Bactericidal effect of water-washing methods on Vibrio vulnificus contaminated in a raw fish Konosirus punctatus: water type, temperature, and pH.

This study aimed to evaluate a method for effectively reducing Vibrio vulnificus contamination in fish based on the type of washing water and method. Texture profiles and sensory evaluations were performed to determine the effect of the developed method on the quality and preference of the samples. The selected fish sample was Konosirus punctatus, which is mainly consumed in Asian countries. Various factors that could affect the survival rate of V. vulnificus were reviewed, including water type, temperature, exposure time, organic acids, pH, and washing methods. As a result, immersion and washing with filtered water with pH adjusted to 4.0 using acetic acid showed a high bactericidal effect of 2.5 log MPN/100 g. Furthermore, this method showed no statistically significant effect on the texture and sensory characteristics of fish. The results of the present study suggest a simple and effective method for preventing V. vulnificus infection in raw fish.

A review on gold nanoparticles as an innovative therapeutic cue in bone tissue engineering: Prospects and future clinical applications.

Bone damage is a complex orthopedic problem primarily caused by trauma, cancer, or bacterial infection of bone tissue. Clinical care management for bone damage remains a significant clinical challenge and there is a growing need for more advanced bone therapy options. Nanotechnology has been widely explored in the field of orthopedic therapy for the treatment of a severe bone disease. Among nanomaterials, gold nanoparticles (GNPs) along with other biomaterials are emerging as a new paradigm for treatment with excellent potential for bone tissue engineering and regenerative medicine applications. In recent years, a great deal of research has focused on demonstrating the potential for GNPs to provide for enhancement of osteogenesis, reduction of osteoclastogenesis/osteomyelitis, and treatment of bone cancer. This review details the latest understandings in regards to GNPs based therapeutic systems, mechanisms, and the applications of GNPs against various bone disorders. The present review aims to summarize i) the mechanisms of GNPs in bone tissue remodeling, ii) preparation methods of GNPs, and iii) functionalization of GNPs and its decoration on biomaterials as a delivery vehicle in a specific bone tissue engineering for future clinical application.

Exercise mitigates flow recirculation and activates metabolic transducer SCD1 to catalyze vascular protective metabolites.

Exercise promotes pulsatile shear stress in the arterial circulation and ameliorates cardiometabolic diseases. However, exercise-mediated metabolic transducers for vascular protection remain under-investigated. Untargeted metabolomic analysis demonstrated that wild-type mice undergoing voluntary wheel running exercise expressed increased endothelial stearoyl-CoA desaturase 1 (SCD1) that catalyzes anti-inflammatory lipid metabolites, namely, oleic (OA) and palmitoleic acids (PA), to mitigate NF-κB-mediated inflammatory responses. In silico analysis revealed that exercise augmented time-averaged wall shear stress but mitigated flow recirculation and oscillatory shear index in the lesser curvature of the mouse aortic arch. Following exercise, endothelial Scd1-deleted mice (Ldlr-/- Scd1EC-/-) on high-fat diet developed persistent VCAM1-positive endothelium in the lesser curvature and the descending aorta, whereas SCD1 overexpression via adenovirus transfection mitigated endoplasmic reticulum stress and inflammatory biomarkers. Single-cell transcriptomics of the aorta identified Scd1-positive and Vcam1-negative endothelial subclusters interacting with other candidate genes. Thus, exercise mitigates flow recirculation and activates endothelial SCD1 to catalyze OA and PA for vascular endothelial protection.

Subchronic inhalation exposure to ultrafine particulate matter alters the intestinal microbiome in various mouse models.

Exposure to ultrafine particles (UFPs) has been associated with multiple adverse health effects. Inhaled UFPs could reach the gastrointestinal tract and influence the composition of the gut microbiome. We have previously shown that oral ingestion of UFPs alters the gut microbiome and promotes intestinal inflammation in hyperlipidemic Ldlr-/- mice. Particulate matter (PM)2.5 inhalation studies have also demonstrated microbiome shifts in normolipidemic C57BL/6 mice. However, it is not known whether changes in microbiome precede or follow inflammatory effects in the intestinal mucosa. We hypothesized that inhaled UFPs modulate the gut microbiome prior to the development of intestinal inflammation. We studied the effects of UFP inhalation on the gut microbiome and intestinal mucosa in two hyperlipidemic mouse models (ApoE-/- mice and Ldlr-/- mice) and normolipidemic C57BL/6 mice. Mice were exposed to PM in the ultrafine-size range by inhalation for 6 h a day, 3 times a week for 10 weeks at a concentration of 300-350 µg/m3.16S rRNA gene sequencing was performed to characterize sequential changes in the fecal microbiome during exposures, and changes in the intestinal microbiome at the end. PM exposure led to progressive differentiation of the microbiota over time, associated with increased fecal microbial richness and evenness, altered microbial composition, and differentially abundant microbes by week 10 depending on the mouse model. Cross-sectional analysis of the small intestinal microbiome at week 10 showed significant changes in α-diversity, ß-diversity, and abundances of individual microbial taxa in the two hyperlipidemic models. These alterations of the intestinal microbiome were not accompanied, and therefore could not be caused, by increased intestinal inflammation as determined by histological analysis of small and large intestine, cytokine gene expression, and levels of fecal lipocalin. In conclusion, 10-week inhalation exposures to UFPs induced taxonomic changes in the microbiome of various animal models in the absence of intestinal inflammation.

Antimicrobial, antibiofilm, and antivirulence properties of Eisenia bicyclis-extracts and Eisenia bicyclis-gold nanoparticles towards microbial pathogens.

Nanomaterials derived from seaweed have developed as an alternative option for fighting infections caused by biofilm-forming microbial pathogens. This research aimed to discover potential seaweed-derived nanomaterials with antimicrobial and antibiofilm action against bacterial and fungal pathogens. Among seven algal species, the extract from Eisenia bicyclis inhibited biofilms of Klebsiella pneumoniae, Staphylococcus aureus, and Listeria monocytogenes most effectively at sub-MIC levels. As a result, in the present study, E. bicyclis was chosen as a prospective seaweed for producing E. bicyclis-gold nanoparticles (EB-AuNPs). Furthermore, the mass spectra of E. bicyclis reveal the presence of a number of potentially beneficial chemicals. The polyhedral shape of the synthesized EB-AuNP with a size value of 154.74 ± 33.46 nm was extensively described. The lowest inhibitory concentration of EB-AuNPs against bacterial pathogens (e.g., L.monocytogenes, S. aureus, Pseudomonas aeruginosa, and K. pneumoniae) and fungal pathogens (Candida albicans) ranges from 512 to >2048 µg/mL. Sub-MIC of EB-AuNPs reduces biofilm formation in P. aeruginosa, K. pneumoniae, L. monocytogenes, and S. aureus by 57.22 %, 58.60 %, 33.80 %, and 91.13 %, respectively. EB-AuNPs eliminate the mature biofilm of K. pneumoniae at > MIC, MIC, and sub-MIC concentrations. Furthermore, EB-AuNPs at the sub-MIC level suppress key virulence factors generated by P. aeruginosa, including motility, protease activity, pyoverdine, and pyocyanin, whereas it also suppresses the production of staphyloxanthin virulence factor from S. aureus. The current research reveals that seaweed extracts and a biocompatible seaweed-AuNP have substantial antibacterial, antibiofilm, and antivirulence actions against bacterial and fungal pathogens.

Hypoxia stabilizes SETDB1 to maintain genome stability.

Von Hippel-Lindau (VHL) is a tumor suppressor that functions as the substrate recognition subunit of the CRL2VHL E3 complex. While substrates of VHL have been identified, its tumor suppressive role remains to be fully understood. For further determination of VHL substrates, we analyzed the physical interactome of VHL and identified the histone H3K9 methyltransferase SETBD1 as a novel target. SETDB1 undergoes oxygen-dependent hydroxylation by prolyl hydroxylase domain proteins and the CRL2VHL complex recognizes hydroxylated SETDB1 for ubiquitin-mediated degradation. Under hypoxic conditions, SETDB1 accumulates by escaping CRL2VHL activity. Loss of SETDB1 in hypoxia compared with that in normoxia escalates the production of transposable element-derived double-stranded RNAs, thereby hyperactivating the immune-inflammatory response. In addition, strong derepression of TEs in hypoxic cells lacking SETDB1 triggers DNA damage-induced death. Our collective results support a molecular mechanism of oxygen-dependent SETDB1 degradation by the CRL2VHL E3 complex and reveal a role of SETDB1 in genome stability under hypoxia.

Detection of Red Pepper Powder Adulteration with Allura Red and Red Pepper Seeds Using Hyperspectral Imaging.

This study used shortwave infrared (SWIR) technology to determine whether red pepper powder was artificially adulterated with Allura Red and red pepper seeds. First, the ratio of red pepper pericarp to seed was adjusted to 100:0 (P100), 75:25 (P75), 50:50 (P50), 25:75 (P25), or 0:100 (P0), and Allura Red was added to the red pepper pericarp/seed mixture at 0.05% (A), 0.1% (B), and 0.15% (C). The results of principal component analysis (PCA) using the L, a, and b values; hue angle; and chroma showed that the pure pericarp powder (P100) was not easily distinguished from some adulterated samples (P50A-C, P75A-C, and P100B,C). Adulterated red pepper powder was detected by applying machine learning techniques, including linear discriminant analysis (LDA), linear support vector machine (LSVM), and k-nearest neighbor (KNN), based on spectra obtained from SWIR (1,000-1,700 nm). Linear discriminant analysis determined adulteration with 100% accuracy when the samples were divided into four categories (acceptable, adulterated by Allura Red, adulterated by seeds, and adulterated by seeds and Allura Red). The application of SWIR technology and machine learning detects adulteration with Allura Red and seeds in red pepper powder.

Integrating 4-D light-sheet fluorescence microscopy and genetic zebrafish system to investigate ambient pollutants-mediated toxicity.

Ambient air pollutants, including PM2.5 (aerodynamic diameter d ~2.5 µm), PM10 (d ~10 µm), and ultrafine particles (UFP: d < 0.1 µm) impart both short- and long-term toxicity to various organs, including cardiopulmonary, central nervous, and gastrointestinal systems. While rodents have been the principal animal model to elucidate air pollution-mediated organ dysfunction, zebrafish (Danio rerio) is genetically tractable for its short husbandry and life cycle to study ambient pollutants. Its electrocardiogram (ECG) resembles that of humans, and the fluorescent reporter-labeled tissues in the zebrafish system allow for screening a host of ambient pollutants that impair cardiovascular development, organ regeneration, and gut-vascular barriers. In parallel, the high spatiotemporal resolution of light-sheet fluorescence microscopy (LSFM) enables investigators to take advantage of the transparent zebrafish embryos and genetically labeled fluorescent reporters for imaging the dynamic cardiac structure and function at a single-cell resolution. In this context, our review highlights the integrated strengths of the genetic zebrafish system and LSFM for high-resolution and high-throughput investigation of ambient pollutants-mediated cardiac and intestinal toxicity.

Eucalyptol induces osteoblast differentiation through ERK phosphorylation in vitro and in vivo.

Eucalyptol (EU) is monoterpene oxide that is the main component of the essential oil extracted from aromatic plants such as Eucalyptus globules. EU has therapeutic effects such as antibacterial, anti-inflammatory and antioxidant in chronic diseases including inflammation disorder, respiratory disease, and diabetic disease. However, the effects of EU on osteoblast differentiation and bone diseases such as osteoporosis have not been studied. The present study investigated the effects of EU on osteoblast differentiation and bone formation. EU induces mRNA and protein expression of osteogenic genes in osteoblast cell line MC3T3-E1 and primary calvarial osteoblasts. EU also promoted alkaline phosphatase (ALP) activity and mineralization. Here, the osteoblast differentiation effect of EU is completely reversed by ERK inhibitor. These results demonstrate that osteoblast differentiation effect of EU is mediated by ERK phosphorylation. The efficacy of EU on bone formation was investigated using surgical bone loss-induced animal models. EU dose-dependently promoted bone regeneration in zebrafish caudal fin rays. In the case of ovariectomized mice, EU increased ERK phosphorylation and ameliorated bone loss of femurs. These results indicate that EU ameliorates bone loss by promoting osteoblast differentiation through ERK phosphorylation. We suggest that EU, plant-derived monoterpenoid, may be useful for preventing bone loss. KEY MESSAGES: Eucalyptol (EU) increases osteoblast differentiation in pre-osteoblasts. EU up-regulates the osteogenic genes expression via ERK phosphorylation. EU promotes bone regeneration in partially amputated zebrafish fin rays. Oral administration of EU improves ovariectomy-induced bone loss and increases ERK phosphorylation.

Determination of Freshness of Mackerel (Scomber japonicus) Using Shortwave Infrared Hyperspectral Imaging.

Shortwave infrared (SWIR) hyperspectral imaging was applied to classify the freshness of mackerels. Total volatile basic nitrogen (TVB-N) and acid values, as chemical compounds related to the freshness of mackerels, were also analyzed to develop a prediction model of freshness by combining them with hyperspectral data. Fresh mackerels were divided into three groups according to storage periods (0, 24, and 48 h), and hyperspectral data were collected from the eyes and whole body, separately. The optimized classification accuracies were 81.68% using raw data from eyes and 90.14% using body data by multiple scatter correction (MSC) pretreatment. The prediction accuracy of TVB-N was 90.76%, and the acid value was 83.76%. These results indicate that hyperspectral imaging, as a nondestructive method, can be used to verify the freshness of mackerels and predict the chemical compounds related to the freshness.

Exercise Mitigates Flow Recirculation and Activates Mechanosensitive Transcriptome to Uncover Endothelial SCD1-Catalyzed Anti-Inflammatory Metabolites.

Exercise modulates vascular plasticity in multiple organ systems; however, the metabolomic transducers underlying exercise and vascular protection in the disturbed flow-prone vasculature remain under-investigated. We simulated exercise-augmented pulsatile shear stress (PSS) to mitigate flow recirculation in the lesser curvature of the aortic arch. When human aortic endothelial cells (HAECs) were subjected to PSS ( τ ave = 50 dyne·cm -2 , ∂τ/∂t = 71 dyne·cm -2 ·s -1 , 1 Hz), untargeted metabolomic analysis revealed that Stearoyl-CoA Desaturase (SCD1) in the endoplasmic reticulum (ER) catalyzed the fatty acid metabolite, oleic acid (OA), to mitigate inflammatory mediators. Following 24 hours of exercise, wild-type C57BL/6J mice developed elevated SCD1-catalyzed lipid metabolites in the plasma, including OA and palmitoleic acid (PA). Exercise over a 2-week period increased endothelial SCD1 in the ER. Exercise further modulated the time-averaged wall shear stress (TAWSS or τ ave) and oscillatory shear index (OSI ave ), upregulated Scd1 and attenuated VCAM1 expression in the disturbed flow-prone aortic arch in Ldlr -/- mice on high-fat diet but not in Ldlr -/- Scd1 EC-/- mice. Scd1 overexpression via recombinant adenovirus also mitigated ER stress. Single cell transcriptomic analysis of the mouse aorta revealed interconnection of Scd1 with mechanosensitive genes, namely Irs2 , Acox1 and Adipor2 that modulate lipid metabolism pathways. Taken together, exercise modulates PSS ( τ ave and OSI ave ) to activate SCD1 as a metabolomic transducer to ameliorate inflammation in the disturbed flow-prone vasculature.

Factors Associated with Quality of Life of Clinical Nurses: A Cross-Sectional Survey.

Nurses often have a heavy workload and struggle to maintain a good quality of life (QOL). The aim of this study was to explore the relationship between perceived stresses and sleep disturbance, and quality of life among Korean clinical nurses. A cross-sectional design was used to examine these relationships among 200 clinical nurses at three hospitals in South Korea. Standardized instruments were used, including the Perceived Stress Scale (PSS), Insomnia Severity Index (ISI), Eating Behavior Questionnaire (DEBQ), and the WHOQoL-BREF questionnaire. The data were analyzed using descriptive statistics, the t-test, ANOVA, the Scheffe test, Pearson's correlation coefficient, and multiple regression analysis using SPSS/WIN 24.0 program. Multiple regression analysis showed that clinical nurses who had a subjective health status (ß = 0.29, p = 0.001), perceived stress (ß = -0.32, p < 0.001), and sleep disturbance (ß = -0.21, p = 0.001) were more likely to have a higher quality of life. The explanatory power of the regression model was statistically significant at 36.7%. Multiple regression analysis showed that clinical nurses who had better subjective health status, lower perceived stress, and fewer sleep disturbances were more likely to have a higher quality of life.

Activating P2Y1 receptors improves function in arteries with repressed autophagy.

AIM: The importance of endothelial cell (EC) autophagy to vascular homeostasis in the context of health and disease is evolving. Earlier, we reported that intact EC autophagy is requisite to maintain shear-stress-induced nitric oxide (NO) generation via glycolysis-dependent purinergic signalling to endothelial NO synthase (eNOS). Here, we illustrate the translational and functional significance of these findings. METHODS AND RESULTS: First, we assessed translational relevance using older male humans and mice that exhibit blunted EC autophagy and impaired arterial function vs. adult controls. Active hyperaemia evoked by rhythmic handgrip exercise-elevated radial artery shear-rate similarly from baseline in adult and older subjects for 60 min. Compared with baseline, indexes of autophagy initiation, p-eNOSS1177 activation, and NO generation, occurred in radial artery ECs obtained from adult but not older volunteers. Regarding mice, indexes of autophagy and p-eNOSS1177 activation were robust in ECs from adult but not older animals that completed 60-min treadmill-running. Furthermore, 20 dyne • cm2 laminar shear stress × 45-min increased autophagic flux, glycolysis, ATP production, and p-eNOSS1177 in primary arterial ECs obtained from adult but not older mice. Concerning functional relevance, we next questioned whether the inability to initiate EC autophagy, glycolysis, and p-eNOSS1177in vitro precipitates arterial dysfunction ex vivo. Compromised intraluminal flow-mediated vasodilation displayed by arteries from older vs. adult mice was recapitulated in vessels from adult mice by (i) NO synthase inhibition; (ii) acute autophagy impairment using 3-methyladenine (3-MA); (iii) EC Atg3 depletion (iecAtg3KO mice); (iv) purinergic 2Y1-receptor (P2Y1-R) blockade; and (v) germline depletion of P2Y1-Rs. Importantly, P2Y1-R activation using 2-methylthio-ADP (2-Me-ADP) improved vasodilatory capacity in arteries from (i) adult mice treated with 3-MA; (ii) adult iecAtg3KO mice; and (iii) older animals with repressed EC autophagy. CONCLUSIONS: Arterial dysfunction concurrent with pharmacological, genetic, and age-associated EC autophagy compromise is improved by activating P2Y1-Rs.

Discovery of pan-IAP degraders via a CRBN recruiting mechanism.

Inhibitors of apoptosis proteins (IAPs), defined by the presence of baculovirus IAP repeat (BIR) protein domain, are critical regulators of cell survival and cell death processes. Cellular IAP 1/2 (cIAP1/2) and X-linked IAPs (XIAPs) regulate the innate immune signaling pathway through their E3 ubiquitin ligase activity. Peptidomimetics or small-molecule IAP antagonists have been developed to treat various diseases, such as cancer, infection, and inflammation. In this study, we synthesized and characterized IAP-cereblon (CRBN) heterodimerizing proteolysis-targeting chimera (PROTAC), which induces the degradation of cIAP1/2 and XIAP but not CRBN. We demonstrated that this PROTAC inhibits tumor necrosis factor alpha (TNFα)-induced innate immune response and cancer cell migration and invasion, leading to apoptotic cell death. Our study is the first to demonstrate that both cIAPs and XIAP are degradable when applied to the PROTAC strategy.

Factors influencing bactericidal efficacy using atmospheric cold plasma (ACP) against Escherichia coli in wheat flour.

This study aimed to evaluate operating factors that influence the bactericidal efficacy of atmospheric cold plasma (ACP) using wheat flour contaminated with Escherichia coli. It also investigated how non-optimized operating factors of ACP could affect wheat flour properties. Five operating factors (container volume, flour weight, shaking RPM, treatment time, and gas flow rate) were evaluated for the bactericidal effect of ACP using the Box-Behnken design. In addition, thermal and pasting properties were measured to assess the effect of non-optimized ACP operating conditions on wheat flour quality. ACP operating factors (volume of the container, shaking RPM, and treatment time) had significant effects on reducing E. coli in wheat flour (p < 0.05). The bactericidal effect also depended on the distance and contact area between the plasma jet and sample. The temperature at the flour surface increased (max. 70 ℃) when ACP treatment didn't provide sufficient space and optimized duration. Thermal, pasting, and gelling properties of ACP treated-wheat flour in a 10 mL container increased significantly compared to untreated wheat flour. Large amounts of samples, long processing time, and insufficient space contributed to overheating which leads to denaturation or change of the wheat flour properties. The present study proposed important data for industrial sterilization of wheat flour using ACP.

Description of Microbacterium neungamense sp. nov. isolated from a hot spring.

The Gram-positive, nonmotile, rod-shaped bacterium EF45044T was isolated from a hot spring in Chungju, South Korea. The strain was able to grow at concentrations of 0‒5% (w/v) NaCl, at pH 6.0‒10.0 and in the temperature range of 18‒50 °C. Strain EF45044T showed the highest 16S rRNA gene sequence similarity (98.2%) with Microbacterium ketosireducens DSM 12510T, and the digital DNA‒DNA hybridization (dDDH), average amino acid identity (AAI), and average nucleotide identity (ANI) values were all lower than the accepted species threshold. Strain EF45044T contained MK‒12 and MK‒13 as the predominant respiratory quinones and anteiso‒C17:0, anteiso‒C15:0, and iso‒C16:0 as the major fatty acids. Diphosphatidylglycerol, phosphatidylglycerol, and glycolipid were detected as the major polar lipids. The cell-wall peptidoglycan contained ornithine. The DNA G + C content was 71.4 mol%. Based on the polyphasic data, strain EF45044T (= KCTC 49703T) presents a novel species of the genus Microbacterium, for which the name Microbacterium neungamense sp. nov. is proposed.

Mapping Korean National Health Insurance Pharmaceutical Claim Codes to SNOMED CT.

The objective of this study was to map pharmaceutical claim codes to SNOMED CT and thereby facilitate multicenter collaborative research and improve semantic interoperability. The claim codes were mapped to SNOMED CT using rule-based automated and manual methods. The maps were internally validated by terminologists and a pharmacist. Finally, 80% of all claim codes were mapped to the concepts of Pharmaceutical/biologic product hierarchy in SNOMED CT. Of them, 50.6% of the codes were exactly mapped to one clinical drug branch concept.

Optimization of Atmospheric Cold Plasma Treatment with Different Gases for Reduction of Escherichia coli in Wheat Flour.

In this study we aimed to derive the response surface models for Escherichia coli reduction in wheat flour using atmospheric cold plasma (ACP) with three types of gas. The jet-type atmospheric cold plasma wand system was used with a 30 W power supply, and three gases (argon, air, and nitrogen) were applied as the treatment gas. The operating parameters for process optimization considered were wheat flour mass (g), treatment time (min), and gas flow rate (L/min). The wheat flour samples were artificially contaminated with E. coli at a concentration of 9.25 ± 0.74 log CFU/g. ACP treatments with argon, air, and nitrogen resulted in 2.66, 4.21, and 5.55 log CFU/g reduction of E. coli, respectively, in wheat flour under optimized conditions. The optimized conditions to reduce E. coli were 0.5 g of the flour mass, 15 min of treatment time, and 0.20 L/min of nitrogen gas flow rate, and the predicted highest reduction level from modeling was 5.63 log CFU/g.

miR-96-5p targets PTEN to mediate sunitinib resistance in clear cell renal cell carcinoma.

A multiple receptor tyrosine kinase inhibitor, sunitinib, is a first-line therapy for clear cell renal cell carcinoma (CCRCC). Unfortunately, it has the major challenges of low initial response rate and resistance after about one year of treatment. Here we evaluated a microRNA (miRNA) and its target responsible for sunitinib resistance. Using miRNA profiling, we identified miR-96-5p upregulation in tumors from sunitinib-resistant CCRCC patients. By bioinformatic analysis, PTEN was selected as a potential target of miR-96-5p, which showed low levels in tumors from sunitinib-resistant CCRCC patients. Furthermore, PTEN and miR-96-5p levels were negatively correlated in a large The Cancer Genome Atlas kidney renal clear cell carcinoma cohort and high miR-96 and low PTEN represented poor prognosis in this cohort. Additionally, four-week sunitinib treatment increased miR-96-5p and decreased PTEN only in tumors from a sunitinib-resistant patient-derived xenograft model. We found a novel miR-96-5p binding site in the PTEN 3' UTR and confirmed direct repression by luciferase reporter assay. Furthermore, we demonstrated that repression of PTEN by miR-96-5p increased cell proliferation and migration in sunitinib-treated cell lines. These results highlight the direct suppression of PTEN by miR-96-5p and that high miR-96-5p and low PTEN are partially responsible for sunitinib resistance and poor prognosis in CCRCC.

Procedures to Evaluate the Role of Heparan Sulfate on the Reactivity of Resistance and Conductance Arteries Ex Vivo.

Evidence is emerging that disruption of the endothelial glycocalyx might contribute importantly to arterial dysfunction in the context of diabetes. One approach to assess the integrity of the endothelium and the vascular smooth muscle cell layer, in the absence of neural, humoral, and mechanical influences, is by measuring arterial vasomotion ex vivo. Here we describe a procedure to assess non-receptor-mediated vasoconstriction, receptor-mediated vasoconstriction, and endothelium-dependent and -independent vasodilation, in resistance and conductance arteries pressurized to 60 mmHg. In addition to evaluating vasoreactivity using isobaric approaches, the same experimental set-up can be used to initiate a pressure gradient across the artery such that intraluminal, flow-mediated vasodilation can be measured. After recording endothelium-dependent vasodilation using isobaric or flow-mediated approaches, identical interventions can be completed in the presence of enzymes that cleave biologically active heparan sulfates into inactive disaccharide and oligosaccharide fragments to assess the contribution from: (a) endothelial-derived substances (e.g., nitric oxide via nitric oxide synthase inhibition); or (b) important components of the glycocalyx (e.g., removal of heparan sulfate via heparitinase III treatment). Here, we show that acute disruption of a predominant glycosaminoglycan i.e., heparan sulfate impairs intraluminal flow-mediated vasodilation in murine resistance arteries.