Cobalt chloride, a chemical inducer of hypoxia-inducible factor-1α in U251 human glioblastoma cell line

AbstractTumor hypoxia has been described to increase the resistance of cancer cells to radiation therapy and chemotherapy. Hypoxia-inducible factor-1α (HIF-1α) is the main transcriptional factor activated by hypoxia and it plays a key role in reprogramming tumor growth. We examined in this study whether cobalt chloride induce HIF-1α in different concentrations. U251 human glioblastoma cell line was incubated at 16h under normoxia with or without CoCl2 at 1, 5, 10, 20, 25, 50, 100, 150 and 200μM treatments. In proliferation assay, CoCl2 have shown an increase in cellular induction between 50 and 200μM, proportionally. CoCl2 have also shown at 50μM the maximum induction effect. In addition, CoCl2 at 50μM displayed maximum response at 20,000, 30,000 and 40,000 U251 cells, respectively. In HIF-1 expression assay, CoCl2 increases HIF-1α gene expression between 50 and 200μM. Western analysis revealed sharp protein band at 118KDa which represented the HIF-1α protein with high band density at 50μM CoCl2. The present paper reports the adaptive response of human glioblastoma cells to CoCl2, a chemical hypoxia-mimicking agent. The effects of the treatment were evaluated on cell proliferation, and HIF-1α gene expression

Assessment of HIF-1α expression and release following endothelial injury in-vitro and in-vivo

Background: Endothelial injury is an early and enduring feature of cardiovascular disease. Inflammation and hypoxia may be responsible for this, and are often associated with the up-regulation of several transcriptional factors that include Hypoxia Inducible Factor-1 (HIF-1). Although it has been reported that HIF-1α is detectable in plasma, it is known to be unstable. Our aim was to optimize an assay for HIF-1α to be applied to in vitro and in vivo applications, and to use this assay to assess the release kinetics of HIF-1 following endothelial injury. Methods: An ELISA for the measurement of HIF in cell-culture medium and plasma was optimized, and the assay used to determine the best conditions for sample collection and storage. The results of the ELISA were validated using Western blotting and immunohistochemistry (IHC). In vitro, a standardized injury was produced in a monolayer of rat aortic endothelial cells (RAECs) and intracellular HIF-1α was measured at intervals over 24 hours. In vivo, a rat angioplasty model was used. The right carotid artery was injured using a 2F Fogarty balloon catheter. HIF-1α was measured in the plasma and in the arterial tissue (0, 1, 2, 3 and 5 days post injury). Results: The HIF-1α ELISA had a limit of detection of 2.7 pg/ mL and was linear up to 1000 pg/ mL. Between and within-assay coefficient of variation values were less than 15%. HIF-1α was unstable in cell lysates and plasma, and it was necessary to add a protease inhibitor immediately after collection, and to store samples at -800C prior to analysis. The dynamics of HIF-1α release were different for the in vitro and in vivo models. In vitro, HIF-1α reached maximum concentrations approximately 2h post injury, whereas peak values in plasma and tissues occurred approximately 2 days post injury, in the balloon injury model. Conclusion: HIF-1α can be measured in plasma, but this requires careful sample collection and storage. The carotid artery balloon injury model is associated with the transient release of HIF-1α into the circulation that probably reflects the hypoxia induced in the artery wall

Histological changes in placental Syncytiotrophoblasts of poorly controlled gestational diabetic patients

Corresponding Author: Dr. Majed S. Alokail Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451 Tel. ++9661-467-5943, Fax. ++9661-467-5931 Email: [email protected] seems reasonable to expect that biochemical changes occurring in the pregnant woman with diabetes should be reflected in the placenta structure. However, it has not been possible to correlate placental morphology with glycemic control in a comparison between those with long life diabetes and poorly controlled gestational diabetes. In the present study we have histologically studied the syncytiotrophoblast of human placentae from overt diabetic and poorly controlled gestational diabetic patients. Using specific staining techniques and direct light microscopy we qualitatively studied these placentae and compared them with the normal placentae. We found fibrin thrombi, villous oedema, hyperplasia and thickening of basement membrane in the placentae of poorly controlled gestational diabetic mothers. Direct microscopy revealed that these various changes in syncytiotrophoblast structure were marked in the poorly controlled gestational placenta compared with overt diabetics, and could have been due to the presence of histochemical compounds e.g. general carbohydrates and lipids. These studies may indicate that poor control of diabetes during the gestation as indicated by high level HbAlc may result in the accummulation of carbohydrate compounds and fat droplets in the placental basement membrane, leading to structural changes in the placental cells

Structural and Tribological Characterization of Carbon and Glass Fabrics Reinforced Epoxy for Bushing Applications Safety

This article investigates the effect of geometrical alternatives for fiber directions on the structural and tribological properties of glass and carbon fibers when molded with epoxy as polymeric composite fabrics for the safety and quality of bushing applications. To confirm the best composite fabric direction, scanning electron microscope and tribological analyses were carried out for the glass and carbon fabrics at horizontal and vertical geometrical alternative orientations. The tribological test was applied using a pin-on-disk tribometer at constant bark velocity of 0.520 m/s against different loads, beginning with 5, 10, 15, and 20 N for the investigated composite samples. The structural measurements demonstrated that the carbon fiber had a high ability to merge with the resin epoxy when compared with the glass fiber. The tribological analysis elucidated that the lower wear volume loss and friction coefficient were obtained when molding the resin epoxy horizontally to the fiber-stacking direction compared with the other vertical direction. Accordingly, the study deduced that the carbon fiber composite material achieves superior wear resistance when molded by resin epoxy horizontally to the direction of tribological wear, which is suitable for several advanced bushing applications

Single nucleotide polymorphisms in CXCR1 gene and its association with hepatitis B infected patients in Saudi Arabia

Background/Aim. This study aims to investigate whether the SNPs of CXCR1 gene, could predict the likelihood of viral persistence and/or disease progression.Material and methods. We investigated the association of two different SNPs (rs2234671, and rs142978743) in 598 normal healthy controls and 662 HBV patients from a Saudi ethnic population. The HBV patients were categorized into inactive carriers (n = 428), active carriers (n = 162), cirrhosis (n = 54) and Cirrhosis-HCC (n = 18) sub-groups. Genetic variants in CXCR1 were determined by polymerase chain reaction (PCR)-based DNA direct sequencing.Results. The frequency of the risk allele ‘C’ for the SNP, rs2234671 was found to be insignificant when the patient group was compared to the uninfected control group, however, a significant distribution of the allele ‘C’ of rs2234671 was observed among active HBV carriers + cirrhosis + cirrhosis - HCC vs. inactive HBV carriers with an OR = 1.631 (95% C.I. 1.016-2.616) and p = 0.032. However, no significant association was observed for rs142978743 when the distribution of risk allele was analyzed among the different patient groups (i.e. inactive carriers, active carriers, cirrhosis and HCC). Furthermore, the most common haplotype, Haplo-1 (AG), was found to have an insignificant frequency distribution between HBV cases and controls, while the same haplotype was found to be significantly distributed when active carriers + cirrhosis + cirrhosis - HCC patients were compared to inactive HBV carriers with a frequency of 0.938 and p = 0.0315. Haplo-2 (AC) was also found to be significantly associated with a frequency of 0.058 and p = 0.0163.Conclusion. The CXCR1 polymorphism, rs2234671 was found to be associated with chronic HBV infection and may play a role in disease activity

Enterobacter cloacae from urinary tract infections: frequency, protein analysis, and antimicrobial resistance

Abstract The genus Enterobacter belongs to the ESKAPE group, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. This group is characterized by the development of resistance to various antibiotics. In recent years, Enterobacter cloacae (E. cloacae) has emerged as a clinically important pathogen responsible for a wide range of healthcare-associated illnesses. Identifying Enterobacter species can be challenging due to their similar phenotypic characteristics. The emergence of multidrug-resistant E. cloacae is also a significant problem in healthcare settings. Therefore, our study aimed to identify and differentiate E. cloacae using Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) as a fast and precise proteomic analytical technique. We also tested hospital-acquired E. cloacae isolates that produce Extended-spectrum beta-lactamases (ESBL) against commonly used antibiotics for treating urinary tract infections (UTIs). We used a total of 189 E. cloacae isolates from 2300 urine samples of patients with UTIs in our investigation. We employed culturing techniques, as well as the BD Phoenix™ automated identification system (Becton, Dickinson) and Analytical Profile Index (API) system for the biochemical identification of E. cloacae isolates. We used the MALDI Biotyper (MBT) device for peptide mass fingerprinting analysis of all isolates. We utilized the single peak intensities and Principal Component Analysis (PCA) created by MBT Compass software to discriminate and cluster the E. cloacae isolates. Additionally, we evaluated the sensitivity and resistance of ESBL-E. cloacae isolates using the Kirby Bauer method. Out of the 189 E. cloacae isolates, the BD Phoenix system correctly identified 180 (95.24%) isolates, while the API system correctly identified 165 (87.30%) isolates. However, the MBT accurately identified 185 (98.95%) isolates with a score of 2.00 or higher. PCA positively discriminated the identified E. cloacae isolates into one group, and prominent peaks were noticed between 4230 mass-to-charge ratio (m/z) and 8500 m/z. The ESBL-E. cloacae isolates exhibited a higher degree of resistance to ampicillin, amoxicillin-clavulanate, cephalothin, cefuroxime, and cefoxitin. Several isolates were susceptible to carbapenems (meropenem, imipenem, and ertapenem); however, potential future resistance against carbapenems should be taken into consideration. In conclusion, MALDI-TOF MS is a powerful and precise technology that can be routinely used to recognize and differentiate various pathogens in clinical samples. Additionally, the growing antimicrobial resistance of this bacterium may pose a significant risk to human health