Contribution of Blastocystishominis subtypes and associated inflammatory factors in development of irritable bowel syndrome

Blastocystis hominis with worldwide distribution is a human intestinal protozoa found in all countries. There have been differences in the severity of the pathogenesis of various Blastocystis spp. and a concomitant variation in the plasma concentration of the cytokines in patients with irritable bowel syndrome. In the present study, we aimed to demonstrate the contribution of B. hominis subtypes in the development of irritable bowel syndrome. Stool samples were collected from patients with gastrointestinal disorders. All samples were evaluated through native-lugol method. Total DNA was extracted. A PCR protocol was developed to amplify a specific region of the SSU ribosomal DNA (rDNA) gene. Serum levels of IL-6 and TNF-alpha were determined by immunoassay methods. The ClustalW algorithm was applied to align and blast the nucleotide sequences of the amplified region of the SSU rDNA gene. To evaluate the phylogenetic and molecular evolutionary of the nucleotide sequences, we used the MEGA software. In this study, we found 26 haplotypes of B. hominis in the studied samples which were collectively belong to five subtypes (ST1, ST2 in patients without irritable bowel syndrome vs. ST3 and two unknown subtypes in patients with irritable bowel syndrome). Result of ELISA showed a high level of IL-6 and TNF-alpha in the serum of patients with irritable bowel syndrome. The genetic heterogeneity of B. hominis and the existence of different subtypes of the protozoan in patients with IBS may shed light to the fact that some subtypes of parasites may involve in the pathogenesis of IBS

Contribution of Blastocystishominis subtypes and associated inflammatory factors in development of irritable bowel syndrome

Blastocystis hominis with worldwide distribution is a human intestinal protozoa found in all countries. There have been differences in the severity of the pathogenesis of various Blastocystis spp. and a concomitant variation in the plasma concentration of the cytokines in patients with irritable bowel syndrome. In the present study, we aimed to demonstrate the contribution of B. hominis subtypes in the development of irritable bowel syndrome. Stool samples were collected from patients with gastrointestinal disorders. All samples were evaluated through native-lugol method. Total DNA was extracted. A PCR protocol was developed to amplify a specific region of the SSU ribosomal DNA (rDNA) gene. Serum levels of IL-6 and TNF-alpha were determined by immunoassay methods. The ClustalW algorithm was applied to align and blast the nucleotide sequences of the amplified region of the SSU rDNA gene. To evaluate the phylogenetic and molecular evolutionary of the nucleotide sequences, we used the MEGA software. In this study, we found 26 haplotypes of B. hominis in the studied samples which were collectively belong to five subtypes (ST1, ST2 in patients without irritable bowel syndrome vs. ST3 and two unknown subtypes in patients with irritable bowel syndrome). Result of ELISA showed a high level of IL-6 and TNF-alpha in the serum of patients with irritable bowel syndrome. The genetic heterogeneity of B. hominis and the existence of different subtypes of the protozoan in patients with IBS may shed light to the fact that some subtypes of parasites may involve in the pathogenesis of IBS

Selective Labeling and Identification of the Tumor Cell Proteome of Pancreatic Cancer In Vivo

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers. Dissecting the tumor cell proteome from that of the non-tumor cells in the PDAC tumor bulk is critical for tumorigenesis studies, biomarker discovery, and development of therapeutics. However, investigating the tumor cell proteome has proven evasive due to the tumor’s extremely complex cellular composition. To circumvent this technical barrier, we have combined bioorthogonal noncanonical amino acid tagging (BONCAT) and data-independent acquisition mass spectrometry (DIA-MS) in an orthotopic PDAC model to specifically identify the tumor cell proteome in vivo. Utilizing the tumor cell-specific expression of a mutant tRNA synthetase transgene, this approach provides tumor cells with the exclusive ability to incorporate an azide-bearing methionine analogue into newly synthesized proteins. The azide-tagged tumor cell proteome is subsequently enriched and purified via a bioorthogonal reaction and then identified and quantified using DIA-MS. Applying this workflow to the orthotopic PDAC model, we have identified thousands of proteins expressed by the tumor cells. Furthermore, by comparing the tumor cell and tumor bulk proteomes, we showed that the approach can distinctly differentiate proteins produced by tumor cells from those of non-tumor cells within the tumor microenvironment. Our study, for the first time, reveals the tumor cell proteome of PDAC under physiological conditions, providing broad applications for tumorigenesis, therapeutics, and biomarker studies in various human cancers

Sinteza, antitumorsko i citostatsko djelovanje derivata 6H-indolo[2,3-b]kinoksalina

Various 6-aralkyl-9-substituted-6H-indolo[2,3-b]quinoxalines were synthesized by reaction of 1,5-disubstituted 2,3-dioxo-2,3-dihydroindole with orthophenylene diamine. Appreciable anticancer activity of compounds 5b, 5d, 5g and 5l at various cell lines among 59 human tumor cell panels was observed. All the synthesized compounds were evaluated for cytostatic activity against human Molt 4/C8 and CEM T-lymphocytes as well as for murine L1210 leukemia cells. Compound 5h exhibited an IC50 of 71 μmol mL1 against Molt 4/C8 and 117 μmol mL1 against CEM compared to melphalan 3.2 μmol mL1 and 2.5 μmol mL1, respectively. The IC50 for compound 7i against L1210 was 7.2 μmol mL1 compared to melphalan 2.1 μmol mL1.Reakcijom 1,5-disupstituiranih 2,3-diokso-2,3-dihidroindola s ortofenilen diaminom sintetizirani su različiti 6-aralkil-9-supstituirani-6H-indolo[2,3-b]kinoksalini. Spojevi 5b, 5d, 5g i 5l pokazali su značajno antitumorsko djelovanje na 59 humanih tumorskih stanica. Svi sintetizirani spojevi ispitani su na citostatsko djelovanje na stanične linije Molt 4/C8 i CEM T-limfocite, te na murin L1210 stanice leukemije. IC50 za spoj 5h je 71 μmol mL1 na staničnu liniju Molt 4/C8 i 117 μmol mL1 na CEM, dok su vrijednosti za melfalan 3,2, odnosno 2,5 μmol mL1. IC50 spoja 7i na stanice L1210 je 7,2 μmol mL1, dok je za melfalan 2,1 μmol mL1

Human plasma and serum extracellular small RNA reference profiles and their clinical utility

Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell–specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell–specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies