Validation of the InnoXtract™ DNA extraction method for bone, teeth, and rootless hair.

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high-yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non-probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un-insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express-96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high-throughput, high-yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high-yield and high-quality DNA from compromised bone, teeth, and hair shaft samples.

Immunochemical characterisation of styrene maleic acid lipid particles prepared from Mycobacterium tuberculosis plasma membrane.

Membrane proteins of Mycobacterium tuberculosis (Mtb) can be targeted for the development of therapeutic and prophylactic interventions against tuberculosis. We have utilized the unique membrane-solubilising properties of the styrene maleic acid copolymer (SMA) to prepare and characterise 'styrene maleic acid lipid particles' from the native membrane of Mtb (MtM-SMALPs). When resolved by SDS-PAGE and visualised with coomassie blue, the molecular weights of Mtb membrane (MtM) proteins solubilised by SMA were mostly in the range of 40-70 kDa. When visualised by transmission electron microscopy, MtM-SMALPs appeared as nanoparticles of discrete shapes and sizes. The discoid nanoparticles exhibited a range of diameters of ~10-90 nm, with largest portion (~61%) ranging from 20-40 nm. MtM proteins of a molecular weight-range overlapping with that of MtM-SMALPs were also amenable to chemical cross-linking, revealing protein complex formation. Characterisation using monoclonal antibodies against seven MtM-associated antigens confirmed the incorporation of the inner membrane protein PRA, membrane-associated proteins PstS1, LpqH and Ag85, and the lipoglycan LAM into MtM-SMALPs. Conversely, the peripheral membrane proteins Acr and PspA were nearly completely excluded. Furthermore, although MtM showed an abundance of Con A-binding glycoproteins, MtM-SMALPs appeared devoid of these species. Immune responses of healthcare workers harbouring 'latent TB infection' provided additional insights. While MtM-SMALPs and MtM induced comparable levels of the cytokine IFN-γ, only MtM-SMALPs could induce the production of TNF-α. Antibodies present in the donor sera showed significantly higher binding to MtM than to MtM-SMALPs. These results have implications for the development of MtM-based immunoprophylaxis against tuberculosis.

Distinct and shared B cell responses of tuberculosis patients and their household contacts.

This study was aimed at identifying the B cell responses which could distinguish between 'latent tuberculosis infection (LTBI)' and active TB disease. Study subjects were smear-positive TB patients (n = 54) and their disease-free household contacts (HHCs, n = 120). The sera were used for determination of antibody levels (ΔOD values) against Mycobacterium tuberculosis membrane (MtM) antigens by ELISA and for visualisation of seroreactive MtM antigens by immunoblotting. B cell subsets in whole blood samples were determined by flow cytometry. In TB sera, levels of IgG antibodies were significantly higher than IgM and IgA whereas IgM and IgA antibody levels were comparable. Conversely, HHC sera had significantly higher IgM antibody levels than IgG and IgA. The ratio of IgM to IgG antibodies in HHCs were also significantly higher than in patients. Immunoblotting revealed that some of the MtM antigens (<10, ~12 and ~25 kDa) reacted with TB as well as HHC sera whereas some other antigens (~16, ~36, ~45 and ~60 kDa) reacted with most of TB and a subset of HHC sera. Frequencies of classical memory B cells (cMBCs, CD19+CD27+) were significantly higher, and of IgG+ cMBCs were significantly lower in HHCs than in patients. Frequencies of IgA+ cMBCs in HHCs and patients were comparable but both were significantly higher than the corresponding frequencies of IgG+ cMBCs. Frequencies of IgA+ atypical MBCs (aMBCs, CD19+CD27-) in HHCs and patients were also comparable and significantly higher than the IgG+ aMBCs. The plasmablast (CD19+CD27++CD38++) frequencies in HHCs and patients were comparable. These results suggest that the IgM/IgG antibody ratio, antibody binding to selected MtM antigens and relative frequencies of MBC subsets could indicate protective or pathogenic immune responses following the primary infection with Mtb. Responses that orchestrate protection leading to a 'quiescent' LTBI may provide clues to an effective vaccination strategy against TB.

Development and validation of a novel method "SpermX™" for high throughput differential extraction processing of sexual assault kits (SAKs) for DNA analysis.

The Sperm X method uses a nanotechnology derived polymer membrane that functions as a separation medium to effectively trap sperm cells while enabling efficient flow through of the digested epithelial cell DNA. This specialized membrane enabled development of a method that could significantly increase a forensic laboratory's ability to obtain male sperm fraction DNA profiles. The SpermX device provides a rapid, reproducible procedure that is easy to implement in a single-tube format as well as high-throughput truly automated hands-free workflows. Validation studies, performed using the manual SpermX method, include sensitivity, stability, precision (reproducibility and repeatability), mixtures, and a method comparison to the traditional differential extraction. Sensitivity and method comparison studies demonstrated a wide range of sperm cells, from a high of over 2.78 million cells (9158 ng) to a low of 25 cells (83 pg), can be trapped by the SpermX membrane. Stability studies on various substrates (i.e., carpet, cotton, denim, polyester, and silk) and degraded semen gave the expected male DNA profiles. Data from the same operator and a different operator were consistent with low variance. Mixtures, with ratios ranging from approximately 10:1-18182:1, created to simulate real casework type samples including buccal/semen, vaginal epithelial/semen, and post coital swabs at different time intervals, were tested. A comparison of the SpermX method to the conventional differential extraction method resulted in comparable probative male profile allelic data and associated statistical probabilities. For low level sperm samples, down to 25 sperm cells (83 pg), the SpermX method outperformed the conventional differential extraction with more genotypic information and associated probabilities.

Patterns of T and B cell responses to Mycobacterium tuberculosis membrane-associated antigens and their relationship with disease activity in rheumatoid arthritis patients with latent tuberculosis infection.

This study was aimed at exploring whether latent tuberculosis infection (LTBI) contributes to the pathogenesis of immune-mediated inflammatory diseases in a TB endemic setting. We screened 198 rheumatoid arthritis (RA) patients with tuberculin skin test (TST) and studied 61 (median DAS28-ESR = 6.3) who were positive. Whole blood T cell proliferative responses to Mycobacterium tuberculosis (Mtb) membrane (MtM) antigens, including the latency-induced protein alpha crystallin (Acr), were determined by flow cytometry using Ki67 expression as the marker for nuclear proliferation. Serum antibody levels were determined by ELISA. Follow-up investigations (at 3-6, 9-12 and 15-18 months after baseline) were performed in 41 patients who were classified empirically as 'high' (HR-T/HR-B) or 'low' (LR-T/LR-B) responders based on their dynamic T cell or antibody responses. Significant correlations were seen between baseline T cell responses to MtM and Acr, and between IgG, IgA and IgM antibody responses to MtM. However, no correlation was seen between T and B cell responses. At all time points during the follow-up, T cell responses to both antigens (except for MtM at one point) were significantly higher in HR-T (n = 25) than LR-T (n = 16) patients. Levels of IgA and IgM (but not IgG) antibodies to MtM were also significantly higher in HR-B (n = 13) than LR-B (n = 28) at all time points. Importantly, HR-T patients exhibited significantly higher baseline and follow-up DAS28 scores than LR-T. Ten (of 61) patients had a history of TB and developed RA 6 years (median) after contracting TB. Three new TB cases (1 from TST-positive and 2 from TST-negative groups) emerged during the follow-up. Our results suggest that persistently elevated T cell responses to Mtb antigens may contribute to disease activity in RA.

Immune responses to Mycobacterium tuberculosis membrane-associated antigens including alpha crystallin can potentially discriminate between latent infection and active tuberculosis disease.

Changes in expression of membrane antigens may accompany the transition of Mycobacterium tuberculosis (Mtb) from 'dormant' to 'active' states. We have determined whether antibody and T cell responses to Mtb membrane (MtM)-associated antigens, especially the latency-induced protein alpha crystallin (Acr), can discriminate between latent tuberculosis infection (LTBI) and active TB (ATB) disease. Study subjects comprised a previously described cohort of healthcare workers (HCWs, n = 43) and smear-positive ATB patients (n = 10). HCWs were further categorized as occupational contacts (OC, n = 30), household contacts of TB (HC, n = 8) and cured TB (CTB, n = 5). Levels (ΔOD) of serum antibody isotypes (IgG, IgA and IgM) were determined by ELISA and blood T cell proliferative responses were determined by flow cytometry using Ki67 protein as marker for DNA synthesis. Antibodies to MtM and Acr were predominantly IgG and their levels in HCWs and ATB did not differ significantly. However, HCWs showed a significantly higher level of anti-MtM IgM and a significantly lower level of anti-Acr IgA antibodies than the ATB patients. Also, a larger proportion of HCWs showed a high (>1) ΔODAcr/ΔODMtM ratio for IgG. HCWs also showed a higher, though not significantly different from ATB, avidity of anti-MtM (IgG) antibodies. A higher proportion of HCWs (35% of OC, 62.5% of HC and 20% of CTB), compared with ATB (10%) showed a positive T cell response to Acr along with significant difference (P <0.05) between HC and ATB. A significant correlation (r = 0.60, P <0.0001) was noted between T cell responses of HCWs towards Acr and MtM (reported earlier by us) and both responses tended to decline with rising exposure to the infection. Even so, positive responses to Acr (38.5%) were significantly lower than to MtM (92%). Neither antibody nor T cell responses to either antigen appeared affected by BCG vaccination or reactivity to tuberculin. Results of the study suggest that the levels of IgM antibodies to MtM, IgA antibodies to Acr and proliferative T cell responses to both the antigens can potentially discriminate between LTBI and active TB disease. They also underscore the necessity of SOPs for antibody assays.

Multiplexed real-time polymerase chain reaction cell-free DNA assay as a potential method to monitor stage IV colorectal cancer.

BACKGROUND: Liquid biopsy is a new area in cancer diagnostics that measures cell-free DNA in plasma from tumor that may serve as a monitoring tool in colorectal cancer patients. METHODS: Multiplexed real-time polymerase chain reaction based on multicopy retro-transposable elements (targeting 80 base pair and 265 base pair sequences and an internal-positive-control) was used to evaluate the ability of cell-free DNA concentration and DNA Integrity Index to discriminate cancer from healthy patients. A cohort of 40 healthy controls and 39 stage IV colorectal patient's plasma were interrogated. The potency of each biomarker was measured by using receiver operating characteristic curves and derived area under the curve measures. RESULTS: Significant differences in cell-free DNA concentration and DNA integrity index were observed between controls and stage IV patients with a limit of detection <0.1 pg/µL. Investigation of the ability of both biomarker candidates to differentiate cancer from healthy patients showed an area under the curve of 0.9891 and 0.9859 for 80 base pair and 265 amplicons respectively and 0.8603 for DNA integrity index-265/80. CONCLUSIONS: After establishing differences in cell-free DNA levels between healthy and treated and untreated stage IV patients, the multiplexed real-time polymerase chain reaction measurements of retro-transposable elements in cancer patient plasma potentially possess the ability to monitor therapy responsiveness in near real time.

Synergy between tuberculin skin test and proliferative T cell responses to PPD or cell-membrane antigens of Mycobacterium tuberculosis for detection of latent TB infection in a high disease-burden setting.

Tuberculin skin test (TST) is used most widely for the detection of latent tuberculosis infection (LTBI), even though evidences suggest that it could be underreporting the prevalence of LTBI particularly in high disease-burden settings. We have explored whether in vivo (TST) and in vitro (cell-proliferative) T cell responses to PPD can serve as complementary measures. In addition, we also probed whether in vitro T cell response to cell-membrane antigens (Mem) of Mycobacterium tuberculosis (MTB) can serve as a biomarker for LTBI. Study subjects comprised 43 healthcare workers (HCWs), and 9 smear-positive TB patients served as 'disease control'. To measure proliferative T cell responses, 0.1 ml blood (diluted 1:10) was incubated (5 days) with test or control antigen. Cells were stained with fluorescent antibodies to T cell (CD3+/CD4+/CD8+) surface markers and, after fixation and permeabilization, to nuclear proliferation marker Ki67. Data was acquired on a flow cytometer. HCWs who had an intimate exposure to MTB showed significantly higher TST positivity (85%) than the rest (43%), notwithstanding their BCG vaccination status. The proliferative responses of CD4+ and CD8+ subsets of T cells were comparable. Sixty seven and 100% TST-negative HCWs, respectively, were positive for proliferative T cell response to PPD and MTBMem. Cumulative positivity (TST or in vitro) was 86% with PPD and 100% with MTBMem indicating complementarity of the two responses. As standalone in vitro assay, MTBMem provided a significantly higher positivity (95%) than PPD (67%). T cell responses of TB patients were 'generally' depressed, having implications for the development of immunological assays for 'progressive' LTBI. Altogether, these results demonstrate that in vivo and in vitro T cell responses to PPD are complementary and in vitro response to MTBMem can be developed as a highly sensitive biomarker for LTBI.

Successful nuclear DNA profiling of rootless hair shafts: a novel approach.

Historically, rootless hair shaft samples submitted to a forensic laboratory for DNA analysis are reserved for mitochondrial DNA (mtDNA) analysis due to the presence of highly degraded as well as insufficient amounts of nuclear DNA. Although mtDNA has been very successful in obtaining results from rootless hair, this system has its limitations, namely, it is a lineage marker that cannot differentiate between maternally related genotypes. Given the high incidence of hairs as forensic evidence, there is a need for the use of a nuclear DNA test system capable of producing reliable results for hair shaft forensic evidence. This study reports the utilization of an enhanced DNA extraction methodology for hairs, in combination with a recently developed novel, nuclear DNA typing assay, InnoTyper® 21, to improve the success rate for obtaining informative results from highly compromised, degraded, and trace forensic samples such as rootless hair shafts. The InnoTyper 21 kit is a small amplicon retrotransposon marker typing system compatible with currently used capillary electrophoresis platforms. This system contains 20 Alu element markers, ranging in size from 60 to 125 bp, making the assay highly sensitive for extremely degraded forensic samples and thus enabling recovery of nuclear DNA profiles from samples that would otherwise require mtDNA sequencing. A subset of samples was also tested with the GlobalFiler kit with less success due to the larger amplicon sizes in comparison with InnoTyper 21. Results were variable but very promising, with approximately 40% of the total number of hairs tested producing interpretable nuclear DNA profiles with InnoTyper 21. These results demonstrate the ability of the utilized methodologies to produce nuclear DNA results with high statistical power from rootless hair shafts.

Development and validation of InnoQuant® HY, a system for quantitation and quality assessment of total human and male DNA using high copy targets.

The development and validation of InnoQuant® HY, a real-time PCR system containing four DNA targets-two RE autosomal targets of different sizes, male specific targets, and an internal positive control target-are described herein. The ratio of the two autosomal targets provides a Degradation Index, or a quantitative value of a sample's degradation state. The male specific targets are multi-copy targets located on the Y chromosome, which provides information about a sample's male DNA composition. The experimental results demonstrate InnoQuant HY as a robust qPCR method producing accurate DNA quantitation results even at low dynamic ranges, with reproducibility among population groups. The system is human specific with low level higher primate cross reactivity and is able to consistently and reproducibly detect sub-picogram concentrations of human and human male DNA. The use of high copy number Alu and SVA (>1000 copies per genome) retrotransposable elements as the two autosomal targets significantly enhances both sensitivity and reproducibility of determination of DNA quantitation as well as DNA degradation in forensic samples. The inclusion of a sensitive multi-copy Y-chromosome specific target provides accurate quantitation of DNA from a male in challenging male-female mixtures (i.e. sexual assault samples). Even in the presence of a large excess of DNA from a female, accurate quantitation was achieved with a male to female ratio of 1:128,000. Population database studies reveal an average Short/Y target ratio of the quantification values across all four populations tested was 1.124±0.282, exhibiting the system's reproducibility across multiple populations. The results from InnoQuant HY provide a tool equipping a forensic analyst with crucial data about a sample's DNA quantitation, male:female ratio, degradation state, and the presence or absence of PCR inhibitors. With the information gained from the InnoQuant HY kit, a more streamlined and efficient workflow can be created that minimizes unnecessary sample processing and retesting while maximizing recovery of probative DNA profiles from challenging biological evidence.

Development and validation of a novel multiplexed DNA analysis system, InnoTyper® 21.

We report here a novel multiplexed DNA analysis system consisting of 20 Alu markers and Amelogenin for analysis of highly degraded forensic biological samples. The key to the success of the system in obtaining results from degraded samples is the primer design yielding small amplicon size (60-125bp) for all 20 markers. The markers included in the InnoTyper® 21 system are bi-allelic, having two possible allelic states (insertion or null) and thus termed INNULs. The markers are short interspersed nuclear elements (SINEs), a category of retrotransposable elements (REs) which are non-coding genomic DNA repeat sequences, or "mobile insertion elements," comprising approximately 40% of the human genome. Alu elements are primate specific SINEs that have reached a copy number in excess of one million in the human genome, which makes these markers highly sensitive and desirable for forensic samples with extremely degraded DNA. Until now however, due to the inherent size difference associated with insertion and no insertion alleles, the use of Alu REs has not been practical for forensic applications. The novel primer design described herein has allowed the development of a multiplexed Alu system yielding fragment sizes amenable to degraded DNA samples, as frequently encountered in missing persons cases or forensic samples such as hair shafts. Although use of Alus in human identity has been studied using single marker amplification and reported before, we report for the first time development and validation of a system with multiplexed RE markers. Studies performed include PCR optimization, species specificity, sensitivity, degradation and inhibition, precision and accuracy, nonprobative samples, mixture, and population database studies. A population study using 592 samples including five populations was performed using InnoTyper 21. The data indicated the random match probability for the combination of these 20 Alu markers was greater than 1 in 3.8 million for the populations studied, indicating the greater statistical power of these autosomal nuclear DNA markers over haplotype systems typically used in such degraded samples. Results demonstrate the system is successful in obtaining results from highly degraded DNA. A sensitivity study performed demonstrated at least 95% recovery of alleles from as low as 50pg of total input DNA, and partial profiles from as low as 25pg. This study has demonstrated that the bi-allelic INNULs in the InnoTyper 21 system provide a sensitivity of detection and a power of discrimination that makes them useful for human identification of extremely degraded samples.

S-Enantiomer of the Antitubercular Compound S006-830 Complements Activity of Frontline TB Drugs and Targets Biogenesis of Mycobacterium tuberculosis Cell Envelope.

A synthetic molecule S006-830, belonging to the class of thiophene-containing trisubstituted methanes, had shown good in vitro and in vivo bactericidal activity against drug-sensitive and drug-resistant Mycobacterium tuberculosis (Mtb). The molecule had also shown good druglike pharmacokinetic properties. However, S006-830 is a racemic mixture of two enantiomers, one of which could possess a better pharmacological profile than the other. We purified both the enantiomers on a chiral column and observed that S-enantiomer has a significantly higher inhibitory and cidal activity against Mtb than the R-enantiomer. Action of S-S006-830 was "synergistic" for rifampicin and "additive" for isoniazid and ethambutol. The combination of S-S006-830 and rifampicin produced 100% kill of Mtb within 8 days. In a chemical proteomics approach using matrix-bound compound to pull down its target protein(s) from Mtb membrane, FabG4 (ß-ketoacyl CoA reductase, EC 1.1.1.100) emerged as the most likely target for S-S006-830. In target validation assays, the compound exhibited 2-fold higher inhibitory concentration for an Mtb construct overexpressing FabG4. In addition, it inhibited mycolic acid biosynthesis and formation of biofilms by Mtb. Molecular docking of S-S006-830 with FabG4 was consistent with the experimental data. These results support the development of S-S006-830 as a novel lead against tuberculosis.

Correction to "S-Enantiomer of the Antitubercular Compound S006-830 Complements Activity of Frontline TB Drugs and Targets Biogenesis of Mycobacterium tuberculosis Cell Envelope".

[This corrects the article DOI: 10.1021/acsomega.7b01281.].

Naturally produced opsonizing antibodies restrict the survival of Mycobacterium tuberculosis in human macrophages by augmenting phagosome maturation.

This study investigated the hypothesis that serum antibodies against Mycobacterium tuberculosis present in naturally infected healthy subjects of a tuberculosis (TB) endemic area could create and/or sustain the latent form of infection. All five apparently healthy Indian donors showed high titres of serum antibodies against M. tuberculosis cell membrane antigens, including lipoarabinomannan and alpha crystallin. Uptake and killing of bacilli by the donor macrophages was significantly enhanced following their opsonization with antibody-rich, heat-inactivated autologous sera. However, the capability to opsonize was apparent for antibodies against some and not other antigens. High-content cell imaging of infected macrophages revealed significantly enhanced colocalization of the phagosome maturation marker LAMP-1, though not of calmodulin, with antibody-opsonized compared with unopsonized M. tuberculosis. Key enablers of macrophage microbicidal action--proinflammatory cytokines (IFN-γ and IL-6), phagosome acidification, inducible NO synthase and nitric oxide--were also significantly enhanced following antibody opsonization. Interestingly, heat-killed M. tuberculosis also elevated these mediators to the levels comparable to, if not higher than, opsonized M. tuberculosis. Results of the study support the emerging view that an efficacious vaccine against TB should, apart from targeting cell-mediated immunity, also generate 'protective' antibodies.

Stable Tricyclic Antitubercular Ozonides Derived from Artemisinin.

New, highly stable tricyclic antitubercular ozonides 9 and 10 derived from artemisinin are reported in 39 and 9% yields, respectively. The ozonide groups of 9 and 10 were found to be stable under strong basic and acidic conditions. The absolute configuration of ozonides 9 was confirmed by X-ray crystallography. Ozonide 10 shows promising antitubercular activity against M. tuberculosis H37Ra and M. tuberculosis H37Rv with MIC values of 0.39 and 3.12 µg/mL, respectively.

Percutaneous Transcatheter Closure of Ruptured Sinus of Valsalva Aneurysm: Immediate Result and Long-Term Follow-Up.

There is scarcity of data on closure and long-term follow-up of percutaneous treatment of ruptured sinus of Valsalva aneurysm (RSOVA). In this article, we present our experience in percutaneous closure of this defect. Between December 2009 and July 2014, 11 cases of RSOVA were referred to our hospital. Eight of the 11 cases (72.7%) were considered for percutaneous closure. Seven of the eight (87.5%) patients underwent successful percutaneous closure. There were four females and three males in the age group of 16 to 48 years (mean 24.7 ± 6.1 years). Associated defects were bicuspid aortic valve in one patient, mild preexisting aortic regurgitation in two patients, and healed infective endocarditis in one patient. Echocardiography revealed RSOVA from right coronary sinus (RCS) to right atrium (RA) in one patient (14.3%), RCS to right ventricular outflow in three patients (42.8%), and noncoronary sinus ruptured into RA in three patients (42.8%). All patients were symptomatic in New York Heart Association (NYHA) class II to IV. The defect size ranged from 7 to 10 mm (mean 8.4 ± 1.3 mm). The defects were closed from the venous side with device selection 2 to 4 mm higher than the defect size under fluoroscopy and transesophageal echocardiography guidance. Technical success was 87.5%. The mean device size was 12.0 ± 1.6 mm/10.0 ± 1.6 mm. Six out of seven patients (85.7%) had complete disappearance of shunt before discharge. During 1 to 55 months follow-up, all patients were in NYHA class I. There was no residual shunt, progression of AR or new AR, infective endocarditis or device embolization. Percutaneous closure of RSOVA appears to be a safe alternative to surgical therapy, with high technical success and excellent long-term outcome.

Tetrahydrofuran amino acid-containing gramicidin S analogues with improved biological profiles.

Gramicidin S (GS) is a cyclic cationic antimicrobial peptide (CAP) with a wide spectrum of antibiotic activities whose usage has been limited to topical applications owing to its cytotoxic side effects. We have synthesized tetrahydrofuran amino acid (Taa)-containing GS analogues, and we have carried out conformational analysis and explored their structure activity relationships by evaluating their antitubercular, antibacterial and cytotoxic properties. Two of these analogues showed impressive as well as selective activity against Mycobacterium tuberculosis (MTB) without toxicity towards mammalian Vero cells or human RBCs, and are promising as potential leads.

Thiophene containing trisubstituted methanes [TRSMs] as identified lead against Mycobacterium tuberculosis.

Triarylmethanes (TRAMs) and thiophene containing trisubstituted methanes (TRSMs) have been reported by us, having potential against Mycobacterium tuberculosis and Mycobacterium fortuitum strains, respectively. Further, extension through synthesis and biological evaluation of novel TRSMs resulted into an identified lead 36 (S006-830) [(diisopropyl-(2-{4-[(4-methoxy-phenyl)- thiophen-2-yl-methyl]-phenoxy}-ethyl)-amine)] with MIC: 1.33 mg/L, non-toxic against Vero C-1008 cell line with selectivity index >10, ex vivo efficacy equivalent to first line TB drugs-isoniazid (INH), rifampicin (RFM) and pyrazinamide (PZA) in the mouse and human macrophages, and lung CFU count of 2.2 × 10(7) (approximately 15 fold lesser than untreated mice, 31 × 10(7)) with efficacies comparable to ethambutol (EMB) (1.27 × 10(7)) and PZA (1.9 × 10(7)). Further, S006-830 also showed potent bactericidal activity against multi-drug resistant and single-drug resistant clinical isolates of M. tuberculosis.

Novel, potent, orally bioavailable and selective mycobacterial ATP synthase inhibitors that demonstrated activity against both replicating and non-replicating M. tuberculosis.

The mycobacterial F0F1-ATP synthase (ATPase) is a validated target for the development of tuberculosis (TB) therapeutics. Therefore, a series of eighteen novel compounds has been designed, synthesized and evaluated against Mycobacterium smegmatis ATPase. The observed ATPase inhibitory activities (IC50) of these compounds range between 0.36 and 5.45µM. The lead compound 9d [N-(7-chloro-2-methylquinolin-4-yl)-N-(3-((diethylamino)methyl)-4-hydroxyphenyl)-2,3-dichlorobenzenesulfonamide] with null cytotoxicity (CC50>300µg/mL) and excellent anti-mycobacterial activity and selectivity (mycobacterium ATPase IC50=0.51µM, mammalian ATPase IC50>100µM, and selectivity >200) exhibited a complete growth inhibition of replicating Mycobacterium tuberculosis H37Rv at 3.12µg/mL. In addition, it also exhibited bactericidal effect (approximately 2.4log10 reductions in CFU) in the hypoxic culture of non-replicating M. tuberculosis at 100µg/mL (32-fold of its MIC) as compared to positive control isoniazid [approximately 0.2log10 reduction in CFU at 5µg/mL (50-fold of its MIC)]. The pharmacokinetics of 9d after p.o. and IV administration in male Sprague-Dawley rats indicated its quick absorption, distribution and slow elimination. It exhibited a high volume of distribution (Vss, 0.41L/kg), moderate clearance (0.06L/h/kg), long half-life (4.2h) and low absolute bioavailability (1.72%). In the murine model system of chronic TB, 9d showed 2.12log10 reductions in CFU in both lung and spleen at 173µmol/kg dose as compared to the growth of untreated control group of Balb/C male mice infected with replicating M. tuberculosis H37Rv. The in vivo efficacy of 9d is at least double of the control drug ethambutol. These results suggest 9d as a promising candidate molecule for further preclinical evaluation against resistant TB strains.

Melatonin Reverses Fas, E2F-1 and Endoplasmic Reticulum Stress Mediated Apoptosis and Dysregulation of Autophagy Induced by the Herbicide Atrazine in Murine Splenocytes.

Exposure to the herbicide Atrazine (ATR) can cause immunotoxicity, apart from other adverse consequences for animal and human health. We aimed at elucidating the apoptotic mechanisms involved in immunotoxicity of ATR and their attenuation by Melatonin (MEL). Young Swiss mice were divided into control, ATR and MEL+ATR groups based on daily (x14) intraperitoneal administration of the vehicle (normal saline), ATR (100 mg/kg body weight) and MEL (20 mg/kg body weight) with ATR. Isolated splenocytes were processed for detection of apoptosis by Annexin V-FITC and TUNEL assays, and endoplasmic reticulum (ER) stress by immunostaining. Key proteins involved in apoptosis, ER stress and autophagy were quantified by immunoblotting. ATR treatment resulted in Fas-mediated activation of caspases 8 and 3 and inactivation of PARP1 which were inhibited significantly by co-treatment with MEL. MEL also attenuated the ATR-induced, p53 independent mitochondrial apoptosis through upregulation of E2F-1 and PUMA and suppression of their downstream target Bax. An excessive ER stress triggered by ATR through overexpression of ATF-6α, spliced XBP-1, CREB-2 and GADD153 signals was reversed by MEL. MEL also reversed the ATR-induced impairment of autophagy which was indicated by a decline in BECN-1, along with significant enhancement in LC3B-II and p62 expressions. Induction of mitochondrial apoptosis, ER stress and autophagy dysregulation provide a new insight into the mechanism of ATR immunotoxicity. The cytoprotective role of MEL, on the other hand, was defined by attenuation of ER stress, Fas-mediated and p53 independent mitochondria-mediated apoptosis as well as autophagy signals.