Biomarkers for Clinical and Incipient Tuberculosis: Performance in a TB-Endemic Country

Simple biomarkers are required to identify TB in both HIV(-)TB(+) and HIV(+)TB(+) patients. Earlier studies have identified the M. tuberculosis Malate Synthase (MS) and MPT51 as immunodominant antigens in TB patients. One goal of these investigations was to evaluate the sensitivity and specificity of anti-MS and -MPT51 antibodies as biomarkers for TB in HIV(-)TB(+) and HIV(+)TB(+) patients from a TB-endemic setting. Earlier studies also demonstrated the presence of these biomarkers during incipient subclinical TB. If these biomarkers correlate with incipient TB, their prevalence should be higher in asymptomatic HIV(+) subjects who are at a high-risk for TB. The second goal was to compare the prevalence of these biomarkers in asymptomatic, CD4(+) T cell-matched HIV(+)TB(-) subjects from India who are at high-risk for TB with similar subjects from US who are at low-risk for TB.Anti-MS and -MPT51 antibodies were assessed in sera from 480 subjects including PPD(+) or PPD(-) healthy subjects, healthy community members, and HIV(-)TB(+) and HIV(+)TB(+) patients from India. Results demonstrate high sensitivity (approximately 80%) of detection of smear-positive HIV(-)TB(+) and HIV(+)TB(+) patients, and high specificity (>97%) with PPD(+) subjects and endemic controls. While approximately 45% of the asymptomatic HIV(+)TB(-) patients at high-risk for TB tested biomarker-positive, >97% of the HIV(+)TB(-) subjects at low risk for TB tested negative. Although the current studies are hampered by lack of knowledge of the outcome, these results provide strong support for the potential of these biomarkers to detect incipient, subclinical TB in HIV(+) subjects.These biomarkers provide high sensitivity and specificity for TB diagnosis in a TB endemic setting. Their performance is not compromised by concurrent HIV infection, site of TB and absence of pulmonary manifestations in HIV(+)TB(+) patients. Results also demonstrate the potential of these biomarkers for identifying incipient subclinical TB in HIV(+)TB(-) subjects at high-risk for TB

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Identification of the adenine binding site in the ricin toxin A-chain by fluorescence, CD, and electron spin resonance spectroscopy

CD, electron spin resonance, and fluorescence spectroscopy have been utilized to study the adenine binding site of ricin and its toxic A-subunit. At acidic (4.5) and physiological (7.3) pH, adenine or a spin-labeled analogue of adenine, N6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl) adenine, alters the near uv CD spectra of the ricin A-chain as well as intact ricin, whereas the far uv CD spectra of all proteins remain unchanged. Electron spin resonance data show that the adenine spin-labeled analogue interacts strongly with the A-chain both at pH 4.5 and 7.3, but no or very weak binding is observed for the intact ricin or the isolated B-chain. The adenine spin label gets highly immobilized (2AII = 65.5G) by the A-chain. The apparent dissociation constant Kd for the toxic A-chain ligand complex is 1.55 × 10-4 M and 5.6 × 10-5 M at pH 7.3 and 4.5, respectively. Fluorescence intensity of ricin A-chain bound 1,8-anilinonaphthalenesulfonic acid (ANS) decreases by 55% at pH 4.5 with the addition of the spin-labeled analogue of adenine, implying that both the ANS and adenine spin label (ADSL) bind to the hydrophobic domain of the A-chain. Fluorescence of the only intrinsic tryptophan probe of the A-chain is also efficiently quenched by ADSL, indicating that the tryptophan residue and the hydrophobic adenine binding site are closely located. All spectroscopic measurements indicate that adenine or its spin-labeled analogue has a single binding site adjacent to the TRP211 residue in the A-chain. Expansion of the A-chain globule and subsequent exposure of the hydrophobic binding site seem to be responsible for the increased binding of adenine at pH 4.5

Ricin-membrane interaction: membrane penetration depth by fluorescence quenching and resonance energy transfer

The entry of the plant toxin ricin and its A- and B-subunits in model membranes in the presence as well as absence of monosialoganglioside (GM(1)) has been studied. Dioleoylphosphatidylcholine and 5-, 10-, and 12-doxyl- or 9,10-dibromophosphatidylcholines serve as quenchers of intrinsic tryptophan fluorescence of the proteins. The parallax method of Chattopadhyay and London [(1987) Biochemistry 26, 39-45] has been employed to measure the average membrane penetration depth of tryptophans of ricin and its B-chain and the actual depth of the sole Trp 211 in the A-chain. The results indicate that both of the chains as well as intact ricin penetrate the membrane deeply and the C-terminal end of the A-chain is well inside the bilayer, especially at pH 4.5. An extrinsic probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl) ethylenediamine (I-AEDANS) has been attached to Cys 259 of the A-chain, and the kinetics of penetration has been followed by monitoring the increase in AEDANS fluorescence at 480 nm. The insertion follows first-order kinetics, and the rate constant is higher at a lower pH. The energy transfer distance analysis between Trp 211 and AEDANS points out that the conformation of the A-chain changes as it inserts into the membrane. CD studies indicate that the helicity of the proteins increases after penetration, which implies that some of the unordered structure in the native protein is converted to the ordered form during this process. Hydrophobic forces seem to be responsible for stabilizing a particular protein conformation inside the membrane

Comparison of triamcinolone and mitomycin C nasal pack in functional endoscopic sinus surgery: a randomized, clinical trial

Abstract Aims and Objectives Synechia formation in a postoperative nasal cavity is a major factor for suboptimal clinical outcome in functional endoscopic sinus surgery (FESS). Nasal packs medicated with steroids like triamcinolone and topical agents like mitomycin C have been shown to reduce the formation of synechiae in postoperative patients. This study attempts to compare the efficacy of mitomycin C and triamcinoloneimpregnated nasal pack in the prevention of crusting and synechiae formation in patients undergoing FESS. Methods Ninety-eight consenting patients with chronic rhinosinusitis were randomized into two groups of 49 each to receive either triamcinolone-impregnated or mitomycin C-impregnated nasal pack after FESS. Blinded, postoperative endoscopic examination was done at the first and third week and at first and third month and scoring was done according to the Lund Kennedy scoring system. The primary variable for assessing the outcome was synechiae formation in the postoperative period, while reduction of crusting, discharge, mucosal edema, polypoidal mucosal changes, and maintenance of ostial patency were secondary variables. Results Significant reduction in synechiae formation and crusting at 3 months duration was found in the group receiving triamcinolone nasal pack as compared with mitomycin C nasal pack. Mitomycin C was found to significantly reduce crusting in the first postoperative week. Positive trends in reduction of discharge, mucosal edema, polypoidal changes, and in maintenance of patency of ostia were observed in both the groups, but there was no significant difference between the two groups. Conclusion These results suggest that triamcinolone has a long-term effect in the reduction of synechia formation and crusting in post-FESS patients as compared with mitomycin C, though similar effects were seen in both groups in the immediate postoperative period

Identification of the adenine binding site in the ricin toxin A-chain by fluorescence, CD, and electron spin resonance spectroscopy

CD, electron spin resonance, and fluorescence spectroscopy have been utilized to study the adenine binding site of ricin and its toxic A-subunit. At acidic (4.5) and physiological (7.3) pH, adenine or a spin-labeled analogue of adenine, N<SUP>6</SUP>-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl) adenine, alters the near uv CD spectra of the ricin A-chain as well as intact ricin, whereas the far uv CD spectra of all proteins remain unchanged. Electron spin resonance data show that the adenine spin-labeled analogue interacts strongly with the A-chain both at pH 4.5 and 7.3, but no or very weak binding is observed for the intact ricin or the isolated B-chain. The adenine spin label gets highly immobilized (2A<SUB>II</SUB> = 65.5G) by the A-chain. The apparent dissociation constant K<SUB>d</SUB> for the toxic A-chain ligand complex is 1.55 × 10<SUP>-4</SUP>M and 5.6 × 10<SUP>-5</SUP>M at pH 7.3 and 4.5, respectively. Fluorescence intensity of ricin A-chain bound 1,8-anilinonaphthalenesulfonic acid (ANS) decreases by ~55% at pH 4.5 with the addition of the spin-labeled analogue of adenine, implying that both the ANS and adenine spin label (ADSL) bind to the hydrophobic domain of the A-chain. Fluorescence of the only intrinsic tryptophan probe of the A-chain is also efficiently quenched by ADSL, indicating that the tryptophan residue and the hydrophobic adenine binding site are closely located. All spectroscopic measurements indicate that adenine or its spin-labeled analogue has a single binding site adjacent to the TRP211 residue in the A-chain. Expansion of the A-chain globule and subsequent exposure of the hydrophobic binding site seem to be responsible for the increased binding of adenine at pH 4.5

Repurposing of Antifungal Drug Flucytosine/Flucytosine Cocrystals for Anticancer Activity against Prostate Cancer Targeting Apoptosis and Inflammatory Signaling Pathways

This study aimed to repurpose the antifungal drug flucytosine (FCN) for anticancer activity together with cocrystals of nutraceutical coformers sinapic acid (SNP) and syringic acid (SYA). The cocrystal screening experiments with SNP resulted in three cocrystal hydrate forms in which two are polymorphs, namely, FCN-SNP F–I and FCN-SNP F–II, and the third one with different stoichiometry in the asymmetric unit (1:2:1 ratio of FCN:SNP:H2O, FCN-SNP F–III). Cocrystallization with SYA resulted in two hydrated cocrystal polymorphs, namely, FCN-SYA F–I and FCN-SYA F–II. All the cocrystal polymorphs were obtained concomitantly during the slow evaporation method, and one of the polymorphs of each system was produced in bulk by the slurry method. The interaction energy and lattice energies of all cocrystal polymorphs were established using solid-state DFT calculations, and the outcomes correlated with the experimental results. Further, the in vitro cytotoxic activity of the cocrystals was determined against DU145 prostate cancer and the results showed that the FCN-based cocrystals (FCN-SNP F–III and FCN-SYA F–I) have excellent growth inhibitory activity at lower concentrations compared with parent FCN molecules. The prepared cocrystals induce apoptosis by generating oxidative stress and causing nuclear damage in prostate cancer cells. The Western blot analysis also depicted that the cocrystals downregulate the inflammatory markers such as NLRP3 and caspase-1 and upregulate the intrinsic apoptosis signaling pathway marker proteins, such as Bax, p53, and caspase-3. These findings suggest that the antifungal drug FCN can be repurposed for anticancer activity