Species discrimination from hair using ATR-FTIR spectroscopy: Application in wildlife forensics.

Hair is a commonly encountered trace evidence in wildlife crimes involving mammals and can be used for species identification which is essential for subsequent judicial proceedings. This proof of concept study aims, to distinguish the black guard hair of three wild cat species belonging to the genus Panthera i.e. Royal Bengal Tiger (Panthera tigris tigris), Indian Leopard (Panthera pardus fusca), and Snow Leopard (Panthera uncia) using a rapid and non-destructive ATR-FTIR spectroscopic technique in combination with chemometrics. A training dataset including 72 black guard hair samples of three species (24 samples from each species) was used to construct chemometric models. A PLS2-DA model successfully classified these three species into distinct classes with R-Square values of 0.9985 (calibration) and 0.8989 (validation). VIP score was also computed, and a new PLS2DA-V model was constructed using variables with a VIP score ≥ 1. External validation was performed using a validation dataset including 18 black guard hair samples (6 samples per species) to validate the constructed PLS2-DA model. It was observed that PLS2-DA model provides greater accuracy and precision compared to the PLS2DA-V model during cross-validation and external validation. The developed PLS2-DA model was also successful in differentiating human and non-human hair with R-Square values of 0.99 and 0.91 for calibration and validation, respectively. Apart from this, a blind test was also carried out using 10 unknown hair samples which were correctly classified into their respective classes providing 100 % accuracy. This study highlights the advantages of ATR-FTIR spectroscopy associated with PLS-DA for differentiation and identification of the Royal Bengal Tiger, Indian Leopard, and Snow Leopard hairs in a rapid, accurate, eco-friendly, and non-destructive way.

Surgical Tumor Resection Deregulates Hallmarks of Cancer in Resected Tissue and the Surrounding Microenvironment.

Surgery exposes tumor tissue to severe hypoxia and mechanical stress leading to rapid gene expression changes in the tumor and its microenvironment, which remain poorly characterized. We biopsied tumor and adjacent normal tissues from patients with breast (n = 81) and head/neck squamous cancers (HNSC; n = 10) at the beginning (A), during (B), and end of surgery (C). Tumor/normal RNA from 46/81 patients with breast cancer was subjected to mRNA-Seq using Illumina short-read technology, and from nine patients with HNSC to whole-transcriptome microarray with Illumina BeadArray. Pathways and genes involved in 7 of 10 known cancer hallmarks, namely, tumor-promoting inflammation (TNF-A, NFK-B, IL18 pathways), activation of invasion and migration (various extracellular matrix-related pathways, cell migration), sustained proliferative signaling (K-Ras Signaling), evasion of growth suppressors (P53 signaling, regulation of cell death), deregulating cellular energetics (response to lipid, secreted factors, and adipogenesis), inducing angiogenesis (hypoxia signaling, myogenesis), and avoiding immune destruction (CTLA4 and PDL1) were significantly deregulated during surgical resection (time points A vs. B vs. C). These findings were validated using NanoString assays in independent pre/intra/post-operative breast cancer samples from 48 patients. In a comparison of gene expression data from biopsy (analogous to time point A) with surgical resection samples (analogous to time point C) from The Cancer Genome Atlas study, the top deregulated genes were the same as identified in our analysis, in five of the seven studied cancer types. This study suggests that surgical extirpation deregulates the hallmarks of cancer in primary tumors and adjacent normal tissue across different cancers. IMPLICATIONS: Surgery deregulates hallmarks of cancer in human tissue.

Rapid and non-destructive approach for characterization and differentiation of sealing wax using ATR-FTIR spectroscopy.

Sealing wax is used for maintaining the integrity and authenticity of a document or physical evidence. Any tampering with the seal calls into question the overall integrity and authenticity of the tangible evidence or document. In these circumstances, determining the authenticity of the sealing material (physical and chemical) becomes imperative. In this study, ATR-FTIR spectroscopy supported by chemometrics has been used to differentiate sealing wax samples belonging to 12 different brands available across India. All the samples were first melted, cooled, and then analyzed using ATR-FTIR spectroscopy in the mid-infrared region (4000-600 cm-1 ). The obtained spectra were first examined visually for the presence of different functional groups. Principal component analysis (PCA) and principal component analysis-linear discriminant analysis (PCA-LDA) were employed to analyze the sample clustering patterns and to categorize them into their respective groups, respectively. For classification, a PCA-LDA training model was applied, and it demonstrated 95.83% accuracy. The validation test resulted in an accuracy of 83.33%. PCA-LDA model offered 100% accurate prediction for samples on various substrates, including cloth, cardboard, and paper. A blind study was also performed using five unknown samples, which were accurately classified into their respective groups. PCA-LDA model will be helpful in providing investigative leads by linking a questioned sealing wax sample with its respective group.

Aortic valve replacement reduces mortality in moderate aortic stenosis: a systematic review and meta-analysis.

BACKGROUND: With the introduction of transcatheter aortic valve replacement and an evolving understanding of the natural progression and history of aortic stenosis, the potential for earlier intervention in appropriate patients is promising; however, the benefit of aortic valve replacement in moderate aortic stenosis remains unclear. METHODS: Pubmed, Embase, and the Cochrane Library databases were searched up until 30th of December 2021 using keywords including moderate aortic stenosis and aortic valve replacement. Studies reporting all-cause mortality and outcomes in early aortic valve replacement (AVR) compared to conservative management in patients with moderate aortic stenosis were included. Hazard ratios were generated using random-effects meta-analysis to determine effect estimates. RESULTS: 3470 publications were screened with title and abstract review, which left 169 articles for full-text review. Of these studies, 7 met inclusion criteria and were included, totalling 4,827 patients. All studies treated AVR as a time-dependent co-variable in cox-regression multivariate analysis of all-cause mortality. Intervention with surgical or transcatheter AVR was associated with a 45% decreased risk of all-cause mortality (HR = 0.55 [0.42-0.68], I 2 = 51.5%, P < 0.001). All studies were representative of the overall cohort with appropriate sample sizes, with no evidence of publication, detection, or information biases in any of the studies. CONCLUSION: In this systematic review and meta-analysis, we report a 45% reduction in all-cause mortality in patients with moderate aortic stenosis who were treated with early aortic valve replacement compared to a strategy of conservative management. Randomised control trials are awaited to determine the utility of AVR in moderate aortic stenosis.

DPP inhibition alters the CXCR3 axis and enhances NK and CD8+ T cell infiltration to improve anti-PD1 efficacy in murine models of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer death in the USA by 2030. Immune checkpoint inhibitors fail to control most PDAC tumors because of PDAC's extensive immunosuppressive microenvironment and poor immune infiltration, a phenotype also seen in other non-inflamed (ie, 'cold') tumors. Identifying novel ways to enhance immunotherapy efficacy in PDAC is critical. Dipeptidyl peptidase (DPP) inhibition can enhance immunotherapy efficacy in other cancer types; however, the impact of DPP inhibition on PDAC tumors remains unexplored. METHODS: We examined the effects of an oral small molecule DPP inhibitor (BXCL701) on PDAC tumor growth using mT3-2D and Pan02 subcutaneous syngeneic murine models in C57BL/6 mice. We explored the effects of DPP inhibition on the tumor immune landscape using RNAseq, immunohistochemistry, cytokine evaluation and flow cytometry. We then tested if BXCL701 enhanced anti-programmed cell death protein 1 (anti-PD1) efficacy and performed immune cell depletion and rechallenged studies to explore the relevance of cytotoxic immune cells to combination treatment efficacy. RESULTS: In both murine models of PDAC, DPP inhibition enhanced NK and T cell immune infiltration and reduced tumor growth. DPP inhibition also enhanced the efficacy of anti-PD1. The efficacy of dual anti-PD1 and BXCL701 therapy was dependent on both CD8+ T cells and NK cells. Mice treated with this combination therapy developed antitumor immune memory that cleared some tumors after re-exposure. Lastly, we used The Cancer Genome Atlas (TCGA) to demonstrate that increased NK cell content, but not T cell content, in human PDAC tumors is correlated with longer overall survival. We propose that broad DPP inhibition enhances antitumor immune response via two mechanisms: (1) DPP4 inhibition increases tumor content of CXCL9/10, which recruits CXCR3+ NK and T cells, and (2) DPP8/9 inhibition activates the inflammasome, resulting in proinflammatory cytokine release and Th1 response, further enhancing the CXCL9/10-CXCR3 axis. CONCLUSIONS: These findings show that DPP inhibition with BXCL701 represents a pharmacologic strategy to increase the tumor microenvironment immune cell content to improve anti-PD1 efficacy in PDAC, suggesting BXCL701 can enhance immunotherapy efficacy in 'cold' tumor types. These findings also highlight the potential importance of NK cells along with T cells in regulating PDAC tumor growth.

Combination effects of sorafenib with PI3K inhibitors under hypoxia in colorectal cancer.

AIM: This study reports the influence of hypoxia on response of colorectal cancer cells to anticancer effects of sorafenib in combination with PI3K inhibitors GDC-0941 and BEZ-235. MATERIALS AND METHODS: All hypoxic exposures were carried out at 1% O2/5% CO2. Antiproliferation activity was evaluated by 48 hours propidium iodide and 14 days clonogenic assay. Protein levels were evaluated by fluorescence ELISA. Metabolites lactate and glucose were evaluated biochemically. RESULTS: In the 48-hour proliferation assay, sorafenib acted synergistically with GDC-0941 but not with BEZ-235. In long-term colony-forming assays, both GDC-0941 and BEZ-235 were shown to potentiate the antiproliferative activity of sorafenib. At the molecular level, the synergism is mediated through inhibition of pAKT, pS6, p4EBP1, pERK, cyclin D1, and Bcl-2. No change in hypoxia-inducible factor-1α (HIF-1α) levels was observed in cells treated with the combination of compounds under hypoxia. A significant reduction in glucose uptake and lactate release was observed in cells treated with the combination of compounds under normoxia and hypoxia. CONCLUSION: Combinations of sorafenib with PI3K inhibitors BEZ-235 and GDC-0941 are efficacious under hypoxia. Thus, these anticancer combinations have a potential to overcome the hypoxia-mediated resistance mechanisms to antiproliferative agents in cancer therapy.

Anticancer activity of Ophiobolin A, isolated from the endophytic fungus Bipolaris setariae.

The present work describes the anticancer activity of Ophiobolin A isolated from the endophytic fungus Bipolaris setariae. Ophiobolin A was isolated using preparative HPLC and its structure was confirmed by HRMS, (1)H NMR, (13)C NMR, COSY, DEPT, HSQC and HMBC. It inhibited solid and haematological cancer cell proliferation with IC50 of 0.4-4.3 µM. In comparison, IC50 against normal cells was 20.9 µM. It was found to inhibit the phosphorylation of S6 (IC50 = 1.9 ± 0.2 µM), ERK (IC50 = 0.28 ± 0.02 µM) and RB (IC50 = 1.42 ± 0.1 µM), the effector proteins of PI3K/mTOR, Ras/Raf/ERK and CDK/RB pathways, respectively. It induced apoptosis and inhibited cell cycle progression in MDA-MB-231 cancer cells with concomitant inhibition of signalling proteins. Thus, this study reveals that anticancer activity of Ophiobolin A is associated with simultaneous inhibition of multiple oncogenic signalling pathways namely PI3K/mTOR, Ras/Raf/ERK and CDK/RB.

P7170: A Novel Molecule with Unique Profile of mTORC1/C2 and Activin Receptor-like Kinase 1 Inhibition Leading to Antitumor and Antiangiogenic Activity.

The mTOR pathway is often upregulated in cancer and thus intensively pursued as a target to design novel anticancer therapies. Approved and emerging drugs targeting the mTOR pathway have positively affected the clinical landscape. Recently, activin receptor-like kinase 1 (ALK1), belonging to the TGFß receptor family, has been reported as an emerging target for antiangiogenic cancer therapy. Here, we describe a novel orally efficacious compound, P7170, that inhibits mTORC1/mTORC2/ALK1 activity with a potent cell growth inhibition. In cell-based assays, P7170 strongly inhibited (IC50 < 10 nmol/L) the phosphorylation of p70S6K (T389) and pAKT (S473). In many cancer cell lines, such as prostate, ovarian, colon, and renal, P7170 treatment resulted in marked cell growth inhibition. Furthermore, it induced G1-S cell-cycle arrest and autophagy. In vitro HUVEC tube formation, in vivo Matrigel plug, and rat aorta ring assays demonstrated that P7170 exhibited significant antiangiogenic activity. In addition, ALK1 knockdown studies in HUVEC confirmed that the antiangiogenic activity of P7170 was primarily due to ALK1 inhibition. Strong inhibition of ALK1 in addition to mTORC1/mTORC2 differentiates P7170 in its mechanism of action in comparison with existing inhibitors. In vivo mouse xenograft studies revealed P7170 to exhibit a significant dose-dependent tumor growth inhibition in a broad range of human tumor types when administered orally at 10 to 20 mg/kg doses. The distinctive pharmacological profile with favorable pharmacokinetic parameters and in vivo efficacy makes P7170 an attractive candidate for clinical development. It is currently being tested in phase I clinical studies.

P7170, a novel inhibitor of mTORC1/mTORC2 and Activin receptor-like Kinase 1 (ALK1) inhibits the growth of non small cell lung cancer.

BACKGROUND: Lung cancer is the major cause of cancer-related deaths and many cases of Non Small Cell Lung Cancer (NSCLC), a common type of lung cancer, have frequent genetic/oncogenic activation of EGFR, KRAS, PIK3CA, BRAF, and others that drive tumor growth. Some patients though initially respond, but later develop resistance to erlotinib/gefitinib with no option except for cytotoxic therapy. Therefore, development of novel targeted therapeutics is imperative to provide improved survival benefit for NSCLC patients. The mTOR cell survival pathway is activated in naïve, or in response to targeted therapies in NSCLC. METHODS: We have discovered P7170, a small molecule inhibitor of mTORC1/mTORC2/ALK1 and investigated its antitumor efficacy using various in vitro and in vivo models of human NSCLC. RESULTS: P7170 inhibited the phosphorylation of AKT, S6 and 4EBP1 (substrates for mTORC2 and mTORC1) levels by 80-100% and growth of NSCLC cells. P7170 inhibited anchorage-independent colony formation of NSCLC patient tumor-derived cells subsistent of disease sub-types. The compound also induced apoptosis in NSCLC cell lines. P7170 at a well-tolerated daily dose of 20 mg/kg significantly inhibited the growth of NSCLC xenografts independent of different mutations (EGFR, KRAS, or PIK3CA) or sensitivity to erlotinib. Pharmacokinetic-pharmacodynamic (PK-PD) analysis showed sub-micro molar tumor concentrations along with mTORC1/C2 inhibition. CONCLUSIONS: Our results provide evidence of antitumor activity of P7170 in the erlotinib -sensitive and -insensitive models of NSCLC.

Structure effect of carbon nanovectors in regulation of cellular responses.

Carbon nanostructures such as multiwalled carbon nanotubes (CNT) and graphene (G) are potential candidates in a large number of biomedical applications. However, there is limited understanding and connection between the physicochemical properties of diverse carbon nanostructures and biological systems, particularly with regard to cellular responses. It is also crucial to understand how the structure and surface composition of carbon nanostructures affect the cellular internalization process. Here, through in vitro cellular entry kinetics and cytotoxicity studies using MCF-7 breast cancer cells and H460 human lung cancer cells, we show that the structure and surface composition of CNT and G conjugates with various molecules such as PAMAM dendrimers (G4) and G4-poly(ethylene glycol) (PEG) are directly related to their cellular internalization ability and toxicity. Interestingly, the cellular association of CNT and G nanoconjugates was observed to be structure and surface composition dependent. We found that CNT conjugates internalized more compared to G conjugates. Furthermore, G4 conjugated CNT internalized more compared to G4-PEG conjugated CNT, whereas, higher internalization was found for G4-PEG conjugated G than G4 conjugated G. We have also correlated the cytotoxicity and cellular uptake mechanisms of CNT, G, and their conjugates through zeta potential measurements, fluorescence quenching studies and by fluorescence-activated cell sorting. Altogether these studies suggest different biological activities of the carbon nanostructures, with the shape and surface composition playing a primary role.

Discovery of P3971 an orally efficacious novel anticancer agent targeting HIF-1α and STAT3 pathways.

Hypoxia-inducible factor-1α (HIF-1α) and signal transducer and activator of transcription 3 (STAT3) are transcription factors and are activated in response to hypoxia. Both HIF-1α and STAT3 regulate various aspects of cancer biology such as cell survival, proliferation, angiogenesis etc. and are constitutively expressed in a wide range of human cancers. In the last decade, over expression of HIF-1α and STAT3 has been demonstrated in many common human cancers, thereby emerging as highly compelling anticancer targets for drug discovery. We herein report the design and synthesis of new imidazopyridine based potent dual inhibitors of HIF-1α and STAT3 pathways. The lead compound of this series P3971 has been identified as a potent inhibitor of HIF-1α (200 nM) and STAT3 (350 nM) with significant antiproliferative activity against various cancer cell lines. Moreover, P3971 was also found to be orally efficacious in HCT116 (colon cancer) and H460 (lung cancer) xenograft mice models.

Synthesis and therapeutic evaluation of pyridyl based novel mTOR inhibitors.

A series of novel cyanopyridyl based molecules (1-14) were designed, synthesized and probed for inhibition of mammalian target of rapamycin (mTOR) activity. Compound 14 was found to be a potent inhibitor of mTOR activity as assessed by enzyme-linked immunoassays and Western blot analysis. Most importantly, systemic application (intraperitoneal; ip) of compound 14 significantly suppressed macroscopic and histological abnormalities associated with chemically-induced murine colitis.

A novel mTOR inhibitor is efficacious in a murine model of colitis.

Ulcerative colitis is an autoimmune-inflammatory disease characterized by increased proliferation of colonic epithelial cells, dysregulation of signal transduction pathways, elevated mucosal T cell activation, increased production of proinflammatory cytokines, and enhanced leukocyte infiltration into colonic interstitium. Several compounds that possess antiproliferative properties and/or inhibit cytokine production exhibit a therapeutic effect in murine models of colitis. Mammalian target of rapamycin (mTOR), a protein kinase regulating cell proliferation, is implicated in colon carcinogenesis. In this study, we report that a novel haloacyl aminopyridine-based molecule (P2281) is a mTOR inhibitor and is efficacious in a murine model of human colitis. In vitro studies using Western blot analysis and cell-based ELISA assays showed that P2281 inhibits mTOR activity in colon cancer cells. In vitro and in vivo assays of proinflammatory cytokine production revealed that P2281 diminishes induced IFN-gamma production but not TNF-alpha production, indicating preferential inhibitory effects of P2281 on T cell function. In the dextran sulfate sodium (DSS) model of colitis, 1) macroscopic colon observations demonstrated that P2281 significantly inhibited DSS-induced weight loss, improved rectal bleeding index, decreased disease activity index, and reversed DSS-induced shortening of the colon; 2) histological analyses of colonic tissues revealed that P2281 distinctly attenuated DSS-induced edema, prominently diminished the leukocyte infiltration in the colonic mucosa, and resulted in protection against DSS-induced crypt damage; and 3) Western blot analysis showed that P2281 blocks DSS-induced activation of mTOR. Collectively, these results provide direct evidence that P2281, a novel mTOR inhibitor, suppresses DSS-induced colitis by inhibiting T cell function and is a potential therapeutic for colitis. Given that compounds with anticancer activity show promising anti-inflammatory efficacy, our findings reinforce the cross-therapeutic functionality of potential drugs.

Design and synthesis of novel furoquinoline based inhibitors of multiple targets in the PI3K/Akt-mTOR pathway.

We herein report the design and synthesis of furoquinoline based novel molecules (16-36) and their in vitro multiple targeted inhibitory potency against PI3K/Akt phosphorylation and mTOR using cell based and cell-free kinase assay. In particular, compound 23 in addition to PI3K-mTOR inhibitory potency, it has shown potent inhibition of hypoxia-induced accumulation of HIF-1alpha protein in U251-HRE cell line. The inhibitory activities of compound 23 were confirmed by Western blot analysis, using human non-small cell lung carcinoma H-460 cell line and glioblastoma U251 cell lines.

P276-00, a novel cyclin-dependent inhibitor induces G1-G2 arrest, shows antitumor activity on cisplatin-resistant cells and significant in vivo efficacy in tumor models.

P276-00, a flavone that inhibits cyclin-dependent kinases, has been identified by us recently as a novel antineoplastic agent. In this study, we have selected a panel of human tumor cell lines and xenografts to allow determination of selectivity and efficacy of P276-00. When tested against a panel of 16 cisplatin-sensitive and cisplatin-resistant cell lines, the antiproliferative potential of P276-00 was found to be approximately 30-fold higher than cisplatin. Studies to show tumor sensitivity using clonogenic assay in 22 human xenografts indicated that P276-00 was approximately 26-fold more potent than cisplatin, and further, it was also found to be active against cisplatin-resistant tumors of central nervous system, melanoma, prostate, and renal cancers. Further, we studied the effects of P276-00 on cell cycle progression by flow cytometry using asynchronous and synchronous population of tumor and normal cells. Asynchronous population of human prostate carcinoma (PC-3) and human promyelocytic leukemia (HL-60) cells when exposed to P276-00 showed arrest of slow-growing PC-3 cells in G(2)-M with no significant apoptosis observed up to 72 h. Unlike PC-3, significant apoptosis was seen in fast-growing HL-60 cells at 6 h. However, synchronized human non-small cell lung carcinoma (H-460) and human normal lung fibroblast (WI-38) cells showed arrest of cells in G(1). H-460 cells undergo apoptosis, which increases with longer exposure to the compound and also after exposure to P276-00 for 48 h followed by recovery. In contrast, the normal cells (WI-38) remain arrested in G(1) with no significant apoptosis up to 72 h of exposure and also after 48 h of P276-00 treatment followed by recovery, confirming our previous results that P276-00 was less effective against normal cells compared with cancer cells. After promising in vitro results, P276-00 was checked for in vivo efficacy in murine tumor and human xenograft models. Growth inhibition of murine colon cancer (CA-51) was significant when P276-00 was administered i.p. at 50 mg/kg daily for 20 treatments. However, in murine lung carcinoma model (Lewis lung), an increased dose of 60 mg/kg (30 mg/kg twice daily) administered every alternate day i.p. for seven treatments showed significant inhibition in the growth. Further studies were undertaken to establish the efficacy profile of P276-00 in human tumor xenograft models. In the two xenograft models studied, P276-00 showed potent in vivo antitumor potential. Compound P276-00 at a dose of 35 mg/kg administered daily via the i.p. route for 10 days showed significant (P < 0.05) inhibition in the growth of human colon carcinoma HCT-116 xenograft. Furthermore, P276-00 at a dose of 50 mg/kg once daily and 30 mg/kg twice daily administered via i.p. route for 20 treatments significantly (P < 0.05) inhibited growth of human non-small cell lung carcinoma H-460 xenograft. Thus, the in vitro cellular potency, together with in vivo antitumor activity, confirms the potential of P276-00, a cyclin-dependent kinase inhibitor as an anticancer molecule.