bluuebunny/crossref_metadata_2025 huggingface dataset:
Six‐Year Change in Red Blood Cell Omega‐3 and Trans Fatty Acids in the Framingham Heart Study
Authors: Harris William S , Pottala James V , Vasan Ramachandran S , Larson Martin G , Robins Sander J
Published: June 2021

Higher erythrocyte levels of trans fatty acids (FAs) and lower levels of omega‐3 FAs [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] are associated with increased cardiovascular risk. Over the last decade, the US food industry has reduced levels of trans FAs in fat and oils, but whether this has resulted in reduced tissue trans FA levels is not known. Also, fish oil supplementation (FOS) has become more popular, which should increase erythrocyte EPA+DHA levels. We measured levels of omega‐3 and 18‐carbon trans FAs in erythrocytes collected from 298 participants in the Framingham Heart Study in the Offspring population at both exams 7 (1998–2001) and 8 (2004–2006). Erythrocyte levels of trans FAs decreased by 39% (95% CI: −25% to −50%) over a median of 6.6 years in a model adjusted for age and sex differences. At exam 8, 41 participants reported FOS use, which was associated with a 39% increase in the omega‐3 index (95% CI: 30% to 50%) also adjusted for age and sex. In conclusion, efforts to reduce trans FAs in the US diet are paralleled by lower erythrocyte levels in our community‐based sample, suggesting that these efforts are achieving their intended effect. Additionally, the rise in the omega‐3 index is consistent with the use of FOS. Our observations support the use of erythrocyte FA composition to track dietary FA trends. (Supported by NHLBI: R01 HL089590 and by Contract N01‐HC‐25195.)
The effect of food viscosity on markers of appetite
Authors: Hollis James , Lamsal Buddhi , Zhu Yong
Published: June 2021

Accumulating data indicate that food form can influence appetite. In this study, a cross‐over study design was used to determine the effect of food viscosity on markers of appetite. On two separate occasions, 20 male participants reported to the laboratory first thing in the morning following an overnight fast. They were asked to consume a bowl of one of two soups: normal viscosity or high viscosity. The soups contained acetaminophen as a marker of gastric emptying rate. After consuming the meal, subjective appetite measures were recorded at regular intervals (t0+15, 30, 45, 60, 90, 120 and 180 minutes) using visual analogue scales. At each time‐point, a blood draw was also made. Following the last appetite measurement, an ad libitum lunch meal was served. There was a significant main effect of viscosity on hunger, fullness, desire to eat and prospective consumption (p<0.05). There was a significant main effect of viscosity on plasma acetaminophen concentration (p<0.05). However, there was no significant difference in food intake at the subsequent test meal. Blood samples will be assayed for CCK and ghrelin. This study indicates that increasing the viscosity of a liquid meal can reduce postprandial feelings of hunger. This may be due, in part, to a reduced rate of gastric emptying. However, food viscosity has no effect on food intake at the next meal.

This work was supported by a USDA/Iowa State University NWRC grant
Comparison of the impact of trans fatty acids from ruminants and industrial sources on human cholesterol homeostasis
Authors: Labonté Marie‐Ève , Motard‐Bélanger Annie , Paquin Paul , Chouinard Yvan , Lemieux Simone , Couture Patrick , Lamarche Benoît
Published: June 2021

This double‐blind randomized crossover study compared the impact of dietary trans fatty acids (TFA) from ruminants (rTFA) and from industrial sources (iTFA) on surrogate markers of cholesterol absorption (β‐sitosterol and campesterol) and synthesis (lathosterol) in healthy men. Thirty‐eight men consumed 3 experimental isoenergetic diets in a random order for 4 weeks each. The 3 diets were 1) high in rTFA (10.2g/2500 kcal), 2) high in iTFA (10.2g/2500 kcal) and 3) control diet low in TFA from any source (2.2g/2500 kcal). Plasma LDL‐C concentrations were increased to a similar extent in the iTFA (+4%) and rTFA (+6%) diets compared with the control diet. Plasma concentrations of β‐sitosterol, campesterol and lathosterol were significantly reduced after the iTFA diet compared with the control diet (−11%, −26% and −17% respectively, P < 0.05), but not after the rTFA diet. Analyses were also performed according to baseline concentrations of absorption markers. Low absorbers on the control diet had a 29% increase in plasma LDL‐C concentrations after the rTFA diet (P = 0.01 vs. the control diet) but showed no significant change in LDL‐C after the iTFA diet (+11%, P = 0.23). While very high intakes of rTFA and iTFA appear to have comparable LDL‐C raising effects, our data suggest that underlying mechanisms may be different between the two sources of TFA. Financial support: Dairy Farmers of Canada, Novalait Inc and NSERC
Anti‐aging activity of kimchi during fermentation period against oxidative stress‐induced premature senescence in cellular model
Authors: Kim Boh‐Kyung , Cho Eun‐Ju , Park Kun‐Young
Published: June 2021

The protective activity from oxidative stress and antiaging potential of kimchi during fermentation period were investigated under in vitro and cellular system using LLC‐PK1 renal epithelial cell and WI‐38 human lung fibroblast cells. The methanolic extract from kimchi showed radical scavenging activity as concentration dependent‐manner. In addition, the LLC‐PK1 cells showed the decreases in cell viability by the treatment with free radical generators. However, the kimchis, especially optimally ripened kimchi (pH 4.3), led to the significant increases in the cell viability with dose‐dependence. Furthermore, the cellular aging model of H2O2‐treated WI‐38 fibroblast was employed to investigate the anti‐aging effect of kimchi during fermentation on replicative senescence. H2O2‐treated WI‐38 cells showed the loss of cell viability, the increase of lipid peroxidation and shortening of the cell lifespan, indicating the induction of stress‐induces premature senescence (SIPS). However, the treatment with kimchis, especially optimally ripened kimchi, induced a dose‐dependent increase in the cell viability and inhibition in lipid peroxidation. In addition, the extension of the population doubling level of young, middle‐aged and old WI‐38 cell was also observed, suggesting their promising role as anti‐aging agents. Furthermore, H2O2‐treated WI‐38 cells significantly increased the mRNA and protein expression of COX‐2, iNOS, and Bax, while kimchis significantly down‐regulated the expressions. In conclusion, the present study indicate that kimchi, especially optimally ripened kimchi, protect against cellular senescence by attenuating oxidative damage induced by free radical.
Increased colonic iNOS gene expression from sustained Western‐style diet exposure
Authors: Zimmerly Ella M , Jang Hyeran , Sauer Julia , Choi Sang‐Woon
Published: June 2021

Risk of colorectal cancer has been associated with inflammation. However, the proinflammatory effects of diet on colorectal carcinogenesis have not been elucidated. Overexpression of inducible nitric oxide synthase (iNOS) and cycloxygenase‐2 (COX‐2) are common to inflammation and colon cancer; their inhibition is chemopreventative. Mechanistically, nitric oxide (NO) has been implicated as a signaling molecule to promote tumorigenesis, and intermediates of NO have been shown to be mutagenic through direct DNA damage or decline in DNA repair. iNOS and COX‐2 have been linked through the stimulatory effect of nitric oxide and peroxynitrite on COX‐2 activity. In this study we used a dietary model of sporadic colon cancer to assess diet‐induced changes in inflammation. For six months C57BL/6 mice were fed either a control (AIN‐76A) or Western‐style diet, which is high in fat and calories and low in vitamin D, calcium, methionine and fiber. In the colonic mucosa we used real time RT‐PCR to examine relative gene expression of enzymes related to inflammation, including iNOS and COX‐2. The western‐style diet increased iNOS expression in the colonic mucosa (p<0.05) but not COX‐2 expression. These results indicate a Western‐style diet may exert a proinflammatory effect on colorectal carcinogenesis through increased iNOS expression. Supported by the NIH Grant R01 AG25834

Grant Funding Source: NIH
Maternal Overweight Programs Offspring Insulin and Adiponectin Signaling
Authors: Shankar Kartik , Kang Ping , Harrell Amanda , Zhong Ying , Ronis Martin J , Badger Thomas M
Published: June 2021

Maternal overweight (OW) was induced in rats by overfeeding via total enteral nutrition. Male offspring from OW dams gained greater (p < 0.005) body weight and %fat mass assessed by NMR, X‐ray CT and adipose tissue weights when fed high fat diet (45% fat). Hepatic microarray analyses at postnatal day 21 revealed 147 transcripts were significantly (±1.8‐fold, p < 0.05) altered by maternal OW, including expression of 20 known SREBP‐1‐regulated transcripts. Expression of SREBP‐1 mRNA and lipogenic transcripts was confirmed using real‐time PCR. Increased nuclear levels of SREBP‐1 and recruitment to the FASN promoter were confirmed via ELISA and ChIP analyses, respectively. Decreased expression of 10 PPARα regulated genes including FGF21 was also uncovered. FGF21 serum levels were lower (p< 0.05) in OW dam offspring. Further serum levels of high mol wt adiponectin (p < 0.05) were decreased in OW dam offspring at weaning. While no changes in %fat mass or adipose tissue adiponectin mRNA were observed, hepatic expression of AdipoR1 and R2 were lower in OW dam offspring. Finally, phosphorylation of the adiponectin target AMPK was significantly decreased in livers of OW dam offspring. Our data suggest that maternal OW at conception leads to increased lipogenic gene expression in concert with decreased PPARα‐adiponectin‐AMPK signaling in the liver of young offspring. Support ARS‐CRIS 6251‐51000‐005‐00D and R01‐DK084225.
Repopulation of β‐catenin null liver with β‐catenin positive progenitor cells and tumor formation after long‐term exposure to DDC
Authors: Thompson Michael D , Singh Sucha , Monga Satdarshan
Published: June 2021

Exposure to DDC diet in mice results in biliary injury that induces atypical ductular proliferation, which is followed by the appearance of oval cells and the development of fibrosis. We previously reported a potential role for β‐catenin in oval cell induction and expansion. After short‐term exposure to DDC, β‐catenin knockout liver had less A6‐positive (oval cell/biliary marker) cells than wild type. To examine the role of β‐catenin in response to long‐term injury, we exposed WT and KO mice to DDC for 80 and 150 days. After long‐term exposure, we observed the emergence of cells with scant cytoplasm and oval‐shaped nuclei, but this response was more dramatic in the KO. Contrary to short‐term exposure (<4wk), KO liver harbored significantly more A6 positive cells than WT. This coincided with increased fibrosis and serum bilirubin levels in the KO. Interestingly, as early as 80 days in the KO, and more extensively at 150 days, we find clusters of hepatocyte‐like cells, which are positive for β‐catenin. We also observed tumor formation upon gross examination of KO liver at 150 days, but not in WT. Thus, we observe a β‐catenin progenitor cell population with the KO livers after DDC exposure that undergoes chronic proliferation and results in tumorigenesis.
Pegylated interferon 2‐alpha mediated down‐regulation of Wnt/beta‐catenin pathway via up‐regulation of nuclear export factor RanBP3
Authors: DAR MOHD JAMAL , Thompson Michael D , Monga Satdarshan
Published: June 2021

Pegylated‐Interferon‐α (Peg‐IFN) is widely used to treat chronic HCV infection and may have anti‐tumor properties. Wnt signaling pathway is hyperactive in a large proportion of Hepatocellular cancers (HCC). Here, we investigate the possibility that Peg‐IFN may act through inhibition of Wnt signaling. We exposed β‐catenin overexpressing mice to chemical carcinogen and began treatment 10 weeks later with Peg‐IFN (7,000 or 70,000 U) for six weeks. Livers isolated from these mice showed decreased nuclear β‐catenin and decreased Ki‐67 staining when compared to saline‐treated mice. However, no change in the β‐catenin gene transcription was observed. In order to understand the mechanism, we carried out in vitro studies on human hepatoma cells (HepG2 & Hep3B). Treatment with Peg‐IFN led to a decrease in the active form of β‐catenin in these cells. Likewise, β‐catenin‐dependent transcriptional activity was decreased by up to 50% as measured by a luciferase reporter assay. Real‐time PCR showed an increase in two negative regulators of Wnt signaling, Dkk1 and RanBP3. Overexpression of RanBP3 in HepG2 cells significantly decreased β‐catenin transcriptional activity. Moreover, SiRNA mediated knockdown of RanBP3 ablated the negative effect of Peg‐IFN on β‐catenin transcriptional activity. In conclusion, Peg‐IFN effectively downregulates Wnt/β‐catenin signaling at least partly through expression of RanBP3, which might be a new target for treatment of β‐catenin associated HCC.
VE‐cadherin restoration does not rescue barrier function in endothelial monolayers depleted of p120
Authors: Herron Crystal Rose , Hollister Patricia R , Lowery Anthony M , Vincent Peter A
Published: June 2021

We previously demonstrated that the association of p120‐catenin (p120) with the juxtamembrane domain (JMD) of vascular endothelial (VE)‐cadherin is required to maintain barrier function of endothelial cell monolayers. Specifically, overexpression of the JMD resulted in increased monolayer permeability, however levels of both VE‐cadherin and p120 were decreased. In order to distinguish if decreased barrier function was due to a loss of p120 and not VE‐cadherin, we established a system in which p120 was depleted by shRNA delivered by lentivirus and VE‐cadherin was restored via adenoviral delivery of VE‐cadherin fused to red fluorescent protein (RFP). Loss of p120 resulted in decreased barrier function as assessed by changed in trans‐endothelial resistance (TEER), which was associated with decreased expression of VE‐cadherin, β‐catenin, and α‐catenin. Decreased TEER was rescued by restoration of p120 but not by expression of VE‐cadherin‐RFP, despite localization of VE‐cadherin‐RFP at cell‐cell borders. Expression of VE‐cadherin‐RFP restored levels of β‐catenin and α‐catenin, implicating that a complex of VE‐cadherin, β‐catenin, and α‐catenin requires p120 to form a restrictive barrier. These results demonstrate that p120 is required for maintaining endothelial barrier function, and experiments are underway to determine the mechanism in which p120 maintains endothelial monolayer integrity. RO1‐HL77870
A Truncated Beta‐catenin Species Promotes Hepatocyte Differentiation in Late Embryonic Liver Development.
Authors: Lade Abigale G. , Monga Satdarshan
Published: June 2021

Beta‐catenin is the effector of the canonical Wnt signaling pathway, a pathway regulating phenomena such as stem cell maintenance and renewal, cell proliferation, cell‐cell adhesion, survival, and differentiation. While membrane‐associated beta‐catenin participates in cell‐cell adhesions, beta‐catenin can also translocate to the nucleus to promote gene expression. Beta‐catenin activity is regulated by post‐translational modification events that control its stability, localization, and affinity for its binding partners. Beta‐catenin is highly expressed in developing embryonic mouse liver, and is critical for proper liver development, as mice with hepatoblast‐specific deficiencies die around E14.5 or E16.5 with evidence of defects in both cell expansion and differentiation. While full‐length beta‐catenin expression progressively decreases in concordance with decreasing proliferation, expression of a 75‐kDa, N‐terminally truncated species steadily increases as liver differentiation occurs. The truncated species exhibits some membranous, but predominantly nuclear/cytoplasmic localization, and its expression correlates with expression of differentiation, rather than proliferation‐associated beta‐catenin targets. Thus, truncated beta‐catenin appears to promote hepatocyte differentiation during embryonic liver development.
Functional evidences of fetal endothelial dysfunction as a programmed phenomenon in pregnancy diseases
Authors: Sobrevia Luis , Casanello Paola
Published: June 2021

Human endothelial dysfunction is a common feature in diseases of pregnancy such as gestational diabetes (GD), pre‐eclampsia (PE) and intrauterine growth restriction (IUGR). Metabolic changes include altered synthesis of nitric oxide (NO), and altered membrane transport of L‐arginine and adenosine in primary cultures of human umbilical vein (HUVEC, macrovascular) and placenta microvillous (hPMEC, microvascular) endothelial cells. These alterations are associated with modifications in the expression and activity of endothelial (eNOS) and inducible (iNOS) NO synthases, respectively, an effect maintained up to passage 5 in culture. Expression and activity of the human cationic amino acid transporter 1 (hCAT‐1) and equilibrative nucleoside transporters 1 (hENT1) and hENT2, as well as the corresponding SLC7A1, SLC29A1 and SLC29A2 gene promoter activities, is exhibited by these cell types. Altered gene expression results from increased NO level, PKC, MAPK, hCHOP‐C/EBPα and Sp1 transcription factors activation. Reduced ENTs‐mediated adenosine transport in GD and PE is associated with stimulation of L‐arginine/NO pathway. In addition, hENT2 activity seems to intend to restore the reduced adenosine transport in GD and PE. It is suggested that a common functional characteristic leading to changes in the bioavailability of adenosine is evidenced by human fetal micro and macrovascular endothelium in GD, PE and IUGR. Supported by FONDECYT 1070865/1080534 (Chile).
Teaching old enzymes new tricks
Authors: Hilvert Donald
Published: June 2021

While nature evolves its catalysts over millions of years, enzyme engineers try to do it a bit faster. Enzyme active sites provide highly optimized micro‐environments for the catalysis of biologically useful chemical transformations. Consequently, changes at these centers can have large effects on enzyme activity. The prediction and control of these effects provides a promising way to access new functions. In this talk, several strategies for tailoring the properties of existing enzymes will be presented, together with the insights into function and evolution that emerge from such studies.
Cadaver‐based practicals versus electronic practicals: Same students, different exams
Authors: Daly Frank
Published: June 2021

At the University of New England, physical therapy doctoral students participate in a prosection‐based human cadaver laboratory and take cadaver‐based practical exams as part of their anatomy and neuroscience courses. Some students can't take their exams as scheduled, due to illness, family emergency or scheduling conflicts. To deal with these situations, an alternative electronic practical exam was developed as a substitute for the cadaver‐based practical examinations. All students fully participated in the cadaver‐based laboratory sessions prior to the electronic practical examinations. Students chose to take the electronic practical even though they were fully aware that they would lose the 3 dimensional and realistic size perspective of the laboratory practical. This is a report on the results of those electronic practical examinations compared to the results of the lab practical examinations taken in the laboratory on cadaver‐based practical examinations. When electronic format practical examination scores were compared to cadaver‐based practical examination scores, there was no statistical difference between the student averages taking the two exams.
Relationships Between Pathophysiology Course Performance and Primary Care Didactic and Clinical Courses and NCCPA Board Scores
Authors: McCleary Vikki L , Hosford Charles C , Kuntz Susan J , Larson Annette C , Laxen Mary Ann J , McHugo Jeanie M
Published: June 2021

In 2006, University of North Dakota School of Medicine and Health Sciences (UNDSMHS) Physician Assistant (PA) Program initiated a study to determine if relationships exist between the pathophysiology course performance and the clinical primary and didactic course performance and also on national board scores in a masters level Physician Assistant Program.

Academic student achievement data were collected in a retrospective document review of students admitted into the UNDSMHS PA Pilot Project in 2006 following IRB approval (# 200804‐303). Data were analyzed using PASW version 17.0. Pearson correlation coefficients were produced and interpreted.

Records of twenty‐nine students (12males/17 females) were reviewed for this study. Mean age and mean years of clinical experience upon matriculation were 36.9 years and 10.0 years respectively. Mean baccalaureate science GPA was 3.0, and overall mean GPA was 3.2. Results of the data analysis revealed significant and positive relationships between the pathophysiology performance and primary care didactic and clinical courses and also on the National Commission on Certification of Physician Assistants.

Results of this study may cultivate discussions centering on PA Program prerequisites and admissions criteria. Additional data will be collected to verify these findings.
Investigating the effects of zinc on rat neural crest cells using an in vitro model
Authors: Yang Hsunhui , Keen Carl L , Lanoue Louise
Published: June 2021

Maternal zinc (Zn) deficiency increases the incidence of heart malformation in rat fetuses, particularly in regions and structures derived from the outflow tract (OFT). To test the hypothesis that Zn deficiency kills cardiac neural crest cells (NCC) and result in heart malformations, we fed gestation day (GD) 1.5 pregnant rat dams a Zn‐adequate (ZnA) or a Zn‐deficient (ZnD) diet. On GD 10.5, we isolated the portion of the neural tube containing the NCC and cultured these explants in control (CT)media, in media containing a Zn chelator (TPEN, 3 uM), or in TPEN‐treated media with ZnCl2 (TZNC, 5 uM) for 4, 6 and 8h. Our results show that the NCC population isolated from dams fed a ZnD diet was smaller compared to NCC from dams fed the ZnA or restricted fed. Regarding the effects of TPEN, cell viability assessed by ATP production was reduced at 4h compared to that of CT or TZNC media; these differences grew more noticeable at 6 and 8h. We observed increased expression of active caspase‐3 and cytochrome c, two markers of apoptotic cell death, and decreased fluozin expression, an intracellular Zn probe, in 6h TPEN‐treated NCC, compared to NCC cultured in CT or TZNC media. Data from this in vitro model demonstrate the vulnerability of cardiac NCC to zinc deficiency, and suggest that Zn deficiency‐induced cell death may contribute to reduce the population of NCC leading to abnormal heart development. (Supported by NIH 2 R01 HD01743).
Myogenin expression is independent of agrin treatment in C2C12 cell culture
Authors: Grow Wade A. , Henley Jessica B.
Published: June 2021

Skeletal muscle differentiation is influenced by myogenic regulatory factors, including MyoD and myogenin. Previous studies have reported that MyoD is expressed prior to myogenin. Our objective was to investigate this process using C2C12 cell culture. When C2C12 myoblasts proliferate to 80% density, growth medium (GM) is replaced with differentiation medium (DM). Subsequently, myoblasts fuse into myotubes within 72 hours in DM. During myotube formation, acetylcholine receptors (AChRs) cluster spontaneously. Treatment with motor neuron derived agrin increases the frequency of AChR clusters through an agrin signaling pathway that also clusters other postsynaptic components of the neuromuscular synapse. Using immunofluorescence, we observed MyoD in myoblast nuclei while myoblasts were in GM, with elimination of MyoD within 72 hours in DM. In contrast, we did not observe myogenin while myoblasts were in GM, with or without agrin treatment, but myogenin was observed in myotube nuclei within 24 hours in DM without agrin treatment. We conclude that myogenin expression is independent of agrin treatment in C2C12 cell culture.

Wade A. Grow was supported by Midwestern University intramural funds.
Cryptic emergence of craniofacial secondary myoblasts
Authors: Noden Drew M , Lance‐Jones Cynthia
Published: June 2021

Most maps of myogenesis in amniote embryos focus on the emergence and movements of primary myoblasts. The assumption that progenitors of secondary myoblasts develop congruently has been shown inaccurate in the trunk, where these cells emerge spatially and temporally apart from their predecessors. Two approaches were used to examine this possible dichotomy in the head of chick embryos. First, patterns of gene expression both within and surrounding precursors of extra‐ocular muscles (EOMs) were probed in situ. Next, the effects on myogenesis of elevated periocular fgf8 levels were assessed. Expressions of pitx2, hgf, sema3a, and myoR revealed a population of myf5/myoD‐negative muscle lineage‐committed cells located within paraxial mesoderm spatially and temporally behind aggregated myf5/myoD‐positive primary myocytes en route to their terminal locations. Treatment with fgf8, applied by implanting protein‐releasing beads, retarded primary myogenesis and resulted in the ectopic differentiation of the myoR‐positive muscle progenitors described above. These results suggest the presence of secondary EOM myoblasts that become committed early to the muscle lineage but remain spatially separate and molecularly cryptic during the period of initial EOM differentiation and morphogenesis. Supported by NIH grant EY01597.
The effects of estrogen, parathyroid hormone, and testosterone on activated raw cells
Authors: Anderson Marquissa , Rose LaKeysha , Tucci Michelle , Benghuzzi Hamed , Cameron Joseph A.
Published: June 2021

Females have higher levels of proinflammatory cytokines following sepsis which is thought to enhance or maintain immune responses, compared to males. The goal of this investigation was to activate macrophages with lipopolysacchride (lps) and then stimulate with estrogen (E), testosterone (T), or parathyroid hormone (PTH) to evaluate the cellular and cytological response over a 24, 48, and 72 hour time period. Both T and PTH showed increased numbers of macrophages after 24 hours which was maintained for the duration of experiment. In addition, by 72 hours, there were increased levels of nitric oxide in all cells challenged with hormones and lps when compared with cells treated with lps alone. The most striking changes were seen the cellular morphology. Cytological evaluation revealed evidence of aggregation and sheet‐like cobblestone appearance of cells treated with estrogen by 24 hours which was maintained for the duration of the experiment. Additional studies are needed to define the role of cellular aggregation and enhanced immune response. Supported by NIH‐NIGMS Grant R 25 GM 50117.
ADP‐Ribosylation of Eukaryotic Elongation Factor 2 by Bacterial Toxins Affects Translation Elongation In vivo and In vitro
Authors: Mateyak Maria Karen , Jan Eric , Kinzy Terri Goss
Published: June 2021

Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA‐mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP‐ribosylation (ADPR) of eEF2 by bacterial toxins on a histidine modified to diphthamide inhibits its translocation activity. Mechanistically, it is unclear how ADPR inhibits eEF2 function. We have developed a novel S. cerevisiae system to directly address this question. This system employs eEF2 mutations which display dominant resistance to diphtheria toxin (DT) expression. These eEF2 mutants lack the diphthamide modification and are not a target of DT; however, their expression allows growth in the presence of wild type ADPR‐eEF2. This is an excellent system to study the in vivo effects of ADPR‐eEF2. For example, yeast expressing both mutant and wild type eEF2 display an increase in parmomycin sensitivity specifically upon DT induction demonstrating a link between ADPR‐eEF2 and translation defects in vivo. These effects are due to the presence of the ADPR‐eEF2, as determined by MS analysis. In addition, we conducted a genetic screen of random yeast 35S rDNA mutations to identify those which confer resistance to DT expression as these mutations may provide insight into how ADPR‐eEF2 functionally interacts with the ribosome. Two 25S rRNA mutations were identified and are being validated. This work was supported by the NIH.
Eicosanoid regulation of hemostasis through concomitant signals in the human platelet
Authors: Holinstat Michael , Vesci Joanne , Apopa Patrick , Boutaud Olivier
Published: June 2021

Anti‐platelet therapy has traditionally targeted the surface receptor of the human platelet. These targets often result in an increase in bleeding which in some conditions may be more deleterious to the patient than the clot itself. Recently, eicosanoid metabolites have been shown to play a crucial role in regulation and stabilization of platelet clot formation. We hypothesized that eicosanoid metabolites such as thromboxane (TxA2) and 12‐HETE may play a significant role in stabilization of platelet clots. To address this question, eicosanoid formation was perturbed in washed human platelets and potential signaling deficits were measured in a number of physiological assays. TxA2 was shown to play a significant role not only in regulating thrombin‐mediated TxA2 formation through feedback via the TP receptor on the platelet surface, but the presence of unique regulatory mechanisms were uncovered indicating the potential for multiple pools of arachidonic acid utilized by the thrombin receptors PAR1 and PAR4. Further, inhibition of the 12‐Lipoxygenase pathway revealed novel physiological regulation of dense granules, Rap1, and GPIIbIIIa. Identification of the underlying mechanisms by which these eicosanoids regulate platelet function is the first step in the development of novel therapeutic treatment of uncontrolled clot formation. This work was supported in part by NIH grant HL089457 (MH).
Hemi‐bis(monoacylglycero)phosphate composition and biosynthesis in Escherichia coli
Authors: Garrett Teresa A , Rose Rebecca L.
Published: June 2021

We are interested in hemi‐bis(monoacylglycero)phosphate (hemi‐BMP) molecules formed in the Gram‐negative bacteria Escherichia coli. Hemi‐BMPs contain a glycerophosphoglcyerol backbone with three fatty acyl chains esterified to the glycerol hydroxyls. Depending on the biosynthetic origin the stereochemistry of the sn‐2 position of the two glycerols differ. One hemi‐BMP molecule known to exist in E. coli is acyl‐phosphatidylglycerol (acyl‐PG). The enzyme PldB, in a reaction in which the acyl chain from 2‐acyl‐lyso‐phospatidylethanolamine is transferred to the sn‐3 position of the head group glycerol, catalyzes the formation of acyl‐PG in Escherichia coli. Interestingly, negative‐ion electrospray mass spectrometry analysis of lipid extracts from an E. coli strain lacking PldB revealed measurable amounts of [M‐H]‐ ions with an exact mass and collision induced decomposition products consistent with acyl‐PG or, potentially, a different hemi‐BMP analog. Toward understanding the formation of the hemi‐BMPs remaining in ΔpldB E. coli we have modernized the in vitro enzyme assay for acyl‐PG formation by PldB by replacing the diethylether historically used in the reaction with detergent. Thirteen detergents were tested at concentrations ranging from 1‐0.01%. CHAPS, 1‐s‐octyl‐β‐D‐thioglucopyranoside (TGP), and octyl‐β‐glucoside were found to support the conversion of 32P‐labeled PG to acyl‐PG using E. coli membranes. Optimal acyl‐PG formation occurred with 0.01% with TGP yielding a specific activity of 1.7 nmol/min/mg. Current work focuses on further optimizing the in vitro reaction by varying ionic strength and pH. The hemi‐BMPs present in ΔpldB E. coli will be purified to determine the stereochemistry of the glycerols in order to gain insight into the composition and biosynthetic origin of hemi‐BMPs in E. coli.
Effects of Particle Size and Protein Composition on Structural Stability and Functional Remodeling of Human High‐Density Lipoproteins
Authors: Gao Xuan , Yuan Shujun , Jayaraman Shobini , Gursky Olga
Published: June 2021

High‐density lipoproteins (HDL) are protein‐lipid complexes that prevent atherosclerosis by removing cell cholesterol. During cholesterol transport, HDL undergo fusion and rupture upon remodeling by plasma factors, and form subclasses differing in size, composition and function. Thus, large HDL are more cardioprotective than small HDL, while HDL(A‐I/A‐II) containing proteins A‐I and A‐II are thought to be less cardioprotective than HDL(A‐I) containing only A‐I, since A‐II is thought to inhibit HDL remodeling. To provide the basis for these functional differences, we tested the effects of particle size and composition on HDL stability, fusion and rupture.

Our spectroscopic, light scattering, calorimetric, electron microscopic, and biochemical studies show that HDL stability decreases with increasing particle size. This may enhance cholesterol uptake from large lipid‐loaded HDL. Surprisingly, HDL(A‐I/A‐II) are slightly less stable than HDL(A‐I). Thus, contrary to the existing notion, A‐II promotes HDL fusion and A‐I dissociation; interestingly, A‐II inhibits HDL rupture. This effect, which may be due to deep penetration of the hydrophobic A‐II into the lipid, may shift the population towards larger more cardioprotective HDL. These results shed new light on the role of particle size and protein composition in HDL remodeling, and help reconcile conflicting reports regarding the role of A‐II on HDL. NIH grants: GM067260 and HL026355
By increasing the level of cardiolipin in the inner mitochondrial membrane, a novel anti‐aging small molecule modulates many longevity‐ and disease‐related processes in mitochondria
Authors: Titorenko Vladimir , Goldberg Alexander , Bourque Simon , Beach Adam , Burstein Michelle , Richard Vincent , Kyryakov Pavlo , Gregg Christopher , Boukh‐Viner Tatiana , Juneau Mylène
Published: June 2021

We use the yeast Saccharomyces cerevisiae as a model to study the mechanisms by which age‐related changes in mitochondrial membrane lipids regulate longevity. We identified a novel anti‐aging small molecule, called LA, which increases the concentration of cardiolipin (CL) in the inner mitochondrial membrane. We: 1) assessed the effect of LA on the life spans of long‐ and short‐lived mutants lacking components of the mitochondrial fission, fusion, and tubulation machines or mitochondrial components involved in the biosynthesis of CL and phosphatidylethanolamine (PE); 2) examined how LA influences the chronology of numerous events characteristic of age‐related, mitochondria‐controlled apoptosis; 3) investigated the effect of LA on the susceptibility of yeast to exogenous pro‐apoptotic compounds targeting mitochondria; and 4) assessed the effect of LA on the age‐dependent dynamics of changes in mitochondrial oxygen consumption, membrane potential, and reactive oxygen species (ROS) generation. Our findings imply that: 1) by increasing CL stability and decreasing the “PE/CL” ratio within the mitochondrial membrane, LA specifically reorganizes the protein machines involved in mitochondrial fusion, fission, apoptosis, respiratory chain, and ROS production, thereby delaying aging; and 2) LA can be used for manipulating mitochondrial dysfunction in aging, neurodegeneration and cardiomyopathy.
Inhibition of SCD activity enhances inflammation in RAW264.7 macrophages but improves cholesterol trafficking
Authors: Liu Xueqing , Joo Diane , Akhtar Zaheer , Ntambi James
Published: June 2021

Obesity is a major risk factor for atherosclerosis. Loss of stearoyl CoA desaturase (SCD) 1 in mice has been shown to protect mice from obesity. However, the role of SCD in atherosclerosis has not yet been fully understood. Macrophage inflammation is a key characteristic of atherosclerosis. Here, we tested the effects of SCD activity on the inflammation and cholesterol transport in RAW264.7 macrophages. Inhibition of the overall SCD activity by A939572, a pharmacological inhibitor targeting the catalytic domain of the enzyme, enhances the induction of inflammatory genes upon LPS treatment in RAW cells, which is prevented by addition of oleate (18:1n9) or palmitoleate (16:1n7), two products of SCD activity. While the total free cholesterol level is elevated in RAW cells treated with A939572, the uptake of oxLDL is, however, reduced. In addition, cholesterol efflux is increased with treatment of A939572 in these macrophages. The above results indicate that although inhibition of SCD activity promotes macrophage inflammation, it improves cholesterol trafficking in RAW macrophages. Thus, SCD might regulate inflammation and cholesterol trafficking through distinct pathways in macrophages.
Thermodynamic and Kinetic Analysis of Membrane Protein Insertion: Case of Diphtheria Toxin T‐Domain
Authors: Ladokhin Alexey S. , Brettmann Joshua , Kyrychenko Alexander , Posokhov Yevgen O. , Rodnin Mykola V. , Thoma Anna
Published: June 2021

The pH‐triggered membrane insertion pathway of the diphtheria toxin T‐domain was studied using site‐selective fluorescence labeling with subsequent application of several spectroscopic techniques. FRET‐based kinetic binding measurements demonstrate rapid association which is largely lipid‐independent. In contrast, the transmembrane insertion kinetics is significantly slower, and is also both pH‐ and lipid‐dependent. Kinetic analysis of binding and insertion indicates the presence of several interfacial intermediates on the insertion pathway of the T‐domain, from soluble W‐state to transmembrane T‐state. Fluorescence correlation spectroscopy measurements of surfactant‐chaperoned membrane insertion indicate that the free energies of stabilizing the interfacial intermediate and the final transmembrane states are −8 and −12 kcal/mole, respectively. We conclude that the acid‐induced membrane action of the T‐domain is modulated by the following two titration transitions with the staggered pH‐dependencies: (a) formation of the membrane‐competent state in bulk solution and (b) formation of the insertion‐competent state on the membrane interface. Anionic lipids not only assist in formation of the insertion‐competent form, but also lower the kinetic barrier for the final insertion. NIH GM069783.
Initiation of lipid binding in Locusta migratoria apolipophorin III
Authors: Phung Yin Mei , Rodriguez Johana , Martinon Daisy , Abdullahi Gezman , Weers Paul M. M.
Published: June 2021

Apolipophorin III (apoLp‐III) from Locusta migratoria is an exchangeable apolipoprotein used as a model to study the structure and function of apolipoproteins. The NMR solution structure of apoLp‐III shows a 5‐helix bundle with an additional 4‐residue helix (helix 4’) located between helix‐4 and helix‐5. We hypothesize that this mini helix initiates lipid binding. An alternative mechanism is that the 1st or 5th helix makes the initial contact with the lipid surface. To test both mechanisms, a site‐directed mutagenesis approach was employed. Helix 4’ was converted into a type I β‐turn, and in separate single mutants Trp‐130 was changed into Gln and Ala‐131 into Ser. In addition, two 4‐helix apoLp‐III mutants were designed by deleting the coding regions for helix‐1 or helix‐2. Based on DNA sequencing, the first round of mutagenesis produced the two single mutations in helix 4’ and the mutant without helix‐1. These three mutant proteins were successfully expressed and purified from E. coli. However, no recombinant protein was found in E. coli for the type I β‐turn and the helix‐5 deletion mutants. Circular dichroism and guanidine‐HCl denaturation analysis will be used to assess the secondary structure and stability of the mutant proteins. Changes in the lipid binding properties will be studied using phospholipid liposomes and binding parameters will be determined by isothermal calorimetry.
The Role of Syndecan‐1 in Arterial Mechanotransduction
Authors: Baker Aaron B , Chitalia Vipul , Steyer Benjamin G , Hirji Sameer , Edelman Elazer R
Published: June 2021

Hemodynamic forces induced by blood flow are potent regulators of vascular homeostasis and arterial structure. While previous work has focused on the role of integrin receptors in mechanotransduction, little is known about the role of cell surface proteoglycans in these processes. This work focuses specifically on the role of the cell surface heparan sulfate proteoglycan syndecan‐1 in vascular cell mechanotransduction. We isolated vascular smooth muscle cells from syndecan‐1 knockout mice and tranfected them with wild‐type and mutated forms of syndecan‐1 using lentiviral vectors. Wild‐type, syndecan‐1 knockout and mutated syndecan‐1 overexpressing mouse vascular smooth muscle cells were cultured in plates with collagen coated silicone membranes. The cells were then subjected to 10% strain at 1 Hz biaxial or uniaxial loads for up to two hours. We found that syndecan‐1 knockout or deletion of the cytoplasmic region of syndecan‐1 caused an increase in actin stress fibers and focal adhesion sites in response to mechanical strain. Further, knockout of syndecan‐1 led to enhanced ERK and Src phosphorylation in response to mechanical strain. These findings support that syndecan‐1 is an important mediator of mechanotransduction in vascular cells.
Chemoenzymatic synthesis of sialyl Lewis x containing different sialic acid forms
Authors: Huang Ronald Wei , Lau Kam , Sugiarto Go , Thon Vireak , Li Yanhong , Ding Li
Published: June 2021

Found in many animals and microorganisms, sialyl Lewis X tetrasaccharide plays essential roles in inflammatory responses and cancer metastasis. In order to better understand the physiological and pathological importance of sialyl Lewis X, it is essential to obtain sialyl Lewis X with substantial purity. A general, convenient, and efficient enzymatic approach for producing sialyl Lewis x tetrasaccharide is attempted. Initially, a one‐pot three‐enzyme approach, using enzymes alpha‐D‐glucose‐1‐phosphate uridylyltransferase (GalU), UDP‐galactose‐4‐epimerase (GalE), and a beta‐1,4‐glycosyltransferase (LgtB), was employed to form disaccharide LacNAc‐beta‐propyl azide from the monosaccharide substrate GlcNAc‐beta‐propyl azide in the galactosylation reaction. Using a one‐pot two‐enzyme system containing Neisseria meningitidis CMP‐sialic acid synthetase (NmCSS) and Pasteurella multocida sialyltransferase (PmST1), we produced Sialyl‐alpha‐2,3‐LacNAc‐beta‐propyl azide. This sialylation reaction was useful in producing different sialic acid forms. In the final step, sialyl Lewis X was produced using a one‐pot two‐ enzyme fucosylation reaction utilizing alpha‐1,3‐fucosyltransferase (alpha1,3FucT) and L‐fucokinase pyrophosphorylase (FKP). The products will be used to generate glycan arrays for glycomics studies.
Design development and application of a fluorescent probe to study changes in hERG channel density and trafficking; a mechanistic basis for cardiac arrhythmia
Authors: Bergeron Zachary L , Collier Abby , Cummins Theodore , Bingham Jon‐Paul
Published: June 2021

Using a novel probe, we will investigate the impact of specific drugs on the surface expression of the hERG potassium ion channel in relation to the cardiac arrhythmias, Torsade de Pointes (TdP) and Long‐QT Syndrome (LQTS) using in vitro models.

Specific Aims

Using BeKm2‐NBDX, we aim to prove that over time, drugs causing aqLQTS by direct block, and drugs causing aqLQTS through inhibition of trafficking events are manifesting the same type of Density‐Related aqLQTS.

Methods

(1) Utilize Solid Phase Peptide Synthesis (SPPS) to bioengineer a fluorescent probe based on the framework of the scorpion toxin peptide BeKm‐1. (2A) Validate the stable expression of the HEK293/hERG over‐expressing cell line by western blot analysis of both transfected and wild type cells and their utility for hERG binding studies with the BeKm2‐NBDX probe. (2B) Demonstrate effective probe conjugation to hERG channel proteins in stably transfected cells using plate‐based assays for quantitation and confocal microscopy for visualization. Further, to optimize environmental variables for maximal binding and fluorescence as well as for extracellular fluorescence inhibition in order to functionalize BeKm2‐NBDX for hERG trafficking and internalization studies. (3) Investigate the impact of (A) channel blocking, and (B) traffic inhibiting drugs known to interact with the hERG ion channel protein. Source of funding, AHA – SDG #0530204N.
A unique DNA binding mode enables human mterf1 to regulate mitochondrial transcription
Authors: Garcia‐Diaz Miguel , Yakubovskaya Elena , Mejia Edison
Published: June 2021

Mitochondria play essential cellular roles that are highly dependent on proper regulation of mitochondrial gene expression. Mitochondrial dysfunction is associated with aging and several human diseases. The mterf family of proteins plays key roles regulating mitochondrial transcription and replication. We have solved the first crystal structure of a member of this family, human mterf1, bound in a sequence‐specific manner to a 22‐mer double stranded regulatory sequence. This 2.2A resolution structure reveals that the mterf proteins adopt a novel DNA binding fold allowing them to bind a relatively long sequence while partially melting the DNA duplex, a feature that is critical for stable binding. This unique binding mode provides insight into the molecular roles of the mterf proteins, explaining how they can positively and negatively modulate transcription. We have further investigated the mechanism of DNA binding through biochemical and structural analysis of different mterf mutants. In addition, our structural observations and equilibrium binding measurements suggest that a human pathogenic mitochondrial mutation that causes Kearns‐Sayre syndrome (KSS), a neuromuscular disorder, interferes with binding of mterf1 to its main recognition sequence, implying that alterations in transcriptional regulation can lead to the development of this disease. This work was supported by NIH R00 ES015421.
Cold‐inducible RNA binding protein modulates proliferation in the mouse mammary epithelium
Authors: Hartley Rebecca S , Mitchell Therese , Guo Xun , Wu Yuehan , Brandt Yekaterina , Hathaway Helen J
Published: June 2021

Cold‐inducible RNA binding protein (CIRP) is upregulated in response to cellular stress, either facilitating or repressing translation of its mRNA targets. We showed that CIRP is upregulated in all breast cancer cell lines examined, while others have shown CIRP overexpression in primary breast tumors. In the MCF‐7 breast cancer cell line, CIRP upregulates the expression of another stress‐induced RNA binding protein, HuR, as well as the cell cycle regulator cyclin E1. Both HuR and cyclin E1 are also overexpressed in breast cancer, with higher expression correlating with a poor prognosis. In order to determine the in vivo function of CIRP in the breast epithelium, we produced a transgenic mouse expressing CIRP in the mammary epithelium under control of the mouse mammary tumor virus (MMTV) regulatory sequences. Real‐time RT‐PCR showed maximal expression of the transgene in the pregnant mouse at gestation day 16, and a second peak of expression after birth, at lactation day 14. Immunohistochemistry for the proliferation marker Ki‐67 showed decreased proliferation in the mammary epithelium of CIRP transgenic mice compared to wild type littermates at gestation day 16 (45% CIRP vs. 56% wild type, p=0.05) and lactation day 1 (7% CIRP vs. 25% wild type, p=0.0068). These results suggest that CIRP negatively regulates proliferation in the intact mammary gland. We are currently examining effects of CIRP expression on apoptosis in the involuting mammary gland. This work was supported by a grant from the National Cancer Institute‐National Institutes of Health to RSH (R01CA095898).