Photodynamic Therapy (PDT) involves the administration of a tumor localizing photosensitizing agent, which upon activation with light of an appropriate wavelength leads to the destruction of the tumor cells. The aim of the present study was to determine the efficacy of erythrosine as a photosensitizer for the PDT of oral malignancies. The drug uptake kinetics of erythrosine in malignant (H357) and pre-malignant (DOK) oral epithelial cells and their susceptibility to erythrosine-based PDT was studied along with the determination of the subcellular localization of erythrosine. This was followed by initial investigations into the mechanism of cell killing induced following PDT involving both high and low concentrations of erythrosine. The results showed that at 37°C the uptake of erythrosine by both DOK and H357 cells increased in an erythrosine dose dependent manner. However, the percentage of cell killing observed following PDT differed between the 2 cell lines; a maximum of ∼80% of DOK cell killing was achieved as compared to ∼60% killing for H357 cells. Both the DOK and H357 cell types exhibited predominantly mitochondrial accumulation of erythrosine, but the mitochondrial trans-membrane potential (ΔΨ(m)) studies showed that the H357 cells were far more resistant to the changes in ΔΨ(m) when compared to the DOK cells and this might be a factor in the apparent relative resistance of the H357 cells to PDT. Finally, cell death morphology and caspase activity analysis studies demonstrated the occurrence of extensive necrosis with high dose PDT in DOK cells, whereas apoptosis was observed at lower doses of PDT for both cell lines. For H357 cells, high dose PDT produced both apoptotic as well as necrotic responses. This is the first instance of erythrosine-based PDT's usage for cancer cell killing.

The role of mitochondrial dysfunction in the development of insulin resistance and type 2 diabetes remains controversial. In order to specifically define the relationship between insulin receptor (InsR) signaling, insulin resistance, hyperglycemia, hyperlipidemia and mitochondrial function, we analyzed mitochondrial performance of insulin-sensitive, slow-oxidative muscle in four different mouse models. In obese but normoglycemic ob/ob mice as well as in obese but diabetic mice under high-fat diet, mitochondrial performance remained unchanged even though intramyocellular diacylglycerols (DAGs), triacylglycerols (TAGs), and ceramides accumulated. In contrast, in muscle-specific InsR knockout (MIRKO) and streptozotocin (STZ)-treated hypoinsulinemic, hyperglycemic mice, levels of mitochondrial respiratory chain complexes and mitochondrial function were markedly reduced. In STZ, but not in MIRKO mice, this was caused by reduced transcription of mitochondrial genes mediated via decreased PGC-1α expression. We conclude that mitochondrial dysfunction is not causally involved in the pathogenesis of obesity-associated insulin resistance under normoglycemic conditions. However, obesity-associated type 2 diabetes and accumulation of DAGs or TAGs is not associated with impaired mitochondrial function. In contrast, chronic hypoinsulinemia and hyperglycemia as seen in STZ-treated mice as well as InsR deficiency in muscle of MIRKO mice lead to mitochondrial dysfunction. We postulate that decreased mitochondrial mass and/or performance in skeletal muscle of non-diabetic, obese or type 2 diabetic, obese patients observed in clinical studies must be explained by genetic predisposition, physical inactivity, or other still unknown factors.

A super reduced Fe(4)S(4) cluster with a sulfur based radical,[NBu(4)](4)[Fe(3)(III)Fe(II)(μ(3)-S)(4)(mnt)(3)(6-)(mnt)(1-)˙](4-)˙,(1)(mnt, maleonitrile dithiolate) which evolves H(2)S gas on treatment with acid under ambient conditions has been synthesized and structurally characterized. The Fe-S distances in 1 are in the range 2.246-2.383 Å, in stark contrast to that of the known n =-2 member of the series based on the [Fe(4)(μ(3)-S)(4)(S(2)C(2)R(2))(4)](n) unit (R = CF(3), Ph) with Fe-S bond lengths of 2.149-2.186 Å. The EPR of 1 displays very weak signals at g, 4.03 and 2.38 along with a strong S-based radical EPR signal at g, 2.003 associated with five structured components tentatively assigned to hyperfine interaction arising out of the naturally abundant (57)Fe with <A>= 88 G. The EPR profile resembles the reduced Fe-S cluster of CO inhibited Clostridium pasteurianum W5 hydrogenase or the Fe(4)S(4) centers of wild-type enzyme, IspH treated with HMBPP or IPP.

Respiratory Syncytial Virus (RSV) is the leading cause of lower respiratory-tract infections in infants and young children worldwide. Despite this, only six complete genome sequences of original strains have been previously published, the most recent of which dates back 35 and 26 years for RSV group A and group B respectively. We present a semi-automated sequencing method allowing for the sequencing of four RSV whole genomes simultaneously. We were able to sequence the complete coding sequences of 13 RSV A and 4 RSV B strains from Milwaukee collected from 1998-2010. Another 12 RSV A and 5 RSV B strains sequenced in this study cover the majority of the genome. All RSV A and RSV B sequences were analyzed by neighbor-joining, maximum parsimony and Bayesian phylogeny methods. Genetic diversity was high among RSV A viruses in Milwaukee including the circulation of multiple genotypes (GA1, GA2, GA5, GA7) with GA2 persisting throughout the 13 years of the study. However, RSV B genomes showed little variation with all belonging to the BA genotype. For RSV A, the same evolutionary patterns and clades were seen consistently across the whole genome including all intergenic, coding, and non-coding regions sequences. The sequencing strategy presented in this work allows for RSV A and B genomes to be sequenced simultaneously in two working days and with a low cost. We have significantly increased the amount of genomic data that is available for both RSV A and B, providing the basic molecular characteristics of RSV strains circulating in Milwaukee over the last 13 years. This information can be used for comparative analysis with strains circulating in other communities around the world which should also help with the development of new strategies for control of RSV, specifically vaccine development and improvement of RSV diagnostics.

ObjectivesThe objective of the present study was to evaluate the antitubercular activity of amino and acyl amino derivatives of coumarins when used alone and in combination with isoniazid, rifampicin, streptomycin or ethambutol, and to decipher the mode of action of the most effective agent.MethodsA series of amino and acyl amino coumarins were synthesized and screened for activity against the Mycobacterium tuberculosis H37Rv strain. These compounds were further evaluated by standard assay procedures to determine their MBCs, fractional inhibitory concentration index values and cytotoxicities. The MICs for a susceptible and a multidrug-resistant clinical isolate of M. tuberculosis were also determined. Electron and fluorescence microscopy of the test compound-treated mycobacterial samples were also carried out in an attempt to find out the target of action.Results7-Amino-4-methylcoumarin (7-amino-4-methyl-2H-chromen-2-one; NA5) displayed the lowest MIC of 1 mg/L against not only H37Rv but also the susceptible as well as the multidrug-resistant clinical isolates. Certain acyl amino coumarins were also found to inhibit the aforementioned strains and isolates with MICs in the range of 1.0-3.5 mg/L. They were also found to act in synergy with isoniazid/rifampicin. Electron microscopy revealed the cell-wall-attacking characteristic of these compounds, while fluorescence microscopy indicated that mycolic acid might be the target of action.ConclusionsThe present study clearly demonstrated the in vitro antitubercular potential of the novel drug candidate NA5. Further studies are warranted to establish the in vivo efficacy and therapeutic potential of NA5.

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

Tuberculosis (TB) is the leading cause of death worldwide attributable to a single infectious disease agent. India has more new TB cases annually than any other country. In 2008, India accounted for a fifth of the estimated 9.4 million TB cases globally. There is an overwhelming need for improving TB diagnostics in India through the use of cost effective, patient-friendly methods appropriate to different tiers of the country health system. Substantial progress has been made in India in the field of TB diagnosis and serious efforts have been made to herald the development of diagnostic tests for pulmonary TB, extra pulmonary TB and MDR-TB. Diverse approaches have been attempted towards improving smear microscopy, rapid culture and for differentiation between the Mycobacterium tuberculosis complex and non-tuberculous mycobacteria. Several laboratories have developed in-house PCR assays for diagnosing TB with high accuracy. Approaches for distinguishing M. tuberculosis and/or Mycobacterium bovis infection and disseminated Mycobacterium avium complex infection in HIV-AIDS patients have also been described. Serological tests to detect antigens or antibodies to M. tuberculosis specific components by using cocktails of Excretory/Secretory protein antigens, Ag85 complex antigens, Hsp 65 antigen, RD1 antigens and Rapid Reverse Line Blot Hybridization assays to detect MDR-TB (mutations to rifampicin, isoniazid and streptomycin) have also been developed. Other methods like measurement of adenosine deaminase activity and use of luciferase reporter phages have also been explored for TB diagnosis. These advances in the Indian context are detailed in the present chapter. The validation and application of these methods in laboratory and public health settings is likely to result in improved TB diagnosis and contribute to effective disease management in India.

Ethnic specificity is a key determinant in understanding the association of genetic variants with outcome of disease susceptibility. SP110, a component of the nuclear body, has been subjected to association studies with conflicting results. In this study we probed SP110 variants in pulmonary (PTB) and lymph node tuberculosis (LNTB) cases to explore their role in controlling susceptibility to Mycobacterium tuberculosis infection in north Indians. We genotyped 24 SP110 variants in over 140 north Indian tuberculosis cases and 78 ethnicity-matched controls. The SP110 gene variants were available from public databases. The cases and controls were free of any population stratification when subjected to Eigenstrat principal component analysis. Genotyping was carried out using the Sequenom MassARRAY platform. Applying exclusion criteria, 11 single nucleotide polymorphisms (SNPs) of the LNTB panel and 13 SNPs of the PTB panel passed all filters and were analyzed further. No significant association was observed between SP110 variants and PTB. Surprisingly, we discovered evidence of an association of SP110 variants with LNTB, a form of extrapulmonary tuberculosis, at 3 loci, namely, rs6436915, rs1427294, and rs1346311. When permutations analysis (n = 10,000) of allelic p values was undertaken, only rs1427294 passed the test with its p value remaining statistically significant. The C allele of rs1427294 exhibited a 5-fold risk of developing LNTB. No significant haplotypes were observed. In the pilot study presented here, our results provide evidence for the first time that SP110 may be a risk determinant locus in LNTB while confirming a doubtful role of SP110 in PTB in north Indians. In general, the results might indicate a role of SP110 variants in extrapulmonary tuberculosis rather than PTB.

Glycemic control is improved more after gastric bypass surgery (GBP) than after equivalent diet-induced weight loss in patients with morbid obesity and type 2 diabetes mellitus. We applied metabolomic profiling to understand the mechanisms of this better metabolic response after GBP. Circulating amino acids (AAs) and acylcarnitines (ACs) were measured in plasma from fasted subjects by targeted tandem mass spectrometry before and after a matched 10-kilogram weight loss induced by GBP or diet. Total AAs and branched-chain AAs (BCAAs) decreased after GBP, but not after dietary intervention. Metabolites derived from BCAA oxidation also decreased only after GBP. Principal components (PC) analysis identified two major PCs, one composed almost exclusively of ACs (PC1) and another with BCAAs and their metabolites as major contributors (PC2). PC1 and PC2 were inversely correlated with pro-insulin concentrations, the C-peptide response to oral glucose, and the insulin sensitivity index after weight loss, whereas PC2 was uniquely correlated with levels of insulin resistance (HOMA-IR). These data suggest that the enhanced decrease in circulating AAs after GBP occurs by mechanisms other than weight loss and may contribute to the better improvement in glucose homeostasis observed with the surgical intervention.