bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of water deprivation on inhibitory GABAergic synaptic transmission in the hypothalamic paraventricular nucleus (PVN)
Authors: Yamaguchi Junya , Andrade Mary Ann , Truong Tamara , Toney Glenn M.
Published: April 2020

Presympathetic neurons (PSNs) of the hypothalamic PVN mostly lack basal firing in unstressed animals due to the dominance of GABAergic inhibition, which our findings indicate is actively promoted and defended by the coupling of synaptic glutamate (Glu) release to heightened synthesis and packaging of GABA into synaptic vesicles. Our findings indicate that the latter process, referred to as Glu‐GABA strengthening (GGS), depends on EAAT3‐mediated uptake of Glu into PVN GABA terminals. To investigate the impact of PVN GGS on PSN responses to short‐term exposure to a physiological challenge that increases glutamatergic excitation of the PVN, male C57Bl/6 mice were either euhydrated (EUH) or water deprived (WD) for 48 h. Western blot analysis revealed significantly increased (P<0.05) PVN EAAT3 in WD (132 ± 1.2%) compared to EUH (100 ± 9.5 %) mice (n=18/group). Together with our previous report of reduced PVN expression of the glial Glu transporter GLT‐1 (EAAT2) in WD rats, the present findings suggest that Glu clearance in the PVN is shift during dehydration to favor uptake by neuronal EAAT3. If true, this could indicate that PVN GGS becomes enhanced during dehydration. To explore this further, we determined in brain slices from WD mice (n=4) that the amplitude of evoked IPSCs at baseline (−397 ± 102 pA) was unaffected by bath exposure to Glu (100 μM, 10 min) (−352 ± 89 pA), indicating either that dehydration disrupts PVN GGS or enhances it nearly to its maximum. To distinguish between these possibilities, we generated a cumulative eIPSC amplitude curve from 1000 stimuli delivered at a frequency of 5 Hz to the GABAergic peri‐nuclear zone (PNZ) that surrounds the PVN. The slope (‐pA/stimulus) of this relation in WD mice early during the stimulus train (stimuli 1–100) was similar to that of mice lacking GGS due to EAAT3 knockdown (KD) in PNZ GABA neurons (WD: 186 ± 0.9, EAAT3 KD: 180 ± 0.9). By contrast, the late phase slope (stimuli 901–1000) was 3 time higher for PSNs from WD mice than from the PNZ EAAT3 KD group (WD: 52 ± 0.2, EAAT3 KD: 17 ± 0.1). The latter indicates that eIPSCs in the WD group are resistant to rundown/depression and suggests that PVN GGS is likely near maximally enhanced, not disrupted, during dehydration. We posit that enhanced PVN GGS may be critical for preserving synaptic homeostasis and preventing excessive PSN activation and sympathoexcitation during short‐term dehydration stress.

Support or Funding Information

NIH HL088052 (GMT)
Exploring the Role of Mitochondrial ClpP Activators on Immune Signaling in Breast Cancer
Authors: Aponte-Collazo Lucas J. , Fennell Emily M.J. , Graves Paul R. , Herring Laura , Iwanowicz Edwin J. , Holmuhamedov Ekhson , Graves Lee M.
Published: April 2020

Approximately 20% of breast cancer cases diagnosed yearly are of the triple‐negative breast cancer (TNBC) type. Current treatments for TNBC patients are limited to surgical intervention and traditional chemotherapies. ONC201 is a recently discovered orally‐active imipridone molecule in phase I/II clinical trials for a large variety of aggressive cancers including glioblastoma, breast, endometrial, and others. We have been studying highly potent analogs of ONC201, known as the TR compounds (TRs), for their efficacy in TNBC. To better understand the mechanism of action, we coupled a TR compound to agarose beads and applied proteomics to identify potential targets. Our studies identified the mitochondrial caseinolytic peptidase (ClpP), as a major ONC201 and TRs binding protein. We further demonstrated that these compounds activated the proteolytic activity of ClpP and that this mitochondrial signal was required for subsequent activation of the integrated stress response as determined by increased CHOP and ATF4 proteins. In addition to activation of the integrated stress response, other recent studies have suggested that the antitumor effects of ONC201 may in part be dependent on increased immune cell recruitment and cancer growth inhibition through this mechanism. Thus, one of our major objectives is to understand how activation of ClpP by ONC201 or TRs impacts cellular signaling events that lead to enhanced immune cell recruitment. We hypothesize that activation of ClpP promotes activation of key immune signaling proteins, like NF‐kB, which subsequently leads to an increase in proteins required for immune cell recruitment. Accordingly, our preliminary data shows activation or up‐regulation of proteins connected to innate immune signaling including MAVS, NF‐kB, and TBK1 in response to ONC201 or TRs treatment. Additionally, our qPCR data shows that treatment of TNBC cells with ONC201 or TRs leads to increased mRNA levels of specific Interleukins (IL‐12, IL‐10). Collectively, these results suggest that activation of ClpP by ONC201 or TRs increases innate immune signaling and expression of cytokines, which are crucial events for successful recruitment of immune cells. Future experiments will evaluate if activation of NF‐κB by ONC201 or TRs treatment is ClpP dependent using a ClpP knockout in TNBC cell lines. Lastly, we will also measure changes in levels of known NF‐κB regulated cytokines (TNFα, IL‐6, IL‐18 and IL‐1β) after treating TNBC cells with ONC201 or TRs using flow cytometry. Overall, this work provides valuable new insight into potential mechanisms by which ClpP activators mediate their anti‐cancer effects through immune signaling and recruitment.

Support or Funding Information

L.J.A.‐C was supported by the NIH 5 T32 GM007040
Femoral condyle dimensions: comparing natural anatomy to knee replacement hardware
Authors: Horbaly Haley Elizabeth , Auerbach Benjamin Elizabeth
Published: April 2020

Orthopedics companies have recently pursued sex‐specific total knee replacement (TKR) designs that account for sexual dimorphism to improve post‐operative outcomes for females. Prior research on knee anatomy has shown no significant differences in morphology between the sexes when controlling for size. For knee replacements to be effective they must functionally mimic natural skeletal anatomy, yet the variance in TKR appliances in relation to skeletal variation has not been thoroughly studied. The question remains if scaling knee replacement appliances to the individual sufficiently reflects the functional variation of knee morphology, or if additional variation in shape must be taken into account.

This research analyzes 14 bilateral landmarks on the distal femur and orthopedic hardware for 17 individuals who received unilateral TKRs and 36 control individuals with no TKRs. Landmarks reflect the morphology of the distal articular surfaces, including dimensions of the condyles and their relative spacing. Ten interlandmark distances (ILDs) were extracted for analyses. We performed principal components analysis on the pooled sample. PC1 (63% of variance) describes size differences; all ILDs positively relate to it and it separates females from males. PC2 (14% of variance) indicates that measurements reflecting condylar morphology are inversely related to intercondylar measurements (overall condylar breadth, intercondylar width [ICW], and proximal and distal intercondylar distances [PID, DID]). Of these traits, ICW, which has the weakest relationship with body size, exhibits the strongest association with PC2. Further exploration of intercondylar measures reveals that PID and DID dimensions are significantly different (p<0.05) in males. An ANCOVA reveals that these two dimensions have different scaling relationships, which indicates the medial condyle deviates from a proximodistal orientation (increases its angle) with increased body size. PID and DID are comparable distances in the TKR hardware (p = 0.327) and pooled females (p = 0.159). Overall, no sexual dimorphism is present in the dimensions of the distal femur when accounting for size, and bilateral asymmetry in individuals with unilateral TKR is not outside the range of normal human variation. The only exception is that males show medial condyle asymmetry between their natural femur and replacement hardware. Further, the comparably‐sized PID and DID dimensions in femoral implants mimic the pattern observed in females, while males show significant differences in these dimensions. This analysis indicates that traditional unisex hardware dimensions may be more similar to female distal femur anatomy, and is likely not contributing to reduced post‐operative outcomes for females.
Nanolipoprotein Particles (NLPs) as an In Vivo Delivery Platform for Biomedical Applications
Authors: Gilmore Sean F.
Published: April 2020

Nanoparticles hold great promise for the delivery of therapeutics. One emerging class of these materials are nanolipoprotein particles (NLPs), which are mimetics of naturally occurring high‐density lipoproteins (HDLs). NLPs are attractive for drug or vaccine formulation since they: (1) can be loaded with hydrophobic “cargo” molecules, (2) have high water solubility and (3) are easily modified for targeted delivery to specific tissues. However, their widespread adoption has been limited by poor in vivo stability, likely due rapid exchange with lipoproteins in blood. We report herein progress towards loading NLPs with several classes of therapeutically relevant molecules and drastic improvement of NLP in vivo stability. Additionally, we have evaluated the functionality, stability, cytotoxicity, toxicity, immunogenicity and in vivo biodistribution of NLPs. We found that a wide range of molecules with disparate chemical properties could be reliably conjugated to NLPs, including proteins, single‐stranded DNA, vaccine adjuvants, and small molecule drugs including chemotherapeutics. We also report an optimized NLP formulation that is highly stable in blood serum, with a half‐life greater than 24 hrs and no cytotoxicity to human cell cultures at doses as high as 320 μg/ml. As NLPs are constructed from lipids and recombinant, truncated apolipoproteins, we report that these particles do not possess any inherent immunogenicity. Biodistribution of the particles is route dependent, but when administered intravenously to mice, we observe uptake into the brain as measured through accelerator mass spectrometry. These findings are supportive of NLPs as a versatile platform that holds promise for use as a delivery vehicle for a range of therapeutics.

Support or Funding Information

This work was funded by LLNL awards 15‐LW‐023 and 17‐LW‐051.
The Interplay between Palmitoylation and Phosphorylation of NHE1 in the Regulation of Cell Proliferation.
Authors: Leffelman Hannah , Wallert Mark A
Published: April 2020

The Na+‐H+ Exchanger Isoform 1 (NHE1) is key to the regulation of cell cycle progression and cell proliferation. NHE1 is a 12‐pass transmembrane transport protein that exchanges one extracellular Na+ for one intracellular H+ with the primary function of intracellular pH regulation. This pH regulation is essential to the control of cell proliferation and migration. The exchanger’s transmembrane domain is involved in ion exchange and the extended cytoplasmic tail functions as the protein’s regulatory domain. The regulation of NHE1 is extremely complex and involves protein and lipid binding sites including phosphorylation and palmitoylation sites. NHE1 transport activity is regulated by a series of signaling pathways which include kinases such as MEK, Erk, Rsk, Rock, and Akt. Recently, it was recognized that NHE1 also undergoes reversible palmitoylation. Here we evaluate the impact of the inhibition of phosphorylation and palmitoylation of NHE1 on cell proliferation to identify the interplay between these two covalent modifications of NHE1. We hypothesize that the combined impact of inhibition of palmitoylation and phosphorylation will have a synergistic impact inhibiting cell growth. Cell proliferation was evaluated utilizing Chinese hamster lung fibroblasts (PSN cells), which express human NHE1, in the presence of the inhibitors Mk‐2206 (Akt), PD98059 (MEK), Y27632 (Rock), 2‐BP (Palmitoylation), and EIPA (NHE1).
The Shape Solidity of the Glenoid Fossa of the Scapula is Sexually Dimorphic: A New Approach to Understanding the Glenoid Fossa
Authors: Bolden Alexys M. , Burkett Jesse T. , Shockley J. Mason , Alsharaf Fatemah , Zdilla Matthew J. , Lambert H. Wayne
Published: April 2020

Sexual dimorphism in the size of the glenoid fossa of the scapula is well‐established; however, there remains little information with regard to differences in glenoid shape between sexes. Therefore, this study assessed 71 glenoid fossae from dry scapula. Three differential equations were utilized to ensure that scapulae were appropriately sexed and, accordingly, included in the study. When all three equations were in agreement with regard to sex, the scapula was included for study. A total of 64 scapulae (45 females and 19 males) met inclusion criteria. Shape parameters that were assessed included circularity (4π*area/perimeter2), roundness (4*area/π*major axis2), aspect ratio of a bet‐fit ellipse (major axis/minor axis), and solidity (area/area of a convex hull). Shape parameters such as circularity, aspect ratio, and roundness were not significantly different between sexes; however, solidity was significantly different between females and males (p=0.0475). The results demonstrate a difference in the concavity of the boundary of the glenoid fossa between sexes. Further study should elucidate the functional differences in glenohumeral joint biomechanics with regard to the solidity of the glenoid fossa while taking sex into consideration.

Support or Funding Information

WV Research Challenge Fund [HEPC.dsr.17.06]
Effects of in Vitro Exercise‐Induced Endothelial Shear Stress on Oxidative Stress and Vasoconstriction Gene Expression
Authors: Garcia Mario , Morales-Acuna Francisco , Conde Daniel , Gurovich Alvaro N.
Published: April 2020

Background

Sedentary lifestyle contributes to endothelial dysfunction through increasing oxidative stress and endothelin‐1, a potent vasoconstrictor that contributes to the pathogenesis of vascular disease. Exercise‐induced endothelial shear stress (ESS) elicits a mechanical stimulus leading to intracellular signaling transduction regulating cellular function that might protect against endothelial dysfunction through promotion of the expression of anti‐oxidative and pro‐vasodilatory genes. Although it has been reported that increments on steady ESS has led to improvements in endothelial function in vivo, the role of pulsatile exercise induced ESS and varying exercise intensities in vitro has not been explored yet. Therefore, the purpose of this study was to determine the effects of pulsatile exercise‐induced ESS based on in vivo exercise intensity on anti‐oxidative (superoxide dismutase, SOD2) and vasoconstrictive (endothelin 1, EDN‐1) gene expression.

Methods

Commercially available human carotid artery endothelial cells (Sigma‐Aldrich Comp.) were cultured until 95–100% confluence and exposed to pulsatile resting ESS (18 dynes/cm2) for 5 hours, followed by 1 hour at resting ESS, low‐intensity exercise‐induced ESS (35 dynes/cm2), or high‐intensity exercise‐induced ESS (80 dynes/cm2) on parallel fluidic units (Ibidi USA, Inc.). Ibidi pump software (PumpControl Software 1.5.4) was used to control shear stress intensity. Total RNA was extracted and reverse transcribed followed by measurement of EDN‐1 and SOD2 mRNA expression via qRT‐PCR. qRT‐PCRs were performed in duplicates and changes in gene expression were calculated using the ΔΔ‐CT method with GAPDH used as the normalizing control gene. Statistical analysis was performed using graphpad prism 8 software (Graphpad Software.). Significance was considered at p<0.05.

Results

mRNA expression of SOD2 increased 2.5‐fold after exposure to low‐intensity exercise‐induced ESS (p<0.05). EDN‐1 showed a 5‐fold increase following exposure to low‐intensity exercise‐induced ESS (p<0.05) in comparison to resting conditions. High‐intensity pulsatile exercise‐induced ESS decreased the expression of EDN‐1 in comparison to resting and low‐intensity conditions (p<0.05).

Conclusion

Pulsatile exercise‐induced ESS improved anti‐oxidative properties in human carotid artery endothelial cells. It seems that higher exercise intensities are required to downregulate the expression of vasoconstrictive genes.
Lipid composition and organization helps plasma membrane repair and offers novel therapeutic target to treat degenerative muscle disease
Authors: Jaiswal Jyoti K. , SenChandra Sreetama , Bittel Daniel , Horn Adam
Published: April 2020

The plasma membrane of all cells is routinely injured, with considerable investigative effort focused on understanding how proteins help repair this injury. Despite being the most abundant component of the plasma membrane, the role of lipids in repairing injured mammalian cells has remained unexplored. Through systematic live cell imaging we have examined the involvement of lipids in plasma membrane repair following injury. This shows that change in composition and localization of plasma membrane lipids is crucial for repair. Specifically, we find that cell injury results in a rapid change in local cholesterol and sphingomyelin content, and failure of which increases membrane fluidity and compromises membrane repair. Plasma membrane injury also alters the local composition of phospholipids including PIP2 and diacylglycerol (DAG), which is required for the repair of the injured plasma membrane. Failure of these lipid changes in muscle fibers injured by mechanical or other activity, results in myofiber death and Limb Girdle Muscular Dystrophy (LGMD2B). To target this deficit we developed a gene therapy approach to exogenously modify the plasma membrane lipid composition in a mouse model of LGMD2B. This approach improved the repair of the dystrophic muscle, attenuated myofiber death and improved muscle strength and histopathology. These findings provide direct evidence of the diverse roles of lipids in not only maintaining the integrity of the plasma membrane, but also in re‐establishing its integrity following injury. Here, we establish the clinical relevance of the structural and signaling roles of lipids in cell repair, and identify therapeutic approaches that address these deficits in muscle diseases.

Support or Funding Information

This work is supported by R01AR055686 from NIH/NIAMS.
Independent and Combined Effects of Bisphenol A and Diethylhexyl Phthalate on Gestational Outcomes and Offspring Development in Sprague‐Dawley Rats
Authors: Dagher Josephine Bou , Campbell Elyssa , Kaimal Amrita , Hahn-Townsend Coral , Al Mansi Maryam Hazim , Tran Diane , Laurent Christian , Bacak Caleb , Cambric Celexis , Henriquez Joseph , Spivey Jared , Buechter Hannah , Chuang Yen-Jun , Mohankumar Puliyur , Mohankumar Sheba
Published: April 2020

Background

Bisphenol A (BPA) and Diethylhexyl Phthalate (DEHP) are common plastic‐derived environmental contaminants that act as endocrine disrupting chemicals (EDCs) and pose risks for expecting mothers and offspring. Research addressing the effect of a mixture of these EDCs is limited.

Objective

To assess the sex‐specific consequences of developmental exposure to individual as well as mixtures of environmentally relevant doses of BPA/DEHP in male and female Sprague‐Dawley rats.

Methods

From gestational day 6 till 21, dams were orally administered either saline (control), BPA (5mg/Kg BW/day), low‐dose (LD) DEHP (5mg/Kg BW/day), high‐dose (HD) DEHP (7.5 mg/Kg BW/day), a combination of BPA and LD DEHP (B+LD‐D), or a combination of BPA and HD DEHP (B+HD‐D). Gestational weights and number of abortions were tracked. Litter size and weights, number of live births and stillbirths were counted. Morphometric parameters at birth were measured. Body weighs, food and water intakes were monitored weekly from postnatal weeks 3–12. Male and female offspring were sacrificed at 16–24 weeks of age and organs were dissected and weighed.

Results

The abortion rate of dams exposed to HD DEHP and the mixtures, B+LD‐D and B+HD‐D were higher at 8, 14 and 23% respectively. Gestational index was reduced in these groups as well. Prenatal exposure to BPA or HD DEHP significantly decreased relative thymus weight in male but not female offspring at 16 weeks. Relative heart weight increased in B+HD‐D exposed male offspring compared to the other groups. No programming effects in relative organs weights were detected in female offspring.

Conclusion

The results indicate that a mixture of BPA and DEHP, even at low doses, induced a pronounced effect on pregnancy outcomes. Male offspring appear to be more susceptible to the programming effects of these EDCs or their mixture. This suggests a great need to reconsider the possible additive, antagonistic or synergistic effects of EDC mixtures to which pregnant individuals are commonly exposed to.

Support or Funding Information

Supported by UGA research foundation.
Ex‐vivo Effects of Limonene in Airways and Vasculature in Allergic Adenosine A2A receptor Knock‐out and Wild‐type mice
Authors: Rajalingam Sahith , Bhavsar Riya , Patel Mehaben , Mustafa S. Jamal , Ledent Catherine , Ponnoth Dovenia S.
Published: April 2020

Limonene, a mono‐terpene flavonoid, has anti‐inflammatory effects in asthma by activating A2A adenosine receptors. We studied the effects of inhaled d‐limonene on isolated aortic and tracheal rings from asthmatic mice using tissue/organ bath system. We used mice in which the A2A receptor was genetically knocked out (A2AKO) and C57BLJ/6 (wild‐type; WT) for the study. The mice were divided into control (CON) and allergen‐induced sensitized (SEN), control mice treated with limonene through aerosol route (CON+LIM) and allergen‐induced sensitized mice treated with limonene (SEN+LIM). Mice were sensitized i.p. on days 1, 6 with 20μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Control groups were given an i.p injection of vehicle (1,6 days) and the aerosol challenge of normal saline (11–13 days). SEN +LIM and CON +LIM groups were given 0.01% limonene in the form of aerosol 90 min prior to the OVA and saline challenge on days 11,12,13. Trachea and aorta were isolated on day 14 for further experiments. Tissue responses were studied in the form of contraction and relaxations in all groups. Treatment with limonene significantly reduced the tracheal reactivity (80.68±20% in SEN+LIM vs SEN; p<0.05) to methacholine (MCh) in WT mice that were allergic. MCh induced tracheal contractions were not altered in A2AKO limonene treated sensitized group compared to non‐treated A2AKO sensitized group (209.87 ± 41.33 A2AKO SEN+LIMSEN+LIM Vs 212.51 ± 43.18 in A2AKO SEN) In presence of A2A‐antagonist (SCH58651), a significant increase in the MCh induced contractility in WT SEN was observed(341.65 ± 2.664 SEN+SCH Vs 225.99 ± 67.34 SEN, pP<0.05). A2A agonist CGS 21680 induced relaxation in WT CON was abolished in WT SEN but restored in WT SEN+LIM (37.8± 16.4% Vs 1.2± 11.1 in SEN, p<0.05). The CGS‐induced tracheal relaxation in WT SEN+LIM and SEN groups was increased in the presence of the NADPH inhibitor apocynin (60.58 ± 17.20 SEN; 21.28 ± 16.02 SEN). In the aorta, acetylcholine‐induced aortic relaxation (endothelium‐dependent response) in WT SEN group was significantly lower than controls (29.82 ± 5.62 WT SEN Vs 51.51 ± 30.13 WT CON, p<0.05) with limonene restoring the response (61.56 ± 11.34 WT‐SEN+LIM). CGS 21680 induced aortic relaxation in WT SEN was completely blunted compared to WT CON (5.62±3.72 WT SEN Vs 66.91 ± 6.8 WT‐CON, *p<0.05). Treatment with limonene restored CGS 21680‐induced relaxation in asthmatic mice (63.8 ± 6.32 WT SEN+LIM Vs 5.62± 3.72 SEN) In conclusion, it appears that limonene restores the tracheal and aortic relaxation induced by adenosine‐mediated pathways by activating the A2A receptor in asthmatic mice.

Support or Funding Information

Supported by LIU start‐up funds (DSP), HL027339 (SJM)
Mechanisms of Pisiform Reduction in Mammals
Authors: Kjosness Kelsey M. , Gavazzi Lia M. , Reno Philip L.
Published: April 2020

Most mammalian pisiforms are large, elongated carpal bones with two ossification centers and an associated growth plate, similar to the calcaneus in the ankle. Short pisiforms are rare among mammals, but can be found in humans, orangutans (Primates: Pongo sp.), two‐toed sloths (Pilosa: Choloepus sp.), and three‐toed sloths (Pilosa: Bradypus sp.). These groups utilize diverse forms of locomotion; humans are bipedal, orangutans are quadrumanous climbers, and sloths are suspensory quadrupeds. Human pisiforms form from a single ossification center and have likely lost a primary ossification center and the associated growth plate. Given the rarity of short pisiforms among mammals, little is known about the functional implications or developmental mechanisms underlying this convergent morphology in species with such diverse locomotor modes and evolutionary histories. The purpose of this study is to compare ossification patterns in orangutans and sloths with the mechanism underlying human pisiform truncation. We compare pisiform ossification data from human, orangutan, and sloth museum specimens. Unlike humans, orangutan pisiforms retain two centers of ossification but appear to form the secondary center of ossification later than other apes, possibly shortening the growth period for the pisiform. Sloths form a primary center of ossification with an unusual, pitted surface, which may represent an abnormal subchondral surface. In non‐human primates (including orangutans) and giant anteaters (Pilosa: Myrmecophaga tridactyla), which represent the closest living taxonomic group to sloths, pisiform and calcaneus ossification is generally correlated with epiphyses forming at similar times within individuals. In contrast, we found only a single ossification center corresponding to sloth pisiforms at all stages of calcaneal epiphyseal formation. Thus, the single pisiform ossification center appears to precede calcaneal epiphysis formation. This indicates that the sloth pisiform is homologous to the primary center of ossification of the calcaneus and other mammalian pisiforms, and that it fails to form a secondary center. This is in contrast to human pisiforms, which fail to form the primary ossification center. Humans, orangutans, and sloths are all unusual among mammals for having short pisiforms and each group achieves pisiform reduction through unique mechanisms.

Support or Funding Information

This research was funded by the National Science Foundation (BCS‐1540418 and BCS‐1638812).
The effects of Gtf2i and Gtf2ird1 mutations on the skull in Williams‐Beuren Syndrome
Authors: Hill Cheryl A. , Kirkland Nicole M. , Kopp Nathan D. , Dougherty Joseph D.
Published: April 2020

Williams‐Beuren syndrome (WBS) is a neurodevelopmental disorder caused by a 1–8 Mbp deletion on chromosome 7. In addition to hypersocial personalities and visuospatial changes, individuals with WBS also show changes in cardiovascular and craniofacial phenotypes. GTF2I AND GTF2IRD1 have been implicated in producing the changes seen in the craniofacial phenotype. The purpose of this study is to determine if Gtf2ird1 and Gtf2i interact to produce craniofacial features associated with WBS in mouse models.

Using CRISPR/CAS9 technology, we developed two new mouse models loss‐of‐function mutations in both Gtf2ird1 and Gtf2i and wildtype littermates. The study sample includes computed tomography scans acquired from the skulls of mice with differing number of mutations in these genes and their wildtype littermates (N=119). Three‐dimensional landmark coordinate data were collected from scans of the skull and analyzed using geometric morphometrics.

Skulls of mice with only one loss‐of‐function mutation in Gtf2ird1 were not different from wildtype littermates, while mutations in both Gtf2i and Gtf2ird1 show variable results across the two models. Our results indicate that loss‐of‐function mutations in Gtf2ird1 and Gtf2i interact in nuanced ways to affect the craniofacial structure in these mouse models for Williams‐Beuren syndrome. Varying protein expressions within these mice, interactions with other genes in the deleted region of chromosome 7 and other factors lead to changes in the craniofacial phenotype seen in Williams‐Beuren syndrome.

Support or Funding Information

5R01MH107515‐03 to JDD, NSF fellowship DGE‐1745038 to NDK.
Variants of Mre11 induce genomic instability
Authors: Hilton Austin , Gunning Abigail , Kuehn Sydney , Shanle Erin , Ray Sreerupa
Published: April 2020

DNA double‐strand break (DSB) is the most lethal type of DNA damage and if not properly repaired could destabilize the genome. Cells repair DSBs by three repair pathways‐homology‐directed repair (HDR), non‐homologous end‐joining (NHEJ) and a recent back‐up NHEJ pathway has been reported and is referred to as alternate‐NHEJ (Alt‐NHEJ). Mre11 plays a role in all the three DSB repair pathways. Mre11 has single‐strand endonuclease activity and double‐strand‐specific 3′–5′ exonuclease activity which are essential for DNA end‐processing. Mre11 has been shown to be overexpressed in breast cancer and high Mre11 expression was found to be associated with a more malignant behavior in breast cancer. Mutations in Mre11 genes can have a significant impact on cancer prognosis and treatment. Here we show that cells expressing variants of Mre11 are sensitive to DNA damaging agent etoposide. We also found that the Mre11 variants influence response to various DNA damaging agents and induce genomic instability. Work is on‐going to characterize how the Mre11 variants alter DSB repair mechanisms. This will provide mechanistic insights into the role of the Mre11 variants inducing genomic instability and cancer and has potential to influence cancer therapeutics.

Support or Funding Information

This research was supported by Murdock College Research Program for Natural Sciences ‐ Life Sciences grant (#201812085) and Linfield College Faculty Student Research Collaborative Grant (FSRCG) to SR
Advanced Glycated End Products or High Glucose/Palmitate treatment modulate TREM‐1, Arginase and Nitric Oxide Levels in Macrophages.
Authors: Shatanawi Alia , Al Zayadneh Ebaa M. , Lemtalsi Tahira , Fouda Abdelrahman Y. , Caldwell Ruth B. , Caldwell R. William
Published: April 2020

Arginase is ureohydrolase enzyme that has essential actions in the body, most importantly in the urea cycle. Though, increased arginase activity has been demonstrated to contribute to vascular endothelial dysfunction (VED) in a number of disease/pathophysiological conditions, such as diabetes, aging, atherosclerosis and hypertension. Enhanced arginase activity/expression results in reduced availability of L‐arginine for nitric oxide synthase (NOS) and decrease NO production. Vascular dysfunction is a common characteristic of the early vascular complications of diabetes. Inflammation has been shown to be a potent modulator of vascular dysfunction in diabetes. we have previously found that increased inflammatory mediators in diabetic conditions seem to affect arginase levels as well. we hypothesise that advanced glycated end products (AGE), which get elevated in diabetes, can stimulate expression of Triggering Receptor Expressed On Myeloid Cells (TREM‐1). TREM‐1 has a key role in vascular endothelial activation, inflammation and dysfunction. Also AGEs cause an increase in arginase expression and activity in macrophages. Additionally, treating macrophages with high glucose/palmitate (HG/PA); which represents a model of high fat‐high sugar intake; has also resulted in upregulation of TREM‐1 and arginase 2 levels in macrophages. We also looked at NO levels produced under different conditions and have seen that both AGE or HG/PA cause an increase in NO levels from macrophages. Our studies indicate that there is a cross talk between TREM‐1 receptor and arginase under different diabetic conditions. Our earlier findings indicate the importance of controlling levels of arginase in diabetes to prevent vascular dysfunction. Regulating inflammatory receptors such as TREM‐1 can also have a powerful impact on controlling vascular dysfunction and complications in diabetes.
A Conserved Tyrosine Residue of EcSpeG is Absolutely Critical for Enzymatic Activity
Authors: Lim Ee Qi , Le Van , Dang Joseph , Law Melissa , Kuhn Misty L.
Published: April 2020

The Spermine/Spermidine N‐acetyltransferase from Escherichia coli (EcSpeG) is an allosteric dodecameric enzyme that belongs to the Gcn5‐related N‐acetyltransferase (GNAT) superfamily of enzymes. Under stressful conditions, SpeG acetylates the polyamines spermine (spm) and spermidine (spd), which bind to the active site and allosteric site of the protein. These polyamines are important polycationic molecules for many life processes including cell growth, protein synthesis, and metabolic regulation. GNATs commonly use a general acid base catalytic mechanism to acetylate their substrates and most have a key tyrosine residue that acts as the general acid in the reaction. It was previously shown that mutating this conserved residue in EcSpeG to phenylalanine did not abrogate the catalytic activity. In our experience, mutating this conserved residue in other GNATs has shown it is absolutely critical for activity. Therefore, we created multiple point mutations to test whether the identity of the residue at this position altered catalytic activity. Similar to our previous results with other GNATs, we found this tyrosine residue was critical for activity.

Support or Funding Information

Research reported in this work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM133506 (to MLK).
Incorporating LGBTQ+ Inclusivity and Gender‐Neutral Considerations into Medical School Anatomical Education: First Steps
Authors: Carter Yasmin
Published: April 2020

In recent years there has been increasing recognition of the need to incorporate diversity and inclusion into medical education both in the material taught and the teaching environment, in order to develop and support a more diverse and inclusive student body, professional workforce and patient population. Additionally, it is vital to build a comprehensive understanding of related biological phenomena, and to address gaps in medical knowledge that have arisen due to a historical lack of LGBTQ+ concepts in research. The question of how to do this appropriately in anatomical education and the role of anatomists in this has been a source of interest and debate. Most can agree that terminology and basic concepts surrounding sex and gender are made concrete in the first year of medical school with anatomists playing a key role in these early discussions. With this in mind, we began to create a short presentation and supplementary materials to be provided to medical students during their introduction to the anatomy course. The goal of these materials was to define terminology and highlight the importance of incorporation of LGBTQ+ inclusivity and gender‐neutral considerations into their learning. Additionally, this presentation models respectful language for future discussions of LGBTQ+ staff, students, patients, and academic research. Here, we describe some of the specific challenges to the creation of these focused learning materials and their integration into the anatomy curriculum, including attracting decision‐making stakeholders, student interest, and retention versus momentum. Version 1.0, consisting of a short PowerPoint presentation and a glossary handout was presented to students in the first week of the Development, Structure and Function course at the University of Massachusetts Medical School. The results of student review confirmed their interest in these topics but highlighted the most effective way to stress the importance of this subject is to include it in testing materials. Based on this, the Educational Materials Collection (EMC) was expanded to include a growing MCQ bank. Additionally, materials diving deeper into gender history and bias have been included. Leveraging the power of social media to overcome geographical limitations, Version 2.0 of the lecture and supplementary materials have been shared with anatomical educators worldwide. We will continue to build inclusive and gender‐neutral language into all aspects of the anatomical curriculum and develop training for visiting clinical educators. We plan to continue the dissemination, while also expanding the materials to include more clinical correlates, as well as materials for all levels and fields of anatomical education.
High carbohydrate diets increase respiratory quotients above 1 due to lipid synthesis
Authors: Talal Stav , Cease Arianne , Harrison Jon
Published: April 2020

The respiratory quotient (RQ, carbon dioxide emission/oxygen consumption) is believed to range from 0.7–1 depending on the fuels used for catabolism. However, RQ can rise above 1 when CO2 is released without oxygen consumption. This can occur when hexose sugars are catabolized to pentose sugars via the pentose phosphate pathway to produce NADPH for glutathione regeneration. It is also hypothesized RQs > 1 can occur under lipid synthesis when glucose is catabolized by glycolysis to acetylCOA. However, to our knowledge, no prior studies have assessed animal RQ during periods of carbohydrate conversion to lipid. We used stop‐flow respirometry to measure RQ values for nymphal South American locusts (Schistocerca cancellata) that had been consuming artificial diets varying in carbohydrate:protein ratio for 3–5 days. For locusts measured during the hour after a meal, RQ values increased as the carbohydrate:protein content of the previously‐consumed diet increased, to a maximal value of 1.2. Locusts consuming diets high in carbohydrate:protein ratio synthesized fat at high rates, consistent with the hypothesis that an RQ above 1 is an indicator of synthesis of lipid from carbohydrate.

Support or Funding Information

This research was supported by NSF IOS‐1826848 and BARD FI‐575‐18.
Hypoxia Susceptibility Related to the Expression of Long Noncoding RNA as Regulators of Abraxas2 and Camk2d in the Heart Tissue of a Hypoxic Mouse Model
Authors: Phan Sheshanna , Hassan Omron , Yermian Narineh , Fomicheva Ekaterina , Bondarenko Vladimir
Published: April 2020

Long noncoding RNAs (lncRNAs) play a regulatory role in hypoxic disease states, and identifying regulators that control gene expression in a hypoxic mouse model will introduce therapeutic targets and biomarkers of susceptibility to hypoxia. Previous studies show that CD‐1 and C57BL/6J mice are sensitive and resistant to hypoxic stress, respectively.

CD‐1 and C57BL/6J mice were subjected to an intraperitoneal injection of SE‐175 (0.7 mg/kg) to induce a hypoxic response or 1x phosphate‐buffered saline (PBS) and then sacrificed after five hours in normoxic condition. RNA was extracted from heart tissue and analyzed in a microarray. Genomic BLAST was performed between the human genome and mice lncRNAs with ±2 fold change in expression (p < 0.05). The expression of several genes coding sequences and their associated lncRNAs were analyzed by qPCR.

We identified two lncRNA 1500002F19Rik and LOC108168899 that were found in close proximity to the genes Abraxas2 and Camk2d, respectively. Microarray results suggested that 1500002F19Rik and LOC108168899 may play a role in the expression of Abraxas2 and Camk2d, respectively. The expression levels of Abraxas2, Camk2d, and their corresponding lncRNA were analyzed using qPCR. In mice treated with SE‐175, Abraxas2 and 1500002F19Rik were both upregulated in C57BL/6J mice, but neither displayed a significant change in CD‐1 mice. Camk2d and LOC108168899 were both upregulated in C57BL/6J mice, while Camk2d was upregulated in CD‐1 mice, but LOC108168899 showed no significant change.

Because of the cytoprotective effects of Abraxas2 against oxidative stress, its upregulation and that of 1500002F19Rik in C57BL/6J mice provides a possible mechanism for their resistance to hypoxic stress. Camk2d is implicated in oxidative stress in cardiomyocytes and was upregulated to a higher magnitude in CD‐1 mice than in C57BL/6J mice, while LOC108168899 only showed increased expression in C57BL/6J mice. This suggests that LOC108168899 can be involved in downregulation of Camk2d that prevents further oxidative stress in C57BL/6J mice, so its lower expression in CD‐1 mice can lead to higher expression of Camk2d which may contribute to susceptibility to hypoxia.

Support or Funding Information

This project was supported by Touro University Nevada research grant.
Obesity Increases Microvascular Contractility in Response to Sympathetic Nerve Stimulation
Authors: Souza Gean Domingos , White Carl
Published: April 2020

Obesity impairs vasodilation and increases contractility in small mesenteric resistance arterioles. The mesenteric microvasculature is innervated by sympathetic and sensory perivascular nerves; however, it isn’t known if changes in innervation contribute to microvascular dysfunction in obesity. The current aim was to characterize the sympathetic and sensory nerve‐mediated responses of isolated mesenteric arterioles from lean and obese mice. We placed 6‐week‐old mice (C57BL/6) on either low or high‐fat diets for 12 weeks. We then isolated third order mesenteric arterioles and used pressure myography to record the effect of perivascular nerve activation by electrical field stimulation (EFS). EFS (20 V, 3 Hz, 2 ms pulse width) caused vasoconstriction which was attenuated by the voltage‐gated Na+ channel blocker tetrodotoxin (TTX, 1 μM). Vasoconstriction (% maximal constriction) was larger in vessels from obese (50% ± 2) mice compared to those from lean (22 ± 5%) animals (P<0.001, n=3, t‐test). To assess the effect of obesity on sensory innervation, vessels were treated with guanethidine 10 μM to suppress sympathetic nerves, and precontracted with 1 μM phenylephrine. Under these conditions, EFS evoked similar relaxations (% maximal relaxation in zero calcium and sodium nitroprusside) in the lean (36% ± 11) and obese (45% ± 16) groups (P>0.5, n=3, t‐test). In conclusion, these data show that during obesity the contractile response to sympathetic innervation is increased without change in the dilatory response to sensory innervation.

Support or Funding Information

This work was supported by NIH grant HL142906.
Glucose Limiting Conditions Can Lead to Activation of No‐Go Decay and Subsequent P‐body Assembly
Authors: McManus Gretchen , Segal Scott P.
Published: April 2020

The budding yeast, Saccharomyces cerevisiae preferentially uses glucose as a primary carbon source. When glucose is abundant, it is metabolized anaerobically, leading to the production of ethanol. However, as glucose becomes depleted, yeast will enter diauxic shift. Under this condition, growth will slow, and glucose becomes metabolized aerobically. One effect of the upregulation of aerobic metabolism will be a subsequent increase in the generation of intracellular reactive oxygen species (ROS). The increase of ROS can lead to oxidation of a number of different molecules in the cell, including of nucleic acids. This can lead to the formation of 8‐oxoguanine bases (8‐oxoG). The formation of 8‐oxoG bases in DNA can cause a G to A transition. However, in mRNA the presence of 8‐oxoG will elicit ribosome stalls, resulting in quality control by the No‐Go decay pathway. No‐Go decay is mediated by the proteins Hbs1p and Dom34p, which act to remove stalled ribosomes, and will promote cleavage of the mRNA at the stall site. The remaining 5′ fragment is subject to 3′–5′ decay by the cytoplasmic exosome, while the 3′ fragment is degraded 5′‐3′ by exonuclease Xrn1p in P‐bodies, which are sites of mRNA degradation.

Upon growing yeast into diauxic shift, an increase in P‐body assembly was observed. However, stress granules, which act as sites for mRNA storage during physiological stress, failed to assemble. The same result is seen upon directly exposing yeast to conditions in which glucose is limiting (0.5% glucose). This observed increase in P‐body assembly appears to be due to an upregulation of No‐Go Decay. In strains lacking the proteins Dom34p and Hbs1p, P‐body assembly is abrogated as compared to the wild type strains. These data suggest that glucose limiting stress can potentially lead to an increase in ROS, which can damage mRNA leading to activation of No‐Go Decay and subsequent P‐body assembly.
The Effects of Physical Activity on Burnout and Stress in Second‐Year Medical Students
Authors: Tung Shouli , Khamisani Azmeer , Chirico Erica N.
Published: April 2020

Medical students experience higher than average levels of burnout and stress as they learn to adapt to the rigors of medical school. Due to the demanding structure of medical education, students may forego exercise, healthy diets, social interactions, and sleep. Healthy habits such as sleep, diet, and exercise have been shown to impact academic performance and future functions as physicians. In addition, as exercise training has been shown to reduce oxidative stress, it is through this pathway that the cortisol‐induced stress response may be blunted. We hypothesize that exercise can reduce the self‐perceived levels of stress in second‐year medical students. Fitbits were distributed to second‐year medical students and were used to monitor the activity, steps, and heart rate during the second half of the year as well as during their United States Medical Licensure Exam (USMLE) Step 1 dedicated test preparation period. Each person was also given a stress survey to fill out during the week of their exam, as well as during the week of their USMLE Step 1. Students self‐reported whether they were a low, moderate, or high exercise activity person, which coincided with their resting heart rates, as confirmed by literature. Our results showed that students who had self‐reported low exercise activity had higher stress scores in the Women’s Health, Ears‐Nose‐Throat, and one of two Neurology‐psychiatry blocks, as well as during the USMLE Step 1 detected test preparation period. Stress levels in general increased as the year progressed to the Step 1 exam. There was also a significant negative correlation between sleeping hours and stress levels. In conclusion, these results demonstrate that those students who perform higher levels of physical activity report lower levels of stress, which could possibly occur via a reduction in the oxidative stress‐induced cortisol response. In addition, the reduction of sleep time may have altered the circadian cycle‐responding stress hormone, cortisol, resulting in the perceived level of increased stress that the students reported.