bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Transcriptional Regulators for a Novel Glyoxylate Production Pathway in Methylobacterium extorquens AM1 (LB255)
Authors: Van Fosson Julie , Ngo Richard , Skovran Elizabeth
Published: June 2021

Methylobacterium extorquens AM1 is of interest as a platform for production of biofuels and biodegradable plastics from methanol. Biofuels such as 1‐butanol can be derived from intermediates in the Ethylmalonyl‐CoA pathway; however, sequestration of these intermediates for biofuel production limits the cell’s ability to produce glyoxylate, a metabolite required to power carbon assimilation. A second glyoxylate production pathway, the Oxalyl‐CoA Reductase pathway, exists but is mainly operational during oxalate growth. Use of this pathway may compensate for loss of glyoxylate during 1‐butanol production but an understanding of pathway regulation is needed to enhance cell engineering efforts. Expression studies showed upregulation of this pathway by exogenous oxalate and glyoxylate and QscR was identified as a potential transcriptional activator using transposon mutagenesis. Subsequent studies showed direct binding to the Oxalyl‐CoA Reductase pathway promoter by QscR. Interestingly, growth with succinate prevented upregulation in vivo and neither oxalate nor glyoxylate affected binding in vitro, consistent with the presence of an additional regulator. Future studies will use transposon mutagenesis to identify mutations that cause an increase or decrease in fluorescence from an Oxalyl‐CoA Reductase pathway promoter fusion identifying any additional transcriptional regulators. These studies will aid our ability to engineer M. extorquens to use the alternative glyoxylate production pathway to supply cellular glyoxylate, freeing the Ethylmalonyl‐CoA pathway for biofuel and bioplastic production.

Grant Funding Source: Supported by California State University Program for Education and Research in Biotechnology (CSUPERB) New Investigator Award and Louis Stokes Alliances for Minority Participation (LSAMP)
Characterizing purified Siw14 from Saccharomyces cerevisiae (LB261)
Authors: Steidle Elizabeth , Rolfes Ronda
Published: June 2021

Yeast strains with mutations in siw14Δ are defective in endocytosis, stress responses and linking nutrient status to cell cycle progression. Siw14 is a member of a family of dual‐specificity phosphatases found in fungi, protists, and plants. A model of the structure of the protein demonstrated that the active site is basic and shallow, and contains the consensus sequence HCxxGxxR, which includes the signature catalytic residues C214 and R220 as well as two residues, H213 and G217, conserved in the subfamily. Siw14 shows extensive homology with the Arabidopsis protein At1g5000 that dephosphorylates PI(3)P and PI(5)P, and has limited homology with the mammalian protein PTEN which dephosphorylates PI(3,4,5)P3. To test the hypothesis that Siw14 dephosphorylates one or more of the phosphorylated phosphatidylinositides (PIPn), a GST‐tagged form of the protein was purified from E. coli and assayed with the generic substrate p‐nitrophenylphosphate and each of the PIP¬ns. Recombinant GST‐Siw14 exhibited the highest activity in a buffer containing Tris‐HCl, pH 6.0, 10 mM DTT at 37°C, and activity was linear over 90 min. The enzyme exhibits the highest activity with PI(3,5)P2 but shows activity with other PIPs.
3D‐printing (Vizua) in the teaching of anatomy (LB27)
Authors: LE FLOCH‐PRIGENT Patrice , Vaniet Chloé , Bouhelal Omar , Ordureau Sylvain
Published: June 2021

The creation of gross‐anatomical models has always presented two major difficulties : the veracity and the material. Nowadays the 3D printing is quite easy to get and this two pitfalls can be successfully avoided as the anatomical models can be realized from series of joined CT‐scanned cross‐sections of real anatomical objects : bones, vascular injections, real organs or from normal CT‐scans from radiological examinations. By theses means are performed samples in resin for the oral surgical training where the arteries can be figured in red. The isolated or connected bones can be reproduced in life size but also at any choosen size: enlarged for the isolated bones of the head or of the face, or reduced for the pelvic girdle, for instance. The heart can be reproduced with its real coronary vascularisation. The applications are very numerous in human and in comparative gross‐anatomy. The only limit is the imagination.
Salvage of Coenzyme A breakdown products by the STM4195 gene product in Salmonella enterica (LB276)
Authors: Ernst Dustin , Downs Diana
Published: June 2021

Coenzyme A (CoA) is an essential cofactor required in all living organisms, where it functions primarily as an acyl‐group carrier and activator of carbonyl groups. The biosynthetic pathway generating CoA from pantothenic acid is universally conserved in prokaryotes and eukaryotes. Pantothenate is generated in a pathway that culminates in the condensation of pantoate and β‐alanine. Animals are unable to synthesize pantothenate and must salvage it, or other metabolic intermediates, to generate CoA. In Salmonella enterica, intermediates up to and including pantothenate can be fed exogenously to support CoA biosynthesis. Previous studies have shown that intermediates in CoA biosynthesis beyond pantothenate are not readily salvaged by S. enterica. The work here found that a degradation product of Coenzyme A, derived from boiling CoA, can bypass a genetic block in the final step of pantothenate biosynthesis. The data also showed that the STM4195 gene product is required to salvage this unknown metabolite, and also transports pantothenate precursors, ketopantoate and pantoate. STM4195 is a member of the sodium bile acid symporter family (SBF) of proteins. HPLC separation of a preparation of boiled CoA defined a peak with biological activity, and mass spectrometry and NMR approaches are being used to identify the compound. The results herein provide the first evidence for an active mechanism to transport ketopantoate and pantoate, and further demonstrate a role for STM4195 in the salvage of a unique CoA breakdown product.

Grant Funding Source: Supported by NIH GM095837
Bro1 contributes to cargo selection during Multivesicular Body (MVB) sorting (LB283)
Authors: Dean Shirley , Davies Brian , Azmi Ishara , Payne Johanna , Katzmann David
Published: June 2021

A key step in the lysosomal degradation of membrane proteins is sorting into the Multivesicular Body (MVB), a process wherein a region of the endosomal membrane buds into the lumen of the endosome to form an intraluminal vesicle (ILV). This sorting process is mediated by Endosomal Sorting Complexes Required for Transport (ESCRT‐0, I, II and III). Ubiquitination of MVB cargo and recognition of Ub‐cargo by ESCRT‐0, I and II permit entry into the MVB pathway; however, Ub is typically removed from the MVB cargo prior to cargo inclusion into the ILV. The coordination of deubiquination of MVB cargo with ESCRT‐III disassembly and ILV formation are not clear. Bro1 appears to play a critical role in coordinating cargo de‐ubiquitination and ILV formation through interactions with ESCRT machinery, the Ub peptidase, Doa4, ubiquitin‐modified cargo, and regulation of the ESCRT‐III‐associated AAA ATPase Vps4. These data suggest that members of the Bro1 family may be similarly impacting ESCRT‐dependent processes in a number of contexts.

Grant Funding Source: NIH R01 GM07302
Does alternating dissection effect student performance on practical examinations (LB29)
Authors: Boehm Karl , Fraticelli Nicole , Jordan Rebecca , Riccardi Margaret , Houser Jeremy , Kondrashov Peter
Published: June 2021

In the process of restructuring the Gross Anatomy course for first‐year osteopathic medical students a new lab model was introduced, with teams alternately performing the dissection of midline regions. To evaluate the effectiveness of the new model, we compared practical examination performance from 2 years based on whether or not they were in the dissecting or non‐dissecting lab group. Each practical question was assigned a body region and tissue type. A random intercept logistic regression model was fit to the data and the outcome variable was whether or not the student correctly identified the structure. The random effects were the students, each question asked during the practical exams and the assigned table number. The predictor variables included if the student had dissected the structure in an assigned lab, the region the anatomical structure is found and tissue type.

Statistical analysis of the data indicates that there was no significant difference (p=.34) in student performance between the dissecting and non‐dissecting groups as a whole. Detailed comparisons indicated that in certain areas it might be more beneficial to have the whole class dissecting, as students who were not present for dissection of pelvic and abdominal neurovasculature did not perform as well on these questions. In conclusion students are capable of mastering the lab material in this curricular model.
Characterization of F‐Box Gene At1g61340 in Abiotic Stress Responses of Arabidopsis thaliana. (LB294)
Authors: Gonzalez Lauren , Thines Bryan
Published: June 2021

The Ubiquitin‐26S Proteasome System (UPS) degrades key regulatory proteins in eukaryotic cells. Within the UPS, F‐box proteins recruit specific targets to an SKIP/CULLIN/F‐BOX (SCF) complex for ubiquitylation, the mark for degradation. Plants possess a greatly expanded family of F‐box genes, likely reflecting their particularly critical need to adapt cellular state to external conditions. Using publically available microarray data, our lab has identified F‐box gene At1g61340 as cold induced and for its co‐expression with known stress response regulators. Altered expression of cold responder CBF3 in At1g61340 overexpression lines indicates that At1g61340 impacts the cold response of Arabidopsis, and a partially sterile phenotype in these lines suggests a link between this stress response and reproductive development. At1g61340 may be expressed as two spliceforms, one of which has an interrupted F‐box domain and, perhaps, altered ability to recruit its target for degradation. Continued characterization of At1g61340 and investigation into its alternative splicing may reveal not only a new role for the UPS in plant biology, but also a potentially novel form of UPS regulation.
Gestational weight gain in women receiving prenatal care in rural midwifery clinics (LB313)
Authors: Gonzales‐Pacheco Diana , Cox Kim , Murray‐Krezan Cristina
Published: June 2021

Background: Gestational weight gain (GWG) impacts maternal and fetal outcomes and is the most significant predictor of postpartum weight retention. In 2009, the Institute of Medicine (IOM) released revised GWG recommendations on prepregnancy body mass index (BMI).

Objective: To describe GWG in women receiving prenatal care provided by Certified Nurse Midwives (CNM) and compare their weight changes to the 2009 IOM GWG recommendations.

Design: Secondary data analysis of GWG in pregnant women who participated in a pilot study comparing postpartum weight changes in women receiving group prenatal care vs. individual care provided by CNMs. Women were grouped by pre‐gravid BMI according to World Health Organization categories.

Results: Data for 18 women were included in the analysis. Mean age was 25.17 + 4.97 years and mean gravidity was 1.94 + 1.2. For women in group care, median weight gain was 16.8 kg (Q1‐Q3: 13.6‐27.7 kg) vs. 12.3 kg (Q1‐Q3: 10.0‐22.6 Kg) for women in individual care (p= 0.56). By pregravid BMI, the group care cohort’s median weight gain was 16.4 kg (normal weight), 30.5 kg (overweight), and 15.1 kg (obese). For the individual care cohort, median weight gain by pregravid BMI was 18.1 kg (normal weight), 9.9 kg (overweight), and 11.4 kg (obese).

Conclusions: Median weight gain between groups was not significantly different. Median weight gain by BMI category exceeded the 2009 IOM recommendations with the exception of the overweight individual care group.

Grant Funding Source: Supported by the National Center for Research Resources and the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number 8Ul1TR000041
Lactation influences changes in volumetric bone mineral density and microstructure postpartum (LB315)
Authors: Brembeck Petra , Lorentzon Mattias , Ohlsson Claes , Winkvist Anna , Augustin Hanna
Published: June 2021

Lactation decreases areal bone mineral density (BMD) during the first months postpartum and replenishment occurs especially after lactation has ceased. The aim was to study changes in volumetric BMD (vBMD) and microstructure of cortical and trabecular bone in tibia postpartum using high resolution peripheral quantitative computer tomography (HR‐pQCT). Seventy‐nine postpartum women and 19 controls were measured at baseline (0.5 mo after delivery), 4 and 12 mo thereafter. Lactation and body weight were registered. Women were categorized into 1=controls, and in relation to duration of lactation 2(0‐3.9 mo), 3(4‐8.9 mo), and 4(蠅9 mo). Significant changes were found only in group 3 and 4 in cortical vBMD, area and thickness, and trabecular area (P<0.05). At 4 months, cortical vBMD (3=‐0.3%, 4=‐0.5%), cortical area (3=‐0.7%, 4=‐1.5%) and cortical thickness (4=‐1.5%) decreased, and trabecular area increased (3=+0.1%, 4=+0.3%) compared to baseline. Still at 12 months, cortical vBMD (3=‐0.4%, 4=‐1.2%), cortical area (4=‐2.5%) and cortical thickness (4=‐2.5%) were lower than baseline, while trabecular area was higher than baseline in group 4 (+0.3%). In conclusion, longer duration of lactation is related to significant decreases in tibia cortical vBMD during the first year postpartum. There may be a cortical trabecularization, since cortical area decreases and trabecular area increases during lactation.

Grant Funding Source: This study was supported by the Centre for Environment and Sustainability, Swedish Nutrition Foundation and the Swedish Research Council Formas.
Effects of feeding rats with symbiotic oat‐based frozen yogurt on serum triglycerides and cholesterol (LB338)
Authors: Yang Ruishu , Walsh Helen , Cheng Jianjun , Ye Haoqing , Chang Tiehua , Gao Feng , Guo Mingruo
Published: June 2021

Ice cream is one of the most popular dairy foods. However, it may be not suitable for certain segments of the population due to its high sugar and fat. Oats‐based products are considered as functional foods as they are rich in β‐glucan. The objectives of this study were to develop a symbiotic oat‐based frozen yogurt containing fermented oat, probiotics and inulin (prebiotic) and to evaluate its effects on serum triglycerides (TG) and cholesterol in rats. Fermented oat base was prepared by fermenting cooked and sterilized oat slurry with probiotic cultures at 43oC for 6 h. The oat‐based frozen yogurt was formulated using fermented oat base, inulin, sweeteners, soy protein, vegetable oils, and emulsifiers. The new product was analyzed for chemical composition (%, protein: 4.1+0.1; fat: 8.5+0.2; carbohydrates: 22.0+0.2; soluble fiber: 2.10+0.02; calcium: 0.27+0.02) and probiotic survivability. The initial populations of Bifidobacterium and Lactobacillus acidophilus in the experimental frozen yogurt were 8.4×106 and 5.6×103CFU/ml, respectively. Bifidobacterium remained stable during storage at ‐18oC. Rats (Wistar) were conditioned and their TG and cholesterol levels were measured before and after feeding trials. Rats (3 groups) were fed with diets containing 25% (L), 30% (M), and 35% (H) of the oat frozen yogurt, respectively, compared with the rats fed with a diet containing 30% commercial ice cream (C), and a control group fed with a standard diet (S). The TG levels in the rats of both groups fed with M or H diet were significantly lower than that of group C (p<0.05). There was a very significant difference in total cholesterol (TC) and LDL between rats fed with H diet and those fed with C diet (p<0.01). There was no significant difference in TG between rats fed with standard diet and H diet (p>0.05), but the differences in TC, and LDL were very significant between the groups (p<0.01). Results indicate that consumption of oat‐based frozen yogurt may decrease the levels of serum TG, total and LDL cholesterol in rats.
Diet cost and risk of diabetes: does diet cost mediate the association between socioeconomic status and diabetes risk? (LB355)
Authors: Rehm Colin , Tinker Lesley , LaCroix Andrea , Wang Ching‐Yun , Drewnowski Adam
Published: June 2021

Diet cost has been implicated as an important determinant of diet quality. No studies have evaluated whether diet cost is associated with diabetes risk. A prospective cohort study among 47,683 post‐menopausal women 49‐64 years using data from the Observational Study and the control arm of the Dietary Modification trial of the Women’s Health Initiative was conducted to evaluate the association between diet cost and diabetes risk. This study had two aims: 1) to examine the association between diet cost and diabetes and; 2) to determine the extent by which the socioeconomic gradient in diabetes was explained by diet cost. To estimate diet costs, a national food price database was linked to a food frequency questionnaire. Energy‐adjusted diet costs were log‐transformed to evaluate the impact of a relative increase in diet costs. The outcome was treated type‐2 diabetes. Cox proportional hazards models examined the association between diet cost and diabetes after adjusting for covariates associated with diet cost and/or diabetes (e.g., age, smoking, family history, physical activity, body mass index, and income among others). 2,174 new cases of diabetes were observed over 8 years of follow‐up. After adjusting for covariates, a 50% increase in energy‐adjusted diet costs (e.g., from $4/d‐$6/d) was associated with a 14% reduced risk of diabetes (hazard ratio 0.86; 95% CI 0.78, 0.94). A strong socioeconomic gradient in diabetes risk was also observed. Diet cost explained 15‐19% of the association between income/education and diabetes. As the first report to observe an association between diet cost and diabetes these results need to be compared to other studies. Examining upstream factors associated with risk of diabetes expands our understanding of the consequences of the contemporary food environment on health.

Grant Funding Source: Supported by NIDDK R21‐DK‐085406; The WHI program is supported by contracts from the National Heart, Lung and Blood Institute, NIH.
Dynamic regulation of hepatic vitamin E secretion by the α‐tocopherol transfer protein (LB410)
Authors: Chung Stacey , Thakur Varsha , Atkinson Jeffrey , Parker Robert , Manor Danny
Published: June 2021

Vitamin E, a plant‐derived neutral lipid, is an essential nutrient for all vertebrates that scavenges free radicals in biological membranes, thereby preventing oxidative stress. Of the eight naturally‐occurring forms of vitamin E, α‐tocopherol is the most biologically active. This discrimination is achieved by the selective retention of α‐tocopherol by the hepatic α‐tocopherol transfer protein (α‐TTP), and by the selective degradation of other vitamin E isoforms by the hepatic cytochrome P450 CYP4F2. In hepatocytes, α‐TTP facilitates the secretion of α‐tocopherol to the circulation for uptake by extrahepatic target tissues. α‐TTP has also been shown to bind to phosphatidylinositol phosphates in vitro, which may constitute a mechanism for regulation of the protein’s activity. We aim to understand how the actions of α‐TTP are regulated in vivo. Specifically, we study how α‐tocopherol status affects the intracellular localization of α‐TTP, and whether phosphorylation of tyrosines in α‐TTP affects it activity. Using live‐cell fluorescence imaging, we found that localization of α‐TTP in hepatocytes is dynamic: in the absence of α‐tocopherol, the protein is found in a punctate perinuclear pattern, but upon addition of vitamin E, the protein redistributes to a diffuse cytosolic pattern. In addition, we found that tyrosine residue(s) of α‐TTP are phosphorylated, and that this modification is necessary for α‐TTP’s activity. These findings suggest that trans‐localization and tyrosine phosphorylation of α‐TTP are regulated under physiological conditions. Thus, dynamic and homeostatic mechanisms regulate the body‐wide distribution of α‐tocopherol.
Differential effects of whey protein concentrate and hydrolyzed whey/egg protein blends on post‐prandial markers of insulin signaling and skeletal muscle anabolism in rats (LB439)
Authors: Mobley C. Brooks , Fox Carlton , Pascoe Corrie , Ferguson Brian , Healy James , Lockwood Christopher , Roberts Michael
Published: June 2021

We examined how gavage‐feeding the following dietary proteins (10 human eq. g dissolved in 1 ml of water) acutely affected skeletal muscle insulin signaling and anabolism markers 90‐ and 180‐min post‐feeding in male Wistar rats (~250 g) fasted overnight. Supplementation included: a) 80% whey protein concentrate (WPC, n = 15); b) 70% hydrolyzed whey + 30% hydrolyzed egg albumin (70W/30E, n = 15); c) 50W/50E (n = 15); d) 30W/70E (n = 15); and e) 1 ml of water with no protein (fasted, n = 14). Immunoblotting results from mixed gastrocnemius muscles revealed: 1) rats fed WPC, 70W/30E and 50W/50E typically presented an in phospho‐Akt (Thr308) compared to 30W/70E and fasting rats (p < 0.05); 2) markers of mTOR signaling (p‐mTOR Ser2481, p‐p70s6k Thr389, p‐rpS6 Ser235/236) were typically greater in rats fed 70W/30E and 50W/50E compared to WPC, 30W/70E and fasting rats (p < 0.05); and 3) 180‐min post‐feeding muscle protein synthesis (MPS) analysis demonstrated that, compared to fasted rats, WPC feeding increased MPS by 94% (p < 0.05), 70W/30E feeding increased MPS by 74% (p = 0.065) and 50W/50E and 30W/70E feedings non‐significantly increased MPS by 25% and 34%, respectively (p > 0.05). In summary, 70W/30E feeding elicits similar acute post‐feeding responses to that of WPC. However, replacing hydrolyzed whey with a higher percentage of hydrolyzed egg albumin attenuated these responses.

Source of Research Support: 4LIFE Research, Inc.
A high‐throughput, fluorescence‐based cholesterol efflux assay (LB44)
Authors: Saddar Sonika , Kothambawala Tasnim , Chacos Nick , Saini Preeti , Tchaga Grigoriy , Yan Gordon
Published: June 2021

Numerous studies have established a negative correlation between cellular cholesterol efflux and cardiovascular disease.Cholesterol efflux from peripheral tissues and cells by a process called Reverse Cholesterol Transport (RCT). RCT allows extrahepatic cells, including macrophage‐derived foam cells in arterial atherosclerotic plaque, to transport excessive cholesterol back to the liver for bile acid synthesis and excretion, thus lowering the peripheral lipid burden. Standard protocols for quantitation of cholesterol efflux involve labeling cells with tritiated cholesterol and measuring the release of labeled sterol in a 12 or 24 well plate. This protocol, however is not ideal for high‐throughput screening of large numbers of serum samples or screening compounds/small molecules for their effect on cholesterol efflux.

We report here a simple, convenient and high‐throughput cell‐based assay using fluorescently‐labeled cholesterol in a 96‐well plate format. Our results demonstrate that the percentage cholesterol efflux elicited by 2% ApoB‐depleted normal human serum in J774 macrophage cells is comparable to those published in literature. Our assay method utilizes a positive control for this assay which induces efflux independent of any receptor, that is used as a measure of the reliability of the assay. This assay is highly reproducible with low inter‐assay variability (蠄 10%). We demonstrate that this is an efficient method of measuring cholesterol efflux in a cell‐based assay. The increased sensitivity of the assay coupled with its high‐throughput screening capability enables scientists to screen large libraries of reagents and serum samples and provides a valuable tool for drug discovery in the field of cardiovascular disease research.
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
A cholesterol interaction domain in STIM1 is important for STIM1‐Orai1 coupling (LB53)
Authors: Pacheco Jonathan , Vaca Luis
Published: June 2021

STIM1 and Orai1 are the basic components of the calcium influx pathway known as store‐operated calcium entry (SOCE). STIM1 is a calcium sensor, which oligomerizes and activates Orai channels when calcium levels drop inside the endoplasmic reticulum. The activation of Orai1 requires the direct coupling of STIM1 domain known as SOAR (Stim Orai Activating Region) with the Orai1 C‐terminal region. Other proteins have shown to play an important role in regulation of SOCE, including specialized plasma membrane regions known as lipids rafts, which are rich on sphingolipids and cholesterol. In this work, we identified in the SOAR region from STIM1 a cholesterol recognition/interaction amino acid consensus (CRAC) domain, which appears to be important for STIM1‐Orai1 association. Through mutagenesis of this domain, we find by using FRET microscopy that SOAR mutated in the CRAC domain lost its capability to interact with Orai1. This finding suggests that the association of STIM1 to cholesterol may play an important role for the subsequent STIM1‐Orai1 association.
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