Colorimetric gold nanoparticles-based assay for direct detection of Clostridium difficile in clinical isolates from Qatar

Clostridium difficile infection (CDI) is a significant health problem worldwide. Control and prevention strategies of C. difficile horizontal transmission require assays with fast detection with high specificity and sensitivity. Conventional diagnostic methods are time consuming and costly for clinical field settings. This study aims to develop gold nanoparticles (AuNPs)-based assay for direct qualitative detection of the nucleic acid of C. difficile and its toxins. A colloidal solution of AuNPs with a diameter of 13±1 nm was prepared and characterized. The qualitative colorimetric AuNPs assay was developed for restricted genomic C. difficile DNA detection, and results were confirmed by PCR. One hundred and five positive C. difficile isolates were collected from patients with diarrheal diseases and tested using AuNPs based-assay. Ninety-six samples (91.4%) were detected positive using AuNPs based assay, as indicated by the color change from red to blue within 1 min. All ninety-six positive samples were positive for toxin B. In conclusion, nano-gold assay prototype was developed for direct and inexpensive detection of C. difficile. The developed prototypes are simple, sensitive, rapid and can substitute PCR-based detection. The developed assay may show potential in the clinical diagnosis of C. difficile, especially in developing countries as it is less costly as compared to the commercially available assays.NPRP award (NPRP 4-1215-3-317) from the Qatar National Research Fun

R270C polymorphism leads to loss of function of the canine P2X7 receptor

The relative function of the P2X7 receptor, an ATP-gated ion channel, varies between humans due to polymorphisms in the P2RX7 gene. This study aimed to assess the functional impact of P2X7 variation in a random sample of the canine population. Blood and genomic DNA were obtained from 69 dogs selected as representatives of a cross section of different breeds. P2X7 function was determined by flow cytometric measurements of dye uptake and patch-clamp measurements of inward currents. P2X7 expression was determined by immunoblotting and immunocytochemistry. Sequencing was used to identify P2RX7 gene polymorphisms. P2X7 was cloned from an English springer spaniel, and point mutations were introduced into this receptor by site-directed mutagenesis. The relative function of P2X7 on monocytes varied between individual dogs. The canine P2RX7 gene encoded four missense polymorphisms: F103L and P452S, found in heterozygous and homozygous dosage, and R270C and R365Q, found only in heterozygous dosage. Moreover, R270C and R365Q were associated with the cocker spaniel and Labrador retriever, respectively. F103L, R270C, and R365Q but not P452S corresponded to decreased P2X7 function in monocytes but did not explain the majority of differences in P2X7 function between dogs, indicating that other factors contribute to this variability. Heterologous expression of site-directed mutants of P2X7 in human embryonic kidney-293 cells indicated that the R270C mutant was nonfunctional, the F103L and R365Q mutants had partly reduced function, and the P452S mutant functioned normally. Taken together, these data highlight that a R270C polymorphism has major functional impact on canine P2X7

Inducing breast cancer cell death: The impact of taxodone on proliferation through apoptosis

Breast cancer is the most prevalent form of cancer in women and a major contributor to cancer-related fatalities worldwide. Several factors play a role in the development of breast cancer, encompassing age, hormone levels, etc. Taxodone has shown significant anti-tumor properties in both laboratory experiments and living organisms. However, its impact on the human MCF-7 breast cancer cell line has not been researched. This investigation explores the chemo-preventive potential of taxodone in the MCF-7 breast cancer cells. The anticancer potential of taxodone against MCF-7 cells was determined by MTT assay. Further, the induction of apoptosis in MCF-7 breast cancer cells was confirmed via ELISA, which indicated the increased incidences of chromatin condensation and ssDNA breakage in the MCF-7 apoptotic cells upon 24 h of taxodone treatment. The intracellular reactive oxygen species (ROS) level was evaluated using H2DCFDA fluorescent dye to elucidate the mechanism of action triggered upon taxodone treatment. The increasing intercellular ROS level sequentially activated the caspase-mediated apoptosis pathway. Consequently, the outcomes revealed that taxodone decreased the cell viability of MCF-7 dose-dependently. Taxodone triggers apoptosis in MCF-7 cells by increasing intracellular ROS levels and activating the caspase cascade through the mitochondrial apoptosis-induced channel, an early marker of apoptosis onset. Our results indicate that taxodone exhibits anti-proliferative and apoptotic properties against human MCF-7 breast cancer cells, suggesting it to be a natural anticancer agent

Exosomal miRNAs as a Promising Source of Biomarkers in Colorectal Cancer Progression

Colorectal cancer (CRC) is the third most common type of cancer worldwide amongst males and females. CRC treatment is multidisciplinary, often including surgery, chemotherapy, and radiotherapy. Early diagnosis of CRC can lead to treatment initiation at an earlier stage. Blood biomarkers are currently used to detect CRC, but because of their low sensitivity and specificity, they are considered inadequate diagnostic tools and are used mainly for following up patients for recurrence. It is necessary to detect novel, noninvasive, specific, and sensitive biomarkers for the screening and diagnosis of CRC at earlier stages. The tumor microenvironment (TME) has an essential role in tumorigenesis; for example, extracellular vesicles (EVs) such as exosomes can play a crucial role in communication between cancer cells and different components of TME, thereby inducing tumor progression. The importance of miRNAs that are sorted into exosomes has recently attracted scientists’ attention. Some unique sequences of miRNAs are favorably packaged into exosomes, and it has been illustrated that particular miRNAs can be directed into exosomes by special mechanisms that occur inside the cells. This review illustrates and discusses the sorted and transported exosomal miRNAs in the CRC microenvironment and their impact on CRC progression as well as their potential use as biomarkers

Molecular detection of exosomal miRNAs of blood serum for prognosis of colorectal cancer

Abstract Colorectal cancer (CRC) is the third most common cancer affecting people. The discovery of new, non-invasive, specific, and sensitive molecular biomarkers for CRC may assist in the diagnosis and support therapeutic decision making. Exosomal miRNAs have been demonstrated in carcinogenesis and CRC development, which makes these miRNAs strong biomarkers for CRC. Deep sequencing allows a robust high-throughput informatics investigation of the types and abundance of exosomal miRNAs. Thus, exosomal miRNAs can be efficiently examined as diagnostic biomarkers for disease screening. In the present study, a number of 660 mature miRNAs were detected in patients diagnosed with CRC at different stages. Of which, 29 miRNAs were differentially expressed in CRC patients compared with healthy controls. Twenty-nine miRNAs with high abundance levels were further selected for subsequent analysis. These miRNAs were either highly up-regulated (e.g., let-7a-5p, let-7c-5p, let-7f-5p, let-7d-3p, miR-423-5p, miR-3184-5p, and miR-584) or down-regulated (e.g., miR-30a-5p, miR-99-5p, miR-150-5p, miR-26-5p and miR-204-5p). These miRNAs influence critical genes in CRC, leading to either tumor growth or suppression. Most of the reported diagnostic exosomal miRNAs were shown to be circulating in blood serum. The latter is a novel miRNA that was found in exosomal profile of blood serum. Some of the predicted target genes of highly expressed miRNAs participate in several cancer pathways, including CRC pathway. These target genes include tumor suppressor genes, oncogenes and DNA repair genes. Main focus was given to multiple critical signaling cross-talking pathways including transforming growth factor β (TGFβ) signaling pathways that are directly linked to CRC. In conclusion, we recommend further analysis in order to experimentally confirm exact relationships between selected differentially expressed miRNAs and their predicted target genes and downstream functional consequences