bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intestinal dysbiosis enhances gut microflora‐induced neutrophil extracellular traps (LB517)
Authors: Vong Linda , Yeung William , Pinnell Lee , Sherman Philip 
Published: June 2021

Antibiotic (Abx) therapy alters gut microbial composition and is associated with an increased risk of pathogen infection. However, little is known about the ability of Abx to foster the growth of pathobionts ‐ commensal microbes that, as a result of select environmental stresses, become pathogenic. Here, we measured the ability of Abx‐disturbed gut microflora to induce neutrophil extracellular traps (NET), protease‐rich ‘footprints’ left by neutrophils to capture and kill microbes. C57BL/6 mice were administered vancomycin (40 mg/kg/d) and gentamicin (3 mg/kg/d) once daily for 3d. Fecal pellets were collected prior to Abx and for 16d thereafter, and incubated in either Tryptic Soy (TS), deMan‐Rogosa‐Sharpe (MRS) or Luria‐Bertani (LB) broth (37°C, 16 h) to enrich either all microbes, Lactobacilli, or Escherichia coli, respectively. The capacity to induce NET from bone marrow‐derived neutrophils (BMDN) was quantified by measurement of extracellular DNA and visualized by fluorescence microscopy. Microbial composition was monitored by qPCR and DGGE. Baseline NET‐activating capacity was present in TS and LB cultures, and was inhibited following Abx. At 10d post‐Abx, LB cultures induced significantly more NET release, indicating overgrowth of immuno‐stimulatory microbes. In combination with compromised epithelial function, the growth of NET‐activating microbes may exacerbate intestinal disease.Grant Funding Source: This work is supported by CIHR (operating grants MOP‐89894, IOP‐92890)
Exercise training decreases myocardial collagen III and heart failure signs rate, and improves physical performance in spontaneously hypertensive rats (LB522)
Authors: Pagan Luana , Damatto Ricardo , Cezar Marcelo , Moukbel Yasmin , Lima Aline , Bonomo Camila , Gomes Mariana , Rosa Camila , Okoshi Marina , Okoshi Katashi 
Published: June 2021

The aim of this study was to evaluate the influence of physical exercise on physical performance, heart failure (HF) signs rate, and myocardial collagen I and III and lysyl oxidase protein expression in spontaneously hypertensive rats (SHR). Methods: Sixteen‐month‐old SHR were divided into two groups: sedentary (SED, n=8) and exercise (EXE, n=8). Physical training was performed 5 times per week for 16 weeks in a treadmill. Physical performance was assessed by exercise test on a treadmill with a gradual speed increase. At the moment of euthanasia, HF signs were evaluated: tachypnea in vivo, and ascyte, pleural effusion, atrial thrombi, right ventricular hypertrophy, and liver and lung congestion post mortem. Collagen I and III and lysyl oxidase protein expression were analyzed by Western blot. Statistics: The Student's t test and the Goodman test were used to compare the groups (p<0.05). Results: The EXE group showed greater physical capacity and lower HF signs rate than SED group. Collagen III protein expression was lower in EXE group (1.00±0.58 vs. 0.73±0.43, p<0.05). Collagen I and lysyl oxidase protein expression were not significantly different between groups. Conclusion: Physical exercise improves physical performance and reduces heart failure signs rate and myocardial collagen III expression in spontaneously hypertensive rats.Grant Funding Source: Supported by CAPES and CNPq
Generation of vascular cells from patient derived iPSC to model hypertension (LB537)
Authors: Biel Nikolett , Lim Chae , Santostefano Katherine , Cooper‐DeHoff Rhonda , Johnson Julie , Terada Naohiro 
Published: June 2021

Since their establishment within the last decade, induced pluripotent stem cells (iPSC) have opened the possibility to model human diseases in vitro maintaining disease phenotypes as they are differentiated into disease‐relevant cell types. Utilizing the genome‐wide association (GWA) approach, studies have shown that patient response to antihypertensive drugs may depend on specific genotypes. Our goal is to collect iPSC from high and low responder patients and utilize hypertensive relevant cells such as endothelial (EC) and vascular smooth muscle cells (VSMC) to understand the biology of patient drug response. The current study shows the generation of patient derived iPSC and differentiation to EC and VSMC. Patient iPSC were generated from peripheral blood mononuclear cells and reprogrammed by transduction of the Sendai viral vector SeVdp(KOSM)302L encoding four reprogramming factors (Oct4, Sox2, Klf4, and c‐Myc). Endothelial and VSMC were differentiated using OP9 co‐culture model system for the production of CD31+ CD34+ vascular progenitor cells and further differentiating to EC and VSMC lineages using Vascular Endothelial Growth Factor (VEGF) for EC, Platelet Derived Growth Factor (PDGF‐BB) and Transforming Growth Factor‐B (TGF‐B) for VSMC differentiation. Endothelial and VSMC phenotypes were confirmed using EC and VSMC specific proteins and receptors such as von Willebrand‐Factor (vWF) and Vascular Endothelial Growth Factor Receptor (VEGF‐R) for EC and alpha‐Smooth Muscle Actin (aSMA) and Platelet Derived Growth Factor Receptor (PDGF‐R) for VSMC. Future studies will focus on utilizing these cell types to understand the biology of different patient responses to anti‐hypertensive agents.Grant Funding Source: Supported by NIH
A capillary based vacuum assisted instrument for rapid and efficient collection of individual live cells from adherent cultures for single cell and clonal analysis (LB56)
Authors: Karsten Stanislav , Ma Zhongcai , Kudo Lili 
Published: June 2021

There is a high demand for a method permitting simple and rapid collection of individual cells from adherent cell cultures that may be used from simple selection of transfected cells to single cell analysis. Numerous protocols for single cell analyses have been developed, which include whole genome sequencing and expression analyses. Although current flow sorting technology permits collection of single cells, acquisition of cells directly from cell culture dishes based on their morphology, location or fluorescent label remains challenging. Recently we introduced a novel cell and tissue acquisition system (KuiqpicK) and demonstrated its application for brain tissue microdissection. Here, we examined KuiqpicK’s ability to efficiently collect individual live cells. Collection of cells was performed from various adherent cultures, including neuroblastoma SH‐SY5Y, Chinese hamster ovary (CHO), human melanoma MDA‐MB‐435 cell cultures and primary neural progenitor cells established from embryonic and adult mouse and rat brains. Single and multiple cell acquisitions were performed based on the cell morphology, location in the plate and fluorescent label (e.g. CellTracker probes). To test the viability of collected cells, Trypan blue exclusion test and single cell recultivation experiments were performed using CHO and SH‐SY5Y cells. Trypan blue assay demonstrated survival rates ranging from 80 to 95% (n>300) for all cells tested. The clonal expansion of collected single SH‐SY5Y and CHO cells was demonstrated within 6 and 25 days, respectively. In addition, applicability of the instrument for the collection of single cells grown in three‐dimensional (3D) culture system was shown using MDA‐MB‐435 cells. Moreover, isolation of RNA from collected cells exhibited high integrity and low degradation rates. Subsequent T7 based labeling procedure consistently produced high incorporation yields making it suitable for downstream sequencing and gene expression analyses. To summarize, our experimental data convincingly demonstrated that the capillary based vacuum assisted instrument, KuiqpicK is a convenient approach to collect single and multiple cells (e.g. colonies) from both adherent and 3D cell cultures, capable of providing cells with high survival rate and suitable for downstream functional studies.Grant Funding Source: Supported by NIMH 2R44MH091909
Shear stress increases vasodilator sensitivity to the TRPV4 agonist GSK10167904 in rat cremaster arterioles (LB566)
Authors: Woodman Owen , Darby William , Potocnik Simon , Hollenberg Morley , McIntyre Peter 
Published: June 2021

Shear stress, induced by blood flow, is an important activator of TRPV4 and a stimulant of endothelium‐dependent relaxation. We investigated how an increase in shear stress affects vasodilatation in response to activation of TRPV4 and muscarinic receptors. Responses to the TRPV4 agonist GSK10167904 (GSK, 10 nM‐10 μM) and the muscarinic receptor agonist acetylcholine (ACh, 10 nM‐10 μM) were assessed in cannulated and pressurised rat cremaster arterioles (resting diameter 60‐100 μm). GSK and ACh caused concentration‐dependent dilatation (pEC50, GSK 7.60±0.23 ACh 6.42 ± 0.22) which in each case was abolished by removal of the endothelium. The GSK‐induced dilatation was unaffected by inhibition of nitric oxide synthase and cyclooxygenase with L‐NAME (100 μM) and indomethacin (10 μM) but was abolished by the additional inhibition of SKCa and IKCa with apamin (1 μM) and TRAM‐34 (1 μM).The GSK‐induced dilatation was significantly impaired by the TRPV4 antagonist HC067047 (300 nM; pEC50 6.80±0.12, p<0.05) but responses to ACh were unaffected. When shear stress was generated by causing a flow of 20 μl per min through the lumen there was a significant enhancement of the sensitivity to GSK (pEC50 8.52±0.23, p<0.05) but not to ACh (pEC50 6.34±0.23). In the rat cremaster arteriole activation of TRPV4 causes endothelium‐dependent dilatation that involves opening of SKCa and IKCa but is independent of NO or cyclooxygenase products. Lumenal flow enhanced the dilatation in response to GSK but not ACh. We conclude that shear stress enhances TRPV4 mediated vasodilatation without generally enhancing endothelium‐dependent vasodilatation.
Analysis of the uptake of native versus modified albumin by renal tubular epithelial cells (LB59)
Authors: Ly Dinh Khoi Nguyen , Patterson David , Poronnik Philip 
Published: June 2021

Albumin is most abundant protein in blood and plays important homeostatic roles in the regulation of oncotic pressure and as a carrier of many small proteins, peptides and hormones. Albumin in the circulation can be covalently modified at surface lysine residues and thus the circulation contains a mixture of native albumin (i.e. not modified) and modified albumin. A significant amount of albumin is lost from the blood each day as a result of the filtration occurring in the capillaries of the renal glomeruli. This filtered albumin in the early filtrate is thought to be mostly taken up by and degraded in the early renal proximal tubular epithelial cells, while a small proportion is excreted in the final urine. This degradation of albumin by proximal tubules operates via the megalin scavenger receptor and an endocytic pathway.A major gap in our knowledge is whether the megalin‐based albumin uptake is a non‐selective process, or whether megalin has a significantly different affinity for modified versus native albumin.Using a well‐established albumin uptake assay in Opossum Kidney (OK) cells, modified albumin probes (Alexa488‐BSA and FITC‐BSA) were shown taken up into OK cells via the endocytic pathway. This uptake was efficiently competed by a molar excess of modified albumin, whereas an excess of rHA had little effect upon the uptake, suggesting a difference in binding affinity. The addition of 14C‐rHA to OK cells resulted in a very low percentage (3%) of the probe being taken up by cells compared to a 30% uptake using Alexa488‐BSA. In addition, the uptake of 14C‐rHA was not saturated in a dose‐response assay suggesting non‐specific binding. This was confirmed when an inhibitor of the endocytic pathway failed to inhibit the uptake of 14C‐rHA by OK cells.The results indicate that the megalin pathway has a much greater affinity for modified compared to native albumin. This may be an important mechanism for the removal of modified albumin from the body as it can exert toxic effects.
Physical interaction between CYP1A2 and CYP2B4 causes a disruption of inhibitory CYP1A2 complexes (LB592)
Authors: Connick John , Reed James , Backes Wayne 
Published: June 2021

Previous reports showed that CYP1A2 and CYP2B4 can affect each other’s activity when present in the same vesicles with subsaturating NADPH‐P450 reductase (CPR). Specifically, systems containing both P450s exhibited increased CYP1A2 and decreased CYP2B4 activities when compared with those containing each P450 alone. These effects were consistent with the formation of CYP1A2•CYP2B4 complexes that affected the affinity of each P450 for CPR, and were corroborated in living cells using bioluminescence resonance energy transfer (BRET). Previous results also showed that CYP1A2 forms homomeric complexes whose presence was correlated with inhibition of CYP1A2 activity at subsaturating CPR. The goal of this study was to determine if homomeric P450 complexes were disrupted by the presence of another P450 using BRET. First, the potential for CYP2B4•CYP2B4 complexes was examined. Unlike with CYP1A2, we were unable to detect specific CYP2B4•CYP2B4 complexes. Next, we examined the potential for CYP2B4 to disrupt the inhibitory CYP1A2•CYP1A2 complexes, and facilitate the CPR:CYP1A2 interaction. To do this, we cotransfected HEK293T cells with plasmids encoding both CYP1A2‐GFP and CYP1A2‐Rluc in the absence and presence of unlabeled CYP2B4. The data showed a significant decrease in the maximum CYP1A2•CYP1A2 BRET signal when CYP2B4 was present, indicating that CYP2B4 is capable of disrupting these complexes.Grant Funding Source: Supported by R01 ES004344
UDP‐glucuronosyltransferase 1 (UGT1) knockdown in colon epithelial cells causes inactivation of P53 mediated apoptosis and increases colon tumorigenesis (LB599)
Authors: Liu Miao , Chen Shujuan , Hao Haiping , Wang Guangji , Tukey Robert 
Published: June 2021

Significant evidence demonstrates a pattern of down‐regulation of UGT expression in tumor tissue when compared to surrounding healthy tissue. This indicates the loss of UGT expression may be an early marker in the process of tissue neoplastic transformation. Although the UGTs are usually considered to be a metabolic defense against chemical carcinogens through glucuronidation, the underlying mechanism is still unclear. Here we show that UGT1 silenced by UGT1A siRNA in HT29 and LS180 colon epithelial cells decreases actinomycin D and etoposide‐induced P53 protein expression. Also observed is a reduction in P21, BAX, and caspase‐3, with a lowering of the cytotoxicity of these two chemicals, indicating inactivation of P53 mediated apoptosis. To identify this phenomenon in vivo, intestinal epithelial cell (IEC)‐specific Ugt1 knockout (Ugt1ΔIEC) and control (Ugt1F/F) mice are treated with dextran sulfate sodium (DSS) to induce acute colon inflammation and cell apoptosis. Corresponding with the in vitro data, western blot and TUNEL stain show lower protein expressions of P53, P21, BAX, caspase‐3 and less apoptotic cells in the colon of Ugt1ΔIEC mice. However, there is no difference in inflammatory response between Ugt1ΔIEC and Ugt1F/F samples. Moreover, the treatment of azoxymethane plus DSS causes higher tumor incidence and larger tumor size in the colon of Ugt1ΔIEC mice, suggesting that the inactivation of P53 mediated apoptosis increases colon tumorigenesis. In summary, our experiments verify that the presence of UGT1 in colon epithelial cells is important in the activation of P53 mediated apoptosis and protects normal tissue from neoplastic transformation.Grant Funding Source: Supported by US Public Health Service Grants R21CA171008, ES010337 and GM100481, Natural Science Foundation of China (Grants 91029746), and Chiha Scholarship Funding
FSP27 interacts with ATGL to regulate lipolysis and insulin sensitivity in human adipocytes (LB60)
Authors: Grahn Tan Hooi Min , Kaur Rajween , Yin Jun , Schweiger Martina , Sharma Vishva , Lee Mi‐Jeong , Yasuo Ido , Smas Cynthia , Zechner Rudolph , Lass Achim , Puri Vishwajeet 
Published: June 2021

In adipocytes, lipolysis is a highly regulated process involving hormonal signals, lipid droplet‐associated proteins and lipases. The discovery of new lipid droplet‐associated proteins added complexity to the current model of lipolysis. In the present study, we used cultured human adipocytes to demonstrate that Fat Specific Protein 27 (FSP27), an abundantly expressed protein in adipocytes, regulates both basal and stimulated lipolysis by interacting with adipose triglyceride lipase (ATGL). We identified a core domain of FSP27, aa 120‐220, that interacts with ATGL to inhibit its lipolytic function and promote triglyceride storage. We also defined the role of FSP27 in free fatty acid‐induced insulin resistance in adipocytes. FSP27 depletion in human adipocytes increased lipolysis and inhibited insulin signaling by decreasing AKT phosphorylation. However, reducing lipolysis by either depletion of ATGL or expression of exogenous full length FSP27 or aa 120‐220 protected human adipocytes against the adverse effects of free fatty acids on insulin signaling. In embryonic fibroblasts derived from ATGL‐KO mice, exogenous free fatty acids did not affect insulin sensitivity. Our results demonstrate a crucial role for FSP27‐ATGL interactions in regulating lipolysis, TG accumulation, and insulin signaling in human adipocytes.Grant Funding Source: Supported by NIH‐NIDDK R56DK094815, NIH‐NIDDK P30DK046200, KL‐2 (8KL2TR000158)
Human simulation exercises help improve medical pharmacology learning (LB630)
Authors: Al‐Mehdi Abu‐Bakr 
Published: June 2021

In order to evaluate the effect of human simulation as an active learning method on student performance, simulation exercises were included as a mandatory component of the medical pharmacology course taught in the second year of medical school in the previous discipline‐based curriculum at the University of South Alabama College of Medicine. Students in groups of 5 or 6 managed cardiovascular, respiratory, infectious, endocrine, CNS, and toxicology emergency cases. In addition to history taking, physical examination, performance of procedures, and laboratory data analysis, each exercise also included writing detailed prescriptions of emergency and chronic treatment for the simulated patients’ conditions. Pathophysiology and pharmacology related to each case were discussed under the guidance of faculty preceptors during the debriefing sessions. Impact of the human simulation exercises were evaluated by student perception of their usefulness in learning and by NBME medical pharmacology subject examination performance. Analysis of student feedback showed that simulation exercises were perceived as a highly effective instructional method by the students. Student performance in the NBME examination improved significantly during the 2009‐2013 simulation years (an average 3‐digit score of 538±9; n=5) compared with 2006‐2008 pre‐simulation years (478±18; n=3; p<0.05). These data suggest that highly immersive human simulation exercises requiring team‐work, clinical decision‐making, prescription writing, and basic science‐based debriefing help improve student satisfaction and pharmacology learning.Grant Funding Source: Supported by intramural funds
Estimation of radiating conditions in the reservoirs located close uranium mining of regions Kazakhstan (LB634)
Authors: Bakhtin Meirat , Kazymbet Polat , Eltokova Manshuk , Altaeva Nursulu , Baydaulet Imanali , Dzhakenova Alisa 
Published: June 2021

Stepnogorsk Mining and Chemical Plant (SMCP) is in operation more than 50 years and is one of the largest complexes on production of suboxide‐oxide of uranium and other rare metals. Radioactive wastes of SMCP are accumulated at the tailings storage with area of 7,82 km2, where the wastes with mass of 44,17 million tons and amount of radioactivity of 146,4 thousand Curie are concentrated. Complex radioecological conditions in river system “Kutunguz” is caused by dump in the river of miner waters with mine №1 near Shantobe. In river water concentration 238U, 226Ra, 210Ро, 230Th and 210Pb makes accordingly on the average ‐ 4,54 Bq/l, 0,11 Bq/l, 2,50 Bq/l, 0,1 Bq/l and 0,37 Bq/l. In the shallow reservoirs located near SMCP and in landlocked reservoirs Мanibay and Sulukamisk of thalwegs concentration radionuclide’s in water, ground deposits in gidrobionts is higher in comparison with parameters of conditionally control reservoir.The level with microkernels at fishes of the river Kutunguz and amphibian Manibay of a thalweg, the river Kutunguz authentically is higher than blood, than similar parameters, characteristic for conditionally control populations.On the basis of the analysis of a level of radioactive pollution of potable water and open reservoirs loading for various groups of the population is designed. On the basis of the received data principles of the organization and use radiobioecologick monitoring of open reservoirs in uranium regions are offered.
Toxic dopamine metabolites and oxidative stress as key contributors to neurotoxicity (LB641)
Authors: Schamp Josephine , Florang Virginia , Doorn Jonathan 
Published: June 2021

Toxic dopamine metabolites and reactive oxygen species (ROS) are hypothesized to contribute to progressive neurodegeneration as seen in Parkinson’s Disease (PD). The objective of this study is to explore the role of these mediators (i.e., ROS, reactive intermediates of dopamine metabolism) in neurodegeneration/neurotoxicity. Dopamine (DA), a neurotransmitter, is metabolized via monoamine oxidase to 3, 4‐ dihydroxyphenylacetaldehyde (DOPAL), a highly reactive aldehyde species, and H2O2.The DA metabolite DOPAL has proven to be toxic to dopaminergic cells both in vitro and in vivo. DOPAL modifies proteins and causes protein aggregation, which may contribute to cell death. We have found a complex interaction between DOPAL and ROS. 1) Like DA, DOPAL undergoes autooxidation to a semiquinone radical and then to an orthoquinone under normal physiological conditions. These quinones are reactive to proteins, as well as produce ROS/oxidative stress (OxS). 2) OxS was determined to increase levels of DOPAL due to inhibition of carbonyl metabolizing enzymes. 3) It has been shown that antioxidants, such as N‐acetylcysteine and ascorbate, can block protein modification by DOPAL. During DOPAL‐mediated protein modification ROS are produced. Using in vitro studies of dopaminergic cells, we found that modulating antioxidant levels regulates the level of DOPAL‐protein adduction. 4) Evidence suggests that the presence of oxygen catalyzes protein modification by DOPAL, resulting in subsequent elevation of ROS. Such data will help elucidate the complex role of OxS, specifically ROS production, in DOPAL‐mediated neurotoxicity and may provide therapeutic targets to prevent or delay neurodegeneration.Grant Funding Source: NIH R01 ES15507; NIH T32 GM067795