Cancer stem cells (CSCs) which are known to be residing deep inside the core of the tumor in its hypoxia niche is responsible for relapse of cancers. Owing to this hypoxic niche, the residing CSCs simultaneously fuel their stemness, cancerous and drug resistance properties. Attributes of CSCs are still not properly understood in its hypoxia niche. Addressing this, we sorted CSCs from Saos-2 (osteosarcoma) cell line using CD133 antibody. The CD133 CSCs exhibited quiescent cell proliferation in DNA doubling, Ca signaling and cell cycle analysis. CD133 CSCs exhibited increased production of ATP and lactate dehydrogenase (LDH) activity under hypoxia. CD133 cells exhibited decreased glucose uptake compared to ATP levels under hypoxia. Moreover, there was only negligible LDH activity in CD133 cells under normoxia which do not rely on Warburg effect. Stemness markers (such as c-Myc, SOX2, Oct4 and TERT), metastasis marker (CD44) and drug resistance marker (ABCG2) were highly expressed in CD133 cells. In summary, both CD133 cells of Saos-2 (osteosarcoma) cell line did not exhibit Warburg effect under normoxic condition. Moreover, this significantly indicates an uncoupling between stemness and Warburg effect in CD133. This work provides a novel insight into the metabolic and functional features of CSCs in a hypoxic environment which could open new avenues for therapeutic strategies aimed to target CSCs.
A new strategy for the preparation of 8-quinolyl ethers 3(a-g), 5(a-g), and 7(a-d) was studied by copper (II)-catalyzed methodology in the presence of Cs 2 CO 3 and acetone-water mixture (1:1). Screening of quinolinyl-8-ethers was investigated against anticancer expressive studies to validate new chemical entity in medicinal chemistry. Approaches were evaluated against breast cancer (MCF-7), skin cancer (G-361), and colon cancer (HCT 116) cell lines. Inhibitory potentials against phosphoinositide-3-kinase (PI3K) enzyme responsible for cancer development have been evaluated by competitive ELISA studies. In PI3K assay, 3a-c were inactive (IC 50 > 5 μM), while 3e-g, 5a, 5c-e, 5g, 7a, and 7d showed a moderate activity (IC 50 ≥ 0.05 μM). Compounds (5b, 5f, 7b, and 7c) showed significant activity (IC 50 < 1.0 μM); thus, their anticancer activities were carried out. Anticancer activity was found to be selective towards breast cancer (MCF-7); 5b, 5f, 7b, and 7c showed predominant relative percentage activities of 74.12%, 79.04%, 72.56%, and 78.47%, with IC 50 values of 5b (2.27 ± 0.88 μM), 5f (1.38 ± 0.60 μM), 7b (2.64 ± 0.86 μM), and 7c (1.87 ± 0.68 μM) compared with the standard doxorubicin 73.14% inhibition (IC 50 = 1.98 ± 0.75 μM). Docking study also conducted to find out the binding interactions with p110α (PDB ID: 3T8M) enzyme. Compounds 5b, 5f, 7b, and 7c showed best docking score into the active site of PI3K 12.59, 10.51, 56.52, and 8.61 nM. Structure-activity relationship studies demonstrated that the synthesized compounds are the potential PI3K inhibitors to treat various cancer-related diseases.
BackgroundCardiosphere derived cells (CDCs) represent a valuable source in stem cell based therapy for cardiovascular diseases, yet poor differentiation rate hinders the transplantation efficiency. The aim of this study is to check the ability of 5-Azacytidine (Aza) alone and in combination with ascorbic acid (Aza+AA) in delineating CDCs to cardiomyogenesis and the underlying Wnt signaling mechanism in induced differentiation.MethodsCDCs were treated with Aza and Aza+AA for a period of 14 days to examine the expression of cardiac specific markers and Wnt downstream regulators by immunofluorescence, real time PCR and western blot.ResultsResults revealed that Aza+AA induced efficient commitment of CDCs to cardiomyogenic lineage. Immunofluorescence analysis showed significant augment for Nkx 2.5, GATA 4 and α-Sarcomeric actinin markers in Aza+AA group than control group (p = 0.0118, p = 0.009 and p = 0.0091, respectively). Relative upregulation of cardiac markers, Nkx 2.5 (p = 0.0156), GATA 4 (p = 0.0087) and down regulation of Wnt markers, β-catenin (p = 0.0107) and Cyclin D1 (p = 0. 0116) in Aza+AA group was revealed by RNA expression analysis. Moreover, the Aza+AA induced prominent expression of GATA 4, α-Sarcomeric actinin and phospho β-catenin while non phospho β-catenin and Cyclin D1 expression was significantly suppressed as displayed in protein expression analysis. Generation of spontaneous beating in Aza+AA treated CDCs further reinforced that Aza+AA accelerates the cardiomyogenic potential of CDCs.ConclusionCombined treatment of Aza along with AA implicit in inducing cardiomyogenic potential of CDCs and is associated with down regulating Wnt signaling pathway. Altogether, CDCs represent a valuable tool for the treatment of cardiovascular disorders.
A bstract Background: Recent advances in nucleic acid amplification technique (NAAT)-based identification of pathogens in blood stream infections (BSI) have revolutionized molecular diagnostics in comparison to traditional clinical microbiology practice of blood culture. Rapid pathogen detection with point-of-care diagnostic-applicable platform is prerequisite for efficient patient management. The aim of the study is to evaluate an in-house developed, lyophilized OmiX-AMP pathogen test for the detection of top six BSI-causing bacteria along with two major antimicrobial resistance (AMR) markers of carbapenem and compare it to the traditional blood culture-based detection. Materials and methods: One hundred forty-three patients admitted to the Medical Intensive Care Unit, Narayana Hrudayalaya, Bangalore, with either suspected or proven sepsis, of either gender, of age ≥18 years were enrolled for the study. Pathogen DNA extracted from blood culture sample using OmiX pReP method was amplified at isothermal conditions and analyzed in real time using OmiX Analysis software. Results: Among the processed 143 samples, 54 were true negative, 83 were true positive, 3 were false negative, and 2 were false positive as analyzed by OmiX READ software. Gram-negative bacteria (91.3%) and gram-positive bacteria (75%) were detected with 100% specificity and 95.6% sensitivity along with the AMR marker pattern with a turnaround time of 4 hours from sample collection to results. Conclusion: OmiX-AMP pathogen test detected pathogens with 96.5% concordance in comparison to traditional blood culture. Henceforth, OmiX-AMP pathogen test could be used as a readily deployable diagnostic kit even in low-resource settings. How to cite this article: Maheshwarappa HM, Guru P, Mundre RS, Lawrence N, Majumder S, Sigamani A, et al . Validation of an Isothermal Amplification Platform for Microbial Identification and Antimicrobial Resistance Detection in Blood: A Prospective Study. Indian J Crit Care Med 2021;25(3):299–304.
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