protection decreased with increasing concentration. Moreover, at the highest tested concentration, marginal radiosensitization was also observed (as indicated by clonogenic assay). In both cancer cells, IA offered significant amount of radiosensitization which was considerably high at higher concentrations. Further experiments were carried out to estimate the Dose Modification Factor (DMF) to quantify and compare relative radiosensitization by IA in cancer and normal cell lines. The DMF was calculated for three different concentrations of IA, 0.5, 1, and 1.5 µg/ml, and corresponding values were found to be 1.26, 1.43, and 1.89 for A549 cancer cells, whereas for normal CHO cells, it was 1.13, 1.13, and 1.24. In conclusion, differential killing and radiosensitizing effects of IA suggest that it may have potential use as a anticancer agent and radiosensitizer in cancer therapy.

Standardization of CalyculinA induced PCC assay and its advantages over Okadaic acid PCC assay in Biodosimetry applications.

OBJECTIVE: In the present study an attempt was made to estimate coefficients of dose response curves for PCC aberrations induced by CalyculinA and Okadaic acid, using 60Co gamma radiation and 8 MeV pulsed electron beam for biodosimetry application. MATERIALS AND METHODS: The modified method outlined by Puig et al. 2013 was used to conduct Calyculin A and Okadaic acid induced PCC assay in human blood lymphocytes.Chemical treatment was given for the last 1 h of a 48 h culture. The study was carried out in the dose range 2.5 to 20 Gy using 60Co gamma rays and 8 MeV pulsed electron beam. RESULTS AND CONCLUSIONS: Results show a linear dose dependent increase with a slope of 0.047 ± 0.001 from Calycalin A PCC and 0.048 ± 0.002 form Okadaic acid PCC. The slope of the fragments curve was 0.327 ± 0.006 from Calyculin A and 0.328 ± 0.006 from Okadaic acid PCC. Further, dose calibration studies were carried out for 8 MeV electron using Calyculin A PCC assay and the obtained slope from ring yield was 0.054 ± 0.002 and 0.427 ± 0.009 from fragment yield.

Chronic myeloid leukemia with pregnancy: Successful management of pregnancy and delivery with hydroxyurea and imatinib continued till delivery.

The concomitant occurrence of pregnancy and chronic myeloid leukemia is uncommon. We describe the successful management of a 30-year-old G3 P0, A2 woman who was diagnosed to have chronic myelogenous leukemia (CML) in the third trimester of her pregnancy with intra-uterine growth retardation and oligohydroamnios. She was started on hydroxyurea and imatinib, and was continued till delivery and beyond. The use of imatinib did not have any adverse effects on the fetus, except for low birth weight and low APGAR at birth, but the later progress of the child was normal. We conclude that imatinib and hydroxyurea can be continued even at the third trimester in a pregnant lady with CML, if necessary.

Sturge-Weber syndrome with congenital glaucoma and cytochrome P450 (CYP1B1) gene mutations.

UNLABELLED: Sturge-Weber syndrome (SWS) is a progressive condition of mesodermal phakomatosis. This preliminary study is the first report of CYP1B1 mutation analysis in SWS with congenital glaucoma. PURPOSE: Mutations in CYP1B1 gene are the major cause of congenital glaucoma. CYP1B1 is involved in metabolism of melatonin, retinol, and other endogenous/exogenous substrates. Mutations in CYP1B1 adversely affect signal transduction pathways and thus impair development/differentiation of anterior segment structures. This results in impaired aqueous outflow. CYP1B1 has higher expression in fetal eyes and plays major role in morphogenesis of iris, ciliary body, and anterior chamber angle. Hence, we decided to evaluate SWS cases with buphthalmos for 6 most prevalent CYP1B1 mutations by polymerase chain reaction-restriction-fragment length polymorphism followed by sequencing. Trabecular meshwork was studied for morphological alterations by scanning electron microscopy. RESULTS: All patients had normal 46, XY karyotype. Polymerase chain reaction-restriction-fragment length polymorphism showed CYP1B1 mutations in 2 of 5 SWS cases. Scanning electron microscopy findings were suggestive of trabecular dysgenesis. DISCUSSION: No CYP1B1 mutation has been reported in any SWS case till date because syndromic cases were not analyzed for mutations in earlier studies. Earlier studies have reported that onset of glaucoma in SWS shows a bimodal pattern. The results from this pilot study show that SWS cases with gyral calcification, buphthalmos, and early onset glaucoma should be analyzed for CYP1B1 mutations. The effect of vascular malformation-induced venous engorgement and raised intraocular pressure may only be additive and may result in a much more severe phenotype. CONCLUSION: SWS with buphthalmos and gyral calcification should undergo CYP1B1 mutation analysis to identify an underlying genetic pathology for glaucoma. This will aid in determining the prognosis and management and will also help to provide comprehensive counseling in such cases.

Mutation spectrum of CYP1B1 in North Indian congenital glaucoma patients.

PURPOSE: Mutations in Cytochrome P450 (CYP1B1) are a predominant cause of congenital glaucoma. This study was planned with the aim to identify the mutation profile of CYP1B1 in North Indian primary congenital glaucoma (PCG) patients. METHODS: After ethical clearance, 50 congenital glaucoma patients and 50 ethnically matched controls were recruited in this study. Genomic DNA was isolated from the blood and trabecular meshwork, and CYP1B1 was screened for the six most prevalent mutations (termination at 223 [Ter@223], Gly61Glu, Pro193Leu, Glu229Lys, Arg368His, and Arg390Cys) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). DNA sequencing was done to identify other mutations and for confirmation of RFLP positive samples. RESULTS: On PCR-RFLP, 21/50 cases (42%) were found positive for one or more of these mutations. However, on sequencing, we found that 23/50 (46%) harbored the CYPIB1 mutations. Ter@223 was found in 18%, p.R390H in 16%, and p.R368H in 8% of cases. Three novel mutations, p.L24R, p.F190L, and p.G329D, were identified by DNA sequencing. Leucine, phenylalanine, and glycine are conserved at the 24th, 190th, and 329th position in the CYP1B1 protein in different species, suggestive of important functions at these loci. Ter@223 was found to be the most prevalent mutation in our patients while p.R368H was most prevalent in southern India. The difference in frequency and mutation profile may be due to the heterogeneous Indian population. Pathogenic CYP1B1 mutations impair anterior chamber development and differentiation by blocking the aqueous outflow and raising intraocular pressure (IOP). CONCLUSIONS: Three novel mutations were identified in this study. Studies of pathogenic sequence variants in CYP1B1 in different populations may contribute to a better understanding of the disease pathogenesis. This may lead to the development of novel therapeutic approaches in the near future.