bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulator of G Protein Signaling Protein 6 (RGS6) Protects the Heart from Ischemic Injury
Authors: Rorabaugh Boyd , Mabe Nathaniel , Seeley Sarah , Bui Albert , Yang Jianqi , Neubig Richard , Fisher Rory
Published: June 2021

Background

Regulator of G protein signaling (RGS) proteins are important modulators of cardiac function. We previously reported that disruption of interactions between RGS proteins and Gαi2 protects the heart from ischemic injury. However, the ability of specific members of the RGS protein family to influence cardiac sensitivity to ischemic injury has not been previously explored. The goal of the present study was to determine how genetic deletion of specific RGS proteins influences myocardial sensitivity to ischemic injury.

Methods

Hearts from RGS1 knockout (RGS1‐/‐), RGS2‐/‐, RGS2‐/‐ RGS4‐/‐ double knockout, RGS5‐/‐, and RGS6‐/‐ mice, and their respective wildtype (+/+) littermates were subjected to 30 min ischemia and 2 hours reperfusion using the Langendorff isolated heart model. Infarct sizes were measured by triphenyltetrazolium chloride staining.

Results

RGS6‐/‐ hearts had significantly larger infarcts than RGS6+/+ hearts (51 ± 2 % and 34 ± 5 % area at risk, respectively). Importantly, deletion of RGS1, RGS2, RGS4, or RGS5 had no effect on infarct size indicating that this effect was specific for RGS6. We also found that RGS6 expression was significantly decreased in ventricles (but not atria) of RGS6+/+ hearts subjected to ischemia / reperfusion injury.

Conclusion

These data demonstrate that expression of RGS6 protects the heart from ischemic injury. Modulating RGS6 may provide a novel therapeutic approach for the treatment of ischemic heart disease.
Extended Biomimetic Culture and Functional Assessment of Recellularized Human Lungs
Authors: Gilpin Sarah , Charest Jonathan , Tapias Luis , Ren Xi , Wu Tong , Ott Harald
Published: June 2021

Rationale

Native lung scaffolds prepared by decellularization provide a novel platform for organ engineering. Recellularization with specific cell types requires ex vivo culture for evaluation and restoration of organ function prior to transplantation.

Methods

Human lung‐derived epithelial and endothelial cells were isolated and expanded in vitro. Immunostaining, flow cytometery, and qPCR confirmed cell identity. Epithelial and endothelial cells were delivered to lung scaffold airways and vasculature respectively (>350x106).

Constructs were cultured for 10 days in a custom automated, closed‐circuit bioreactor, providing physiologic perfusion and negative pressure ventilation. Perfusate was analyzed daily by iSTAT. Tissue was analyzed by histology and qPCR.

Results

Donor‐derived epithelium was a population of primarily basal‐cell phenotype (70% p63+), with 20% CC10+, and minor pneumocytes (<10% SP‐B/C) by flow cytometry. Basal cell ciliogenesis was confirmed by ALI. Endothelium was sorted to CD31 purity.

Biocompatibility to lung matrix confirmed maintenance of cell phenotype, viability, and proliferation (Ki67+) in slice co‐culture.

Recellularized constructs were cultured for 6 days with constant perfusion, plus 4 days with additional fluid or air ventilation. Increasing rate of glucose metabolism and lactate production in the perfusate was quantified over time. Measured pO2, pCO2, and pH remained consistent.

Cell distribution and viability was confirmed by Resazurin metabolism, and by histology/qPCR of serial tissue biopsies.

Conclusion

Intact acellular lung scaffolds can be repopulated with primary tissue‐derived cells and maintained in extended ex‐vivo biomimetic culture, supporting cell viability and function.

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Phosphorylation of Myosin Light Chain (MLC) is Mitochondrial ATP Dependent and Rho‐kinase Independent During Fluctuation‐Driven Mechanotransduction
Authors: Bartolak‐Suki Elizabeth , Suki Bela
Published: June 2021

Rho‐kinase (ROCK) play central roles in cell contractility by stimulating the phosphorylation of MLC. We hypothesized that vascular cells utilize physiological fluctuations in their mechanical environment due to blood pressure variations to maintain proper contractility through ROCK in an ATP dependent manner. Vascular smooth muscle cells (VSMCs) were isolated from bovine aortae. Cells were seeded on elastic membranes, starved for 24 h and stretched equibiaxially at 10% change in area for 4 h without (monotonous stretch: MS) or with adding 25% (VS) physiological cycle‐by‐cycle variability to stretch amplitude. Unstretched (US) cells were controls. The fluorescent probe TMRM was used to visualize mitochondrial (mt) membrane potential (mMP) a surrogate of ATP production. Y27632 was used to inhibit ROCK and oligomycin to block mitochondrial ATP production. Cell ATP levels were measured by the luciferase assay. Western blot was used to detect phosphoMLC (phMLC). Compared to VS, mMP was reduced during both MS (66%) and US (91%). Oligomycin did not change mMP during MS, but reduced during VS (27%) and US (13%) (p<0.01). Y27632 elevated mMP during MS to the level of VS without affecting it during VS or US. ATP was the highest during US (50%) and lowest with MS (25%) compared to VS (p<0.001). The phMLC level was the highest during VS and was reduced during MS (50%) and US (40%), whereas mt ATP inhibition reduced phMLC to the same low level in all conditions (p<0.001). Thus, contractility of VSMCs was stretch and mt ATP dependent but ROCK only affected ATP during MS. We conclude that changes in stretch pattern in hypertension could be involved in the pathogenesis of vascular diseases through ROCK and the mitochondria.
Oxidized low‐density lipoprotein decreases endothelial progenitor cell populations in bone marrow and peripheral circulation independent of ROS production
Authors: Liu Dylan , Cui Yuqi , Liu Jason , Narasimhulu Chandrakala , He Guanglong , Cowan Peter , Parthasarathy Sampath , Zhu Hua
Published: June 2021

Bone marrow (BM)‐derived endothelial progenitor cells (EPCs) make significant contribution to the function and integrity of vasculature. The number of EPCs is significantly decreased in hyperlipidemic patients. Oxidized low‐density lipoprotein (ox‐LDL) is a major component of hyperlipidemia and contributing to atherosclerosis. The present study was to determine if ox‐LDL could change EPC populations and the role of reactive oxygen species (ROS). EPC numbers and ROS formation in BM and blood were determined in male C57BL/6 mice treated with ox‐LDL intraveneously. Flow cytometry analysis and EPR measurement showed that the ROS production in both BM and blood was significantly increased in ox‐LDL‐treated mice that was effectively blocked with concomitant overexpression of copper/zinc superoxide dismutase (SOD1), extracellular SOD3, and glutathione peroxidase or N‐acetylcysteine treatment. Ox‐LDL treatment produced complex changes in EPC populations in BM and blood. In ox‐LDL‐treated mice, the c‐Kit+/CD31+ cell number was significantly decreased in BM and blood, and the Sca‐1+/Flk‐1+ cell number was significantly decreased in blood but not in BM. Ox‐LDL decreased the cell population of c‐Kit+/CD31+ in BM and blood, and the Sca‐1+/Flk‐1+ cell number in blood with a mechanism predominantly independent of ROS production in vivo.
Compartmentalization of ClC‐3 and TNF‐induced Superoxide Production
Authors: Bacon Matthew , Nguyen Hong‐Ngan , Choi Hyehun , Ettinger Nicholas , Lamb Fred
Published: June 2021

ClC‐3 is required for NADPH Oxidase (Nox)‐dependent endosomal superoxide (O2‐) production and NF‐kB activation in response to cytokines. We co‐localized ClC‐3, Rab‐GTPases and O2‐ following TNFa treatment of HEK293 cells. ClC‐3, Rab5(A, B, C), Rab7 (GFP, mCherry, RFP tags) and O2‐ (ClC‐3‐HyPer fusion protein) were visualized by confocal microscopy. Expressed ClC‐3‐GFP was precipitated using bead‐immobilized GFP‐binding protein under detergent‐free conditions and Western analysis assessed ClC‐3‐Rab associations. An NF‐kB luciferase reporter quantified activation. Alone, ClC‐3 localizes to enlarged vesicles. Tagged ClC‐3 associates with Rab5A, B, and C in early endosomes and vesicles expressing both proteins were small. ClC‐3‐GFP pulled down primarily endogenous Rab5A. Dominant negative (S34N) and constitutively active (Q79L) Rab5A altered the distribution of ClC‐3 and drastically reduced NF‐kB activation by TNFa. The S34N mutant induced discrete small vesicles while Q79L yielded tight multi‐vesicle clusters. ClC‐3 and Rab7 co‐expression produced larger late endosomal vesicles and the overlap of ClC‐3 with Rab7 was more extensive than with Rab5. Under resting conditions ClC‐3‐HyPer co‐localized best with Rab7 vesicles and this signal was enhanced by TNFa and quenched by N‐acetylcysteine. Localization of O2‐ with Rab5 is ongoing. These data demonstrate that ClC‐3 is both an early and late endsosomal protein. In early endosomes it is trafficked by Rab5A and altered function of this GTPase disrupts both localization and TNFa signaling. In resting cells, basal O2‐ production occurs primarily in late endosomes (Rab7). Future work aims to determine if O2‐ is produced in endocytic vesicles or early endosomes following activation of TNFa receptors.
Activation of cardiac P2X receptors in hypertensive rats displays exaggerated sympathoexcitatory reflexes
Authors: Fu Liang‐Wu , Longhurst John
Published: June 2021

The concurrence of hypertension and ischemia leads to increased morbidity and fatality in individuals compared to myocardial ischemia alone. Ischemic mediators activate cardiac sympathetic afferents and evoke cardiac sympathoexcitatory reflex (CSR) responses. Recent work has revealed that the ischemic mediator ATP stimulates ischemic‐sensitive cardiac sympathetic afferents mostly through activation of P2X receptors. However, little is known about these mediators in CSR responses of hypertensive animals. We therefore hypothesized that activation of P2X receptors in the cardiac sensory nerves exaggerates CSR responses in hypertensive compared to normotensive animals. Arterial pressure and renal sympathetic nerve activity in response to intrapericardial injection of the P2X receptor agonist α,β‐meATP (31 to 125 nmol) were recorded in four groups of rats (n=5/each group), two of which were hypertensive, including cold‐induced hypertension (CIH) and spontaneous hypertension (SHR). The other two groups included SD and WKY normotensive rats. A larger CSR was evoked in response to intrapericardial α,β‐meATP in hypertensive (CIH and SHR) rats vs. normotensive (SD and WKY) rats in a dose‐dependent manner. In all four groups, intrapericardial injection of vehicle produced no CSR response. These data suggest that cardiac sympathoexcitatory reflexes induced by activation of P2X receptors are exacerbated in hypertensive animals. (Funded by HL‐66217)
Mdivi‐1, a Novel Mitochondrial Fission Inhibitor, Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion (MI/R) Injury
Authors: Stutzman Devon , Schmidgall Carly , Benjamin Israel , Chen Qian , Hatcher Cathy , Barsotti Robert , Young Lindon
Published: June 2021

MI/R results in cardiac contractile dysfunction and increased cell death. MI/R is initiated in part by uncoupling of the electron transport chain in mitochondria, which generates reactive oxygen species (ROS). Increased ROS lead to the loss of mitochondrial membrane potential, which augments mitochondrial fission in MI/R. Mitochondrial fission is in turn associated with shortening of mitochondria, decreased ATP production, and is thought to promote cardiac contractile dysfunction and post‐reperfused cardiomyocyte loss, leading to increased infarct size. Therefore, inhibiting mitochondrial fission may be an effective new strategy to salvage injured cardiac myocytes during MI/R. Mdivi‐1 (MW= 353 g/mol), a mitochondrial fission inhibitor, that acts by selectively inhibiting dynamin related protein 1, a GTPase that promotes mitochondrial fission via interaction with outer mitochondrial membrane proteins, was used to test this strategy. Isolated perfused rat hearts subjected to I (30 min)/R (90 min) were infused with Mdivi‐1 (25 µM) given for 5 min at the beginning of reperfusion. Mdivi‐1 treated hearts (n=6) significantly reduced infarct size to 25% ± 2%, as compared to control I/R hearts (n=9) 44% ± 4% (p< 0.01). However, Mdivi‐1 only transiently improved post‐reperfused cardiac function at this dose, compared to control I/R hearts. These preliminary results suggest that inhibition of mitochondrial fission salvages cardiac tissue in MI/R. Future studies will test whether other Mdivi‐1 treatment regimens will prove to be more effective.
Substrate Utilization Effects on Shortening Properties in Aging Cardiac Myocytes
Authors: Barton Greg , Aiken Judd , Diffee Gary
Published: June 2021

Aging is associated with declining cardiac contractile function as well as changes in substrate metabolism and mitochondrial function. The relationship between age‐related changes in cardiac metabolism and declining cardiac contractile function has not been determined. We explored the role of alterations in substrate metabolism on age‐related changes in contractile function of isolated left ventricular myocytes from Young (Y) and Old (O) FBN rats. Single, intact myocytes were stimulated for ten minutes of continuous contraction at either2Hz or 4Hz while being perfused with Ringers solution containing either Glucose (11mM) or Oleate (0.4mM) as the fuel substrate. Properties of shortening (% and rate of shortening) were measured at the onset (T0) and after 10 minutes (T10) of continuous contraction, and the decline in shortening over time (T10/T0) was determined at each workload. Myocytes from Y hearts generally had better contractile function when glucose was the substrate, with a smaller workload‐dependent decline in function, compared to when oleate was the substrate. In aged myocytes, the opposite was true, with O myocytes showing less workload‐dependent decline in percent shortening and rate of shortening when oleate was the substrate. This data suggests that when forced to use only one substrate, myocytes from young hearts are better able to use glucose, while cells from old hearts are better able to metabolize oleate.

Supported by NIH AG030423
Maturation Decreases Fractional Activation of Myosin Light Chain Kinase in Ovine Common Carotid Arteries
Authors: Williams James , Adeoye Olayemi , Pearce William
Published: June 2021

The present study tests the hypothesis that postnatal changes in contractile protein organization drive changes in MLC20 phosphorylation that contribute to age‐related changes in contractility. In homogenates, MLCK exhibited similar enzyme velocities in fetal and adult ovine carotid arteries. To compare MLCK activity in situ, common carotids were prepared and mounted in a custom‐made organ bath that enabled solenoid control of tissue freezing in methacarn after 0, 1.0, 2.0 or 3.0 s stimulation at optimal voltage. Fixed segments were divided to measure MLC20, ser19 pMLC and MLCK by Westerns and confocal microscopy. Postnatal maturation significantly decreased in situ MLCK activity (F: 1.05±0.28: A: 0.42±0.08 ng pMLC/ng MLCK/sec), while significantly increasing MLCK (∼100%) and MLC20 (∼60%) abundances. The ratio of Mander's colocalization coefficients (m1 to m2), an index of the ratio of MLCK to MLC20 masses, exhibited identical values in adult and fetal arteries. However, when the mass of MLCK used to normalize MLCK activity was corrected to calculate only the mass of MLCK colocalized with MLC20, the corrected estimates of MLCK velocity, in situ, were identical in fetal and adult arteries (F: 1.70±0.12; A: 1.75±0.09 ng pMLC/ng MLCK/sec) at peak activation. These data strongly suggest that postnatal differences in contractility are due to changes in contractile protein organization, that proportionately more of total MLCK is colocalized with MLC20 in fetal than adult arteries, and that maturation significantly decreases fractional activation of MLCK (68.5±5.0 F, 27.3±1.8% A).
Metaboreflex Activation Delays Heart Rate Recovery after Aerobic Exercise
Authors: Peçanha Tiago , Brito Leandro , Fecchio Rafael , Sousa Patricia , Silva‐Júnior Natan , Forjaz Cláudia
Published: June 2021

The role of metaboreflex on heart rate recovery after exercise (HRR) is controversial. It is speculated that this mechanism is active in exercises with larger muscle masses, however it has not been tested after a typical aerobic exercise. The aim of this study was to evaluate the influence of metaboreflex on HRR. 12 healthy middle‐aged men randomly underwent 2 sessions consisting of an exercise (cycle ergometer, 70% VO2peak, 30 min) followed by 5 min of recovery. In each session, the recovery was performed with (occlusion) or without (control) leg circulatory occlusion (cuffs at the hips inflated to supra‐systolic pressure). ECG was registered and HRR was assessed by: heart rate reduction after 60 (HRR60s) and 300s (HRR300s) of recovery; short‐term time constant of HRR (T30); time constant of the HRR after exponential fitting (HRRτ); and mean values of HR in segments of 30s (HR30s). Heart rate variability (HRV) was assessed by the root mean square residual (RMS) and square root of mean squared differences of successive R–R intervals (rMSSD) on subsequent 30‐s segments. Paired T‐test, Wilcoxon test and ANOVA were employed. HRR60s (25 ± 7 vs. 22 ± 12 beats.min‐1, p = 0.69), T30 (284 ± 155 vs. 309 ± 253 s, p = 0.89) and HRRτ (120 ± 45 vs. 107 ± 103 s, p = 0.75) were similar between control and occlusion sessions, respectively. HRR300s was significantly lower in occlusion session (30 ± 13 vs. 38 ± 14, p = 0.05). There was no session vs. time interaction for the rMSSD index (p = 0.46), while HR30s and RMS had significant time vs. session interactions (p = 0.00 and 0.01, respectively), showing slower decrease of HR30s and slower increase of RMS in the occlusion session. In conclusion, metaboreflex activation delays HRR, particularly at the slower phase of HRR.

Funding: FAPESP 2013/044997‐0 and 2013/05519‐4; CAPES PROEX
Exercise Mitigates Hyperhomocysteinemia Induced Vascular Dysfunction and Adverse Skeletal Muscle Remodeling
Authors: Winchester Lee , Veeranki Sudhakar , Pushpakumar Sathnur , Givvimani Srikanth , Tyagi Suresh
Published: June 2021

Intro

Hyperhomocysteinemia (HHcy) is associated with vascular and skeletal muscle dysfunction. HHcy induces MMP‐9 activity, myostatin release, systemic inflammation and may increase M1 macrophages. Previous reports suggest that exercise reduces HHcy and myostatin levels, however the effects of exercise on macrophages and myostatin during HHcy are unclear. We hypothesize that exercise decreases HHcy induced inflammation and muscular dysfunction by decreasing MMP‐9, myostatin and M1 macrophages.

Methods

C57 WT, FVB, MMP‐9‐/‐, CBS‐/+ (HHcy), and CBS‐/+/MMP‐9‐/‐ (Double KO) mice were administered 4 weeks of treadmill running, swimming, or no exercise for controls. Ultrasonography and laser Doppler were used to measure wall to lumen ratio, blood flow and perfusion rate of the femoral artery. Western Blotting, IHC, Histology and PCR were used to analyze mouse tissue samples.

Results

Blood flow rates in control CBS‐/+ mice were approximately 23.9% lower than in control C57 WT mice. This rate was increased by 1.89 fold with exercise. Wall to Lumen ratio was approximately 4.14 fold higher in the CBS‐/+ control mice compared with WT, but became similar to WT after exercise. Laser Perfusion imaging demonstrated a 12.5% lower femoral artery red blood cell flux in CBS‐/+ mice when compared with WT, but was increased by 1.35 fold after exercise. Collagen staining reveals higher collagen deposition in the gastrocnemius of CBS‐/+ mice compared with WT, but was completely reversed with exercise.

Conclusions

We conclude that Exercise Mitigates HHcy Induced Vascular Dysfunction and Adverse Skeletal Muscle Remodeling.
The Effects of Perinatal Androgen Deprivation on Sexual Motivation in Male Rats
Authors: Khan Talha , Yn Alicia , Fickes Dane , Vang Pakou , Jeong KumHwa , Shah Binita , Yousaf Faeeq , Taylor George , Gopalan Chaya
Published: June 2021

Testosterone and its metabolites—estrogen and dihydrotestosterone— are involved in the brain sex differentiation during the perinatal period by binding to androgen receptors (AR) or estrogen receptors (ER). However, in the presence of flutamide (FLU), testosterone that is metabolized to estrogen will only bind to ER. Blockade of AR using FLU during the perinatal period could alter the levels of estrogen, which in turn, may modulate the sex differentiation of the brain. This study tested perinatal androgen deprivation on estrogen levels and in turn, its effects on sexual motivation paradigm in male rats.

Animals (N=37) were divided into two groups: Group 1 received FLU (20 mg/kg; SC) during perinatal period (prenatal day 4 ‐ postnatal day 4) and Group 2 served as control. On postnatal day 65, these groups were divided into 3 subgroups (injected 5 days/week x 7 weeks): A. finasteride (20 mg/kg, SC), an α‐reductase inhibitor, B. letrozole (2 mg/kg, SC), an aromatase inhibitor and C. vehicle. Each male rat had a choice of approaching a cage containing a gonadally‐intact female or an ovariectomized (OVX) rat for 20 minutes. The movement of the male rat was video‐tracked by Any‐Maze. In addition, serum estrogen levels were measured using ELISA.

Animals receiving FLU not only had high serum estrogen levels, but also exhibited no preference for the gonadally‐intact or the OVX female. On the other hand, the control‐vehicle group spent more time with the gonadally‐intact than the OVX rat. These results suggest that perinatal androgen deprivation in male rats results in high levels of estrogen and lack of sexual motivation towards gonadally‐intact female rats.
Protein Methyltransferase Inhibitors as Cancer Therapeutics
Authors: Copeland Robert
Published: June 2021

The protein methyltransferases (PMTs) constitute a class of enzymes that catalyze the methylation of lysine or arginine residues on histones and other proteins. A number of PMTs have been shown to be genetically altered in cancers through, for example, gene amplification, chromosomal translocations, point mutations and synthetic lethal relationships. DOT1L and EZH2 provide two representative examples of altered PMTs that act as genetic drivers of specific human cancers. The enzymatic activity of DOT1L is associated with MLL‐rearranged leukemia. Point mutations in EZH2 are found among non‐Hodgkins lymphoma patients; the enzymatic activity of both wild type and mutant EZH2 are required for pathogenesis in these patients. Also, deletion of the INI1 (SMARCB1 or hSNF5) subunit of the SWI/SNF chromatin‐remodeling complex occurs in nearly all malignant rhabdoid tumors (MRTs), a childhood cancer with a particularly poor prognosis. An antagonistic relationship has been demonstrated between the SWI/SNF complex and EZH2 that is relieved in INI1 deficient cancers. MRTs show increased reliance on EZH2 for proliferation and are selectively killed by EZH2 inhibitiors in cell culture and in mouse xenograft models. Drug discovery efforts have yielded potent, selective inhibitors of each of these targets that have now transitioned into phase 1 clinical trials. These inhibitors affect the appropriate histone methyl marks in cells, lead to selective cell killing that is dependent on genetic alteration of the target enzyme and effect tumor growth inhibition in xenograft models. These data portend the effective use of selective PMT inhibitors as future personalized cancer therapeutics.
Mechanisms for Translesion DNA Synthesis
Authors: Yang Wei , Lee Young‐Sam , Gao Yang , Gregory Mark
Published: June 2021

In humans among the 17 DNA polymerases identified only five are dedicated to normal DNA replication, and more than 12 are specialized DNA polymerases that are involved in repair and translesion DNA synthesis (TLS). The specialized DNA polymerases are mainly belong to the X and Y families, but a few are in the A and B families and homologous to high‐fidelity replicases. In this talk, I will give examples of DNA polymerases in different structural families that bypass DNA lesions by different mechanisms. For example, human DNA pol eta is essential for UV survival. Deficiency of DNA pol h causes the variant form of Xeroderma Pigmentosum (XPV), characterized by sunlight‐induced pigmentation changes and a highly elevated incidence of skin malignancies. DNA pol z is a B‐family member like E. coli DNA pol II, but it is essential for primer extension in translesion synthesis of a variety of DNA lesions. DNA pol n is an A‐family member and another TLS polymerase. These specialized polymerases appear to use different mechanisms to avoid “road blocks” and carry out translesion DNA synthesis. I will present our most recent results on discerning different mechanisms of TLS essential for cell survival.
Metabolites of Blueberry Anthocyanins Suppress Lipotoxicity Induced Endothelial Inflammation
Authors: Cavalcanti Rafaela , Begaye Nathan , Costa Marcella , Bharath LP , Symons JD , Jalili T , Rajasekaran NS , Jia Z , Babu PVA
Published: June 2021

The vascular benefits of anthocyanins might be mediated by their circulating metabolites. This mechanism has not been explored because the metabolites are not commercially available. We synthesized the blueberry (BB) metabolites vanillic acid‐4‐sulfate (V4S), isovanillic acid‐3‐sulfate (IV3S) and benzoic acid‐4‐sulfate (B4S) and verified their purity (>98 %) using HPLC‐mass spectrometry. We tested the hypothesis that a mixture of these BB‐metabolites attenuates endothelial inflammation induced by lipotoxicity. Human aortic endothelial cells (HAEC) were treated ± BB‐metabolite mixture for 6 h and ± 500 μM palmitate for the last 5 h. The BB‐metabolite mixture contained V4S, IV3S, B4S, hydroxyhippuric acid, and hippuric acid at concentrations reported to peak in the blood 4 ‐ 6 h after consuming 240 g of blueberries in humans. Cell adhesion (assessed via human monocytic THP‐1 cells), and mRNA expression of ICAM‐1, VCAM‐1, E‐selectin, MCP1, IL‐8, NFκB‐p65, IKKβ, IKBα (assessed via qPCR), were quantified ± palmitate ± BB‐metabolites. Palmitate‐treatment increased (p<0.05) cell adhesion and increased (p<0.05) mRNA expression of ICAM‐1, VCAM‐1, E‐selectin, MCP‐1, IL‐8, and IKBα without affecting cell viability. All these effects were ameliorated (p<0.05) by BB‐metabolites. BB‐metabolites, at physiologically relevant concentrations suppressed lipotoxicity induced endothelial inflammation, which may be mediated via NFκB pathway. Blueberries might complement existing therapies to improve vascular complications.
Characterizing household food shopping behavior using novel receipt‐based geospatial measures
Authors: Barnes Timothy , French Simone , Mitchell Nathan , Van Riper David
Published: June 2021

Background

In efforts to better understand how geographic food access measures influences dietary behaviors, researchers are developing second‐generation methodologies to explore and track how individuals behave within their food environment.

Methods

Using receipt‐based data collected over four weeks in 90 households in Minneapolis/St. Paul, MN and geographic information systems (GIS), we created two geospatial measures to characterize households' shopping spaces via minimum bounding geometry. Thus, a spatial ‘footprint’ representing the geographic areas households purchased food at supermarkets and restaurants was determined. Statistical correlation with traditional measures of shopping behavior (frequency and distance traveled) were assessed along with associations with food purchasing (in dollars) and dietary intake of fruit, vegetables, and sugar‐sweetened beverages.

Results

On average, households' shopping space for supermarkets and restaurants were 15.9 and 41.8 square miles, respectively. Shopping space was significantly correlated with both frequency of shopping and distance traveled for supermarkets (r=0.59 and r=0.54) and restaurants (r=0.74 and r=0.45). Neither shopping space for supermarkets or restaurants were significantly associated with purchases of fruits and vegetables, however, restaurant shopping space was significantly associated with expenditures for sugar‐sweetened beverages (β=0.06, p=0.004) and vegetable intake (cup servings) during the past 7 days (β=‐0.01, p=0.015).

Conclusions

Findings suggest that household shopping space may capture shopping and purchasing behaviors of interest to researchers examining the relationship between the food environment and dietary intake.
Imaging IGF‐I Uptake in Growth Plate Cartilage Using in vivo Multiphoton Microscopy
Authors: Serrat Maria , Ion Gabriela
Published: June 2021

Bone elongation occurs in cartilaginous growth plates located at the ends of the principal long bones. Linear growth is driven by a cascade of cellular signals initiated by endocrine and paracrine regulators acting on growth plate chondrocytes. Relative to our knowledge of their signaling mechanisms, surprisingly little is known about how these molecules physically reach avascular cartilage (which lacks a penetrating blood supply) or how they are transported from surrounding blood vessels through the dense extracellular matrix. In vivo multiphoton microscopy is a powerful imaging modality that enables the study of intact growth plates with cellular level resolution. This imaging approach has potential to fundamentally advance our understanding of bone lengthening processes by allowing us to track molecular delivery to cartilage in real time. We developed quantitative methods for measuring the uptake of biologically active IGF‐I into tibial growth plates of live 5‐week old mice. We demonstrate that fluorescently‐labeled IGF‐I is readily taken up in growth plate cartilage and localizes to chondrocytes. In vitro bioactivity tests confirmed that the labeled protein is functional, assessed by phosphorylation of its downstream targets. These results are relevant for understanding mechanisms of growth plate regulation and for developing strategies to modulate limb elongation rate through growth factor delivery to cartilage.

Supported by ASBMR GAP, UK‐CCTS (NIH UL1TR000117), and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (R15AR067451‐01).
Comparing arginine flux, catabolism and availability for NO synthesis in American, Indian, and Jamaican women
Authors: Hsu Jean , Kao Christina , Dwarkanath Pratibha , Karnes Jeffrey , Baker Tameka , Bohren Kurt , Badaloo Asha , Thame Minerva , Kurpad Anura , Jahoor Farook
Published: June 2021

We have reported that Indian women do not have increased Arg production during pregnancy like their American or Jamaican counterparts. There has not been a direct comparison of Arg flux, catabolism, and availability for NO synthesis in non‐pregnant American, Indian, and Jamaican women. The objective of this study was to determine whether Indian women are producing less Arg and/or catabolizing more Arg to Orn and therefore have less Arg available for anabolic pathways than their Jamaican and American counterparts. Ten healthy women of childbearing age from each country were given 6h primed, constant intravenous infusions of 15N2‐Arg, 2H2‐Cit, 15N2‐Orn, and 2H5‐Phe in both the fasted and fed states to measure Arg flux, hydrolysis, bioavailability, NO synthesis and Phe flux, an index of protein breakdown.

USA
India
Jamaica
P Value

μmol/kg FFM/h
μmol/kg FFM/h
μmol/kg FFM/h

Fasted

Arg flux
56.1 ± 2.1a
54.2 ± 3.1a
66.1 ± 3.1 b
0.01

Arg hydrolysis
17.6 ± 1.5 a
23.7 ± 1.5 b
23.4 ± 1.6 b
0.01

Arg bioavailability
49.9 ± 1.3 ab
42.0 ± 2.6 a
55.5 ± 3.5 b
0.004

NO Synthesis
0.18 ± 0.01a
0.15 ± 0.01 b
0.21 ± 0.02 a
0.002

Phe flux
39.7 ± 0.9
39.0 ± 2.2
43.8 ± 2.3
0.17

Fed

Arg flux
79.1 ± 3.3 a
80.3 ± 4.9 a
95.9 ± 2.9 b
0.008

Arg hydrolysis
27.3 ± 2.5 a
39.6 ± 3.7 b
37.2 ± 2.0 b
0.01

Arg bioavailability
51.8 ± 1.2 a
40.7 ± 3.5 b
57.5 ± 3.2 a
<0.001

NO Synthesis
0.20 ± 0.01a
0.21 ± 0.03a
0.34 ± 0.04 b
< 0.0001

Phe flux
58.8 ± 1.6
59.4 ± 3.0
63.3 ± 2.7
0.39

Means with different superscript letters are significantly different, P < 0.05 (post‐hoc Tukey's multiple comparison test).

In conclusion, Indian women had increased Arg hydrolysis without a compensatory increase in Arg flux resulting in decreased bioavailability of Arg for the synthesis of proteins and other Arg‐derived biomolecules, including NO synthesis. Though American women also had slower Arg flux compared to Jamaican women, they were able to conserve Arg bioavailability because of decreased Arg hydrolysis. The results suggest that Indian women may need a higher dietary Arg intake.

Supported by the Bill and Melinda Gates Foundation
The Microenvironment of Injured Mucosa Stimulates a Local Pro‐restitutive Microbiota
Authors: Alam Ashfaqul , Leoni Giovanna , Quiros Miguel , Jones Rheinallt , Nusrat Asma , Neish Andrew
Published: June 2021

Restitution of mucosal injury involves induced and coordinated proliferation and migration of intestinal epithelial cells. The commensal microbiota of the intestine is integral to the repair of damaged intestinal mucosa. N‐formyl peptide receptors (FPRs) are widely expressed pattern recognition receptors that specifically respond to host‐derived and microbial peptides in a redox dependent manner. However, little is known about the host‐microbiota crosstalk mediated by FPRs during repair of gut mucosal injuries. We hypothesized that distinct members of the gut microbiota preferentially colonize the restitutive mucosa to promote wound repair processes in a FPR1‐dependent fashion. For this purpose, defined mechanical wounds were inflicted in the mouse distal colon by using an endoscope and forceps. High throughput sequencing of the V4 region of 16s rRNA gene of the bacteria harvested from mucosal wounds revealed a spatiotemporal shift in the composition and diversity of microbiota. We found several FPR1 and NADPH oxidase NOX2‐dependent microenvironmental changes in mucosal wounds, which include rapid induction of a hypoxic microenvironment, and expression of several HIF1a‐regulated genes, including the mucin muc 3. Interestingly, these events were followed by a dramatic enrichment of a distinct commensal species Akkermansia, an anaerobic, mucinophilic bacterium, which stimulates FPR1 on intestinal epithelial cells to generate reactive oxygen species (ROS) via enterocyte NOX1, resulting in enhanced migration & proliferation of enterocytes. These findings identify a novel role of FPR1 to promote microenvironmental changes that enrich a specific mucosa‐associated bacterium to enhance gut mucosal wound restitution.
Mechanism of Liver Injury Caused by Autophagy Deficiency
Authors: Khambu Bilon , Zhang Hao , Li Yong , Chen Xiaoyun , Yin Xiao‐Ming
Published: June 2021

Autophagy is a vital intracellular degradation process which is required for many physiological & pathological processes. Deficiency of autophagy in the liver causes severe injury characterized by increased liver function enzymes in serum & hepatocyte hyperplasia & degeneration. Mice lacking one of the key autophagy genes, such as Atg7, could also be subjected to a high mortality due to liver failure. However it is not clear how autophagy deficiency causes liver injury. The goal of this study is thus to investigate the potential mechanism of injury. A careful examination of the liver histology of the Atg7‐deficient mice indicates that there was inflammation, cell death, & significant ductular reaction. We hypothesized that there was a notable level of cholestasis, which can cause cholestatic hepatitis & thus liver injury. Indeed, further blood chemistry analysis indicated a raised level of total bile acids, cholesterol, & alkaline phosphatase. This exciting new finding suggests a molecular linker from autophagy to cholestasis regulation. Previously it has been reported that anti‐oxidative stress master gene, Nrf2, is involved in autophagy deficiency‐induced liver injury. Indeed, deletion of Nrf2 in Atg7 or Atg5‐deficient mice reversed the liver condition & corrected the injury phenotype, indicating that Nrf2 participates in the pathological changes caused by autophagy deficiency. Remarkably, co‐deletion of Nrf2 in Atg7‐deficient mice also significantly tamed the cholestatic features. These observations support the notion that Nrf2 over‐activation is the key factor in the development of cholestasis, which then cause hepatocyte apoptosis & liver injury.

Autophagy deficiency can cause cholestasis, which could be responsible for the liver injury seen in the autophagy‐deficient mice.
Expression of Carbonic Anhydrase Genes, CA3 and CA12, Transformed with ENO1, Relate to Copy Numbers of EGFR or RNF139 and XIAP, and Gender in Glioblastomas Using PCR Assays
Authors: Beckner Marie , Pollack Ian , Nordberg Mary , Hamilton Ronald
Published: June 2021

Relationships between amplified oncogenes and glycolysis were studied in glioblastomas (GBs) using ENO1 to define supraphysiologic metabolic gene expressions. Copy numbers (CN) of 78 oncogenes were quantified in 24 GBs using multiplex ligation‐dependent probe amplification. Expressions were obtained in 22 by real‐time qPCR for genes encoding lactate dehydrogenases(LDHA, LDHC), carbonic anhydrases(CA3, CA12), monocarboxylate transporter(SLC16A3/MCT4), ATP citrate lyase(ACLY), glycogen synthase(GYS1) & enolase1(ENO1). Expressions were transformed by division with ENO1 expression after routine normalization. All GBs harbored oncogene gains. 20 oncogenes in 18 GBs were amplified at least 3X, up to 6/tumor, most frequently EGFR. Positive correlations with 6 oncogenes were shared by CA3, CA12, LDHC, LDHA, & SLC16A3, whereas + correlations with 5 different oncogenes were shared by ACLY, GYS1, LDHA, & LDHC. CN of XIAP correlated negatively. Differences (Wilcoxon Rank Sum) for >= 2X oncogene CN gains in GBs versus those without occurred for EGFR with CA3 (p<0.03) and RNF139 with CA12 (p<0.004). Difference for tumors with <2X CN gains versus those with gains occurred for XIAP with CA12 (p<0.05). Male gender associated with CA12 (p<0.02). Thus, transformation by ENO1 revealed strong relationships between genes encoding carbonic anhydrases and EGFR, RNF139, XIAP, and gender in GBs. The Pittsburgh Foundation Walter L. Copeland Fund for Cranial Research & Louisiana State Univ Hlth Sci Cen in Shreveport Grant‐in‐Aid.
A New Twist on PBL: A Pilot Study of a Problem‐based Dissection Exercise
Authors: Betts Kimberly , Schmidt‐Bowman Max , Reed James , Lyons Virginia
Published: June 2021

According to the Liaison Committee on Medical Education, active learning has been defined in part as “the process by which a medical student, independently, or collaboratively with his/her peers, identifies his/her learning objectives and seeks the information necessary to meet the objectives.” Engaged learning practices have been incorporated into our gross anatomy course (e.g. flipped classroom, cooperative learning), however these do not satisfy the standard mentioned above. This year a problem‐based dissection (PBD) exercise was piloted in the gross anatomy course for year 1 medical students. For one laboratory, students were given a clinical case in lieu of classic dissection instructions. Each anatomy group was asked to prepare learning objectives to research in order to solve the case. Additionally, each group formulated a dissection plan, completed the dissection, and presented their results to a faculty member in the anatomy laboratory. Preliminary assessments indicate that students felt more prepared and knowledgeable about the dissection for the PBD exercise compared to traditional laboratories. However, results also show that students felt less confident and less enthusiastic about completing the PBD exercise compared to dissections where instructions were provided. The effect on group dynamics, teamwork and communication skills is currently being assessed and will be reported.
Optimizing Communication in an Online Systemic Anatomy Course with a Laboratory
Authors: Attardi Stefanie , Rogers Kem
Published: June 2021

An online (OL) section of a face‐to‐face (F2F) anatomy course with a prosection lab commenced in 2012‐13. Lectures for F2F students were broadcast in live and archived format to OL students using virtual classroom software (Blackboard Collaborate). Labs were delivered OL by a teaching assistant (TA) who manipulated 3‐dimensional computer models in the virtual classroom. Analysis revealed that although course format was unrelated to grades, F2F labs were preferred as it was easier to communicate with the TA. The course was modified in 2013‐14 (138 OL, 354 F2F) to improve OL student‐teacher communication in lab: OL students were divided into lab groups that rotated through virtual breakout rooms, which decreased the student:teacher ratio and gave them the opportunity to communicate with 3 TAs. The study objectives were to compare perceptions of student‐teacher lab communications between OL (N = 101) and F2F (N = 273) survey participants, and determine if the delivery format would influence final anatomy grades. The majority of respondents agreed they were engaged in lab by TAs (60% OL; 82% F2F), could interact socially with TAs (71% OL; 91% F2F), and could ask TAs questions (75% OL; 94% F2F); however, these proportions were significantly higher in the F2F section (p < 0.001). Final grades were statistically identical between sections. There were strong, positive correlations between incoming grade average and final anatomy grade in both F2F (r = 0.71, p < 0.01) and OL (r = 0.70, p< 0.01) sections. These data suggest that prior academic performance, and not delivery format, predicts anatomy grades. While virtual breakout rooms can be used in OL anatomy labs to facilitate student‐teacher communication, they do not adequately replace the F2F lab environment.
Noncoding RNAs: small, large and viral
Authors: Steitz Joan
Published: June 2021

The lecture will trace the history of our appreciation of the pivotal roles played by noncoding RNAs in the regulation of gene expression, first in uninfected and then in virus‐infected vertebrate cells. I shall begin with the unusual basis of the discovery of the splicing snRNPs (small nuclear ribonucleoproteins) and mention the other major classes of noncoding RNAs. The remainder of the lecture will focus on the habit of viruses to acquire components (here noncoding RNAs) from their host cells but then utilize them in unanticipated ways to enhance the viral life cycle. Thus, through studying viral noncoding RNAs, insights are gained into the evolution of both viruses and their host organisms.
How and why embryonic tissue layers move during early morphogenesis in avian embryos?
Authors: Czirok Andas , Akos Zsuzsa , Kosa Edina , Filla Michael , Isai Dona , Little Charles , Dhar Prajna , Rongish Brenda
Published: June 2021

To understand how cells facilitate large‐scale tissue rearrangements during morphogenesis, we utilize high resolution time‐lapse microscopy of early embryonic tissues (cells and ECM) of avian embryos. To localize the engines of tissue movements, we perform microsurgical incisions and inject magnetic microparticles that can be rotated by an external magnetic field. We interpret our empirical data using a novel mathematical/computational model of cell‐resolved tissue mechanics. Our data demonstrate that changes in local material properties, of embryonic tissue, can be mapped with our microrheology approach: tissues harden gradually both (i) in time (at the same anatomical location) as tissues mature, and (ii) at more anterior locations within the same embryo. The posterior embryo is, in particular, a very loose assembly of cells and ECM. The results of microrheology experiments, tissue responses to microsurgical incisions and long‐term behavior of tissue explants can be explained using a computational model representing the mechanically coupled cell layers of the avian embryo. We conclude that the primary means of tissue displacements is not the conventional motility apparatus (migration along an external ECM scaffold) during convergent‐extension movements, formation of the foregut or medial dislocation of the bilateral heart fields. Instead, cells actively rearrange their adhesion mechanics which can create large‐scale tissue flows and deformations.
Moving in and Renovating: Invasion and Remodeling of The Human Erythrocyte by The Malaria Parasite
Authors: Cowman Alan
Published: June 2021

Moving in and renovating: invasion and remodeling of the human erythrocyte by the malaria parasite

Alan F. Cowman

The Walter and Eliza Hall Institute of Medical Research

In humans the most severe form of malaria is caused by Plasmodium falciparum. With a third of the world's population at risk of infection, with 300 million people infected and over 800,000 deaths annually malaria is still a huge problem. Entry of P. falciparum into erythrocytes involves a complex cascade of protein‐protein interactions using several specific ligand‐receptor interactions, that activate penetration and generation of a vacuole that the parasite resides in for subsequent development. Inside the parasite exports hundreds of its own proteins into the host erythrocyte remodelling these terminally differentiated host cells into one in which the parasite can harvest growth nutrients, while providing a protective niche allowing escape from host responses. Such exported proteins must traverse biological barriers including parasite, vacuole and erythrocyte membranes. Understanding function of the proteins involved in parasite invasion and host cell remodelling is important for development of a vaccine and new treatments required to treat and control disease. Malaria has been a companion of humans throughout history and numerous attempts to control it have been defeated by a combination of the ability of the parasite and mosquito vector to adapt. Recently, hope has arisen with strengthening political will to combat this including initiatives funded by the Bill and Melinda Gates Foundation. It is hoped that increased commitment to malaria together with exploitation of the scientific advances, together with our increased knowledge of the P. falciparum genome will bring this enemy under control.
You Need a Game Plan
Authors: Clifford Philip
Published: June 2021

The Merriam Webster dictionary defines a game plan as a strategy for achieving an objective. If sports teams go through the effort to make detailed plans to win trivial competitions, doesn't it make sense for you to have a game plan for something as consequential as your career‾ To advance in a scientific career you need a game plan ‐ a strategy for how you will prepare for success in a highly competitive environment. Industry and government agencies have long used individual development plans (IDPs) to help employees navigate their careers and now the idea has caught on among PhD scientists.

Constructing an IDP is a four‐step process: self‐assessment, exploring careers, setting goals, and implementing your plan. myIDP (myidp.sciencecareers.org) is a free website designed to help guide early career scientists through these steps. It includes exercises to assist you with the self‐assessment process and it will help you determine how well your skills and interests match 20 different scientific career paths. For each career path, there is an extensive list of resources in the form of articles, books, and professional organizations, which can be used to gain a better understanding about unfamiliar careers. Finally, there is a tool to assist you in setting and achieving your goals.

An IDP will help you develop professionally by creating a game plan for a successful career. This session will briefly cover the why and how of creating an IDP.