bluuebunny/crossref_metadata_2025 huggingface dataset:
Student Perceptions of the Importance of Diversity and Inclusion in Anatomical Dissection Teams: a Two Year Study
Authors: Fox Glenn Michael , Alsup B. Kathleen
Published: April 2020

With the marked increase of diversity and inclusivity teaching initiatives at the University of Michigan, the Division of Anatomical Sciences’ faculty developed a research plan to investigate what aspects of diversity and inclusion are of most importance to students. A pre‐ and post‐survey regarding student perceptions of the importance of diversity and inclusion in anatomical dissection teams was distributed to two cohorts of students in the U‐M DENT 545 (Summer 2018 & 2019). The majority of students considered the Anatomy curriculum to be inclusive, but differed considerably in how important they considered diversity to be within a dissection team. Certain aspects of diversity had notably higher scores for providing positive contributions to a dissection team (race/ethnicity, gender/gender identification, culture, and age), which other aspects scored comparatively lower (political perspective, (dis)ability status, and religious commitments). Additionally, some qualitative comments provided actionable insights on how to improve an inclusive environment.
Primary Endothelial Cells from Seals Mount a Rapid and Sustained Response to Acute Hypoxia
Authors: Allen Kaitlin , Vázquez-Medina José Pablo
Published: April 2020

Marine mammals tolerate repeated bouts of hypoxemia while diving, with up to 90% of blood oxygen stores depleted in some species. Accordingly, tissues such as the endothelium experience acute fluctuations in oxygen availability. In terrestrial mammals, hypoxia/reoxygenation events of similar magnitude contribute to inflammatory signaling, reactive oxygen species (ROS) generation, and endothelial dysfunction in pathological settings. Marine mammals, however, appear well protected against oxidant stress and inflammation despite repeated exposure to hypoxia/reoxygenation, though the specific cellular mechanisms driving this protection remain unknown. We isolated endothelial cells (ECs) from placental arteries from northern elephant seals (Mirounga angustirostris), a pinniped species noted for its extreme diving capacity. Seal primary EC cultures proliferate, express the canonical EC markers PECAM‐1 and VE‐cadherin, and take up acetylated LDL. Seal ECs are metabolically active in adherent culture and respond to mitochondrial inhibition and uncoupling and pharmacological NADPH oxidase activation, demonstrating utility as a live, functionally relevant system in which to study the biochemical drivers of hypoxia/reoxygenation tolerance in diving seals.
Seal ECs exposed to 1% O
2 for up to 6 hours upregulated HIF‐1α protein, with maximal expression occurring within 15 minutes (68‐fold increase over normoxic levels). Human ECs matched this magnitude (73‐fold increase) though not until 1 hour, after which expression of HIF‐1α rapidly and progressively declined (22‐fold above normoxic levels at 6 hours). In contrast, seal ECs maintained elevated HIF‐1α protein expression (52‐fold) at 6 hours. Pharmacological stimulation with menadione, an inducer of redox cycling, significantly increased oxidant generation in human (5‐fold, p=0.027) but not seal ECs (2.5‐fold, p=0.18), as did
acute exposure to hypoxia/reoxygenation. Together, our data show that seal ECs mount a rapid, sustained response to hypoxia
/reoxygenation events, and that these cells are a functional system in which to study the mechanisms underlying innate hypoxia and oxidative stress tolerance in diving mammals.

Support or Funding Information

National Science Foundation GRFP
Deletion of Mouse Sf3b4 in Neural Crest Cells Causes Craniofacial Abnormalities
Authors: Kumar Shruti , Alam Sabrina Shameen , Beauchamp Marie-Claude , Majewski Jacek , Jerome-Majewska Loydie
Published: April 2020

The SF3B4 gene encodes a core component of the U2‐major spliceosome complex important during splicing of pre‐mRNA to mRNA. Recently, patients with two craniofacial disorders: Nager syndrome and Rodriguez syndrome were identified to carry rare mutations in this gene. The autosomal dominant nature of these syndromes indicates that this is due to haploinsufficiency for SF3B4. Both disorders affect the face as well as the limb including both hands and feet. Nager syndrome patients survive and exhibit smaller jaw bones, smaller cheek bones, cleft palate, hearing problems as well as downward slanted eyelids. However, Rodriguez syndrome is much more severe, and patients often die before or soon after birth. We hypothesized that craniofacial abnormalities associated with reduced SF3B4 levels is due to tissue‐specific expression and requirement of this gene in neural crest cells. Using in situ hybridization, we showed that in mouse embryos, Sf3b4 was expressed ubiquitously from embryonic day (E)9.5 – E12.5. From E10.5 – E12.5, enriched expression of this gene was found in the maxillomandibular region, limb and tail bud. To generate a conditional mutant mouse line for Sf3b4, we used CRISPR/Cas9 to insert LoxP sequences in intron 1 and 3 of this gene. To test our hypothesis, we mated Sf3b4 conditional mutant mice to Wnt1‐Cre2 transgenic mice to delete exons 2 and 3 specifically in neural crest cells. Heterozygous mutant embryos from these matings were normal. From E9.0 onwards, all homozygous mutant embryos had hypoplasia of the midbrain and pharyngeal arches. Homozygous mutant embryos died by E14.5. To examine neural crest cells and their derivatives in the head and craniofacial region, we used the Rosa26R lacZ reporter line. LacZ staining of mutant embryos showed a reduced number of neural crest cells in pharyngeal arches. Thus, our data suggests that craniofacial malformations due to mutations in SF3B4 are the result of reduced neural crest cells.

Support or Funding Information

Canadian Institutes of Health Research (CIHR)
Re‐Defining the Specificity of Phosphoinositide‐Binding by Human PH Domain‐Containing Proteins
Authors: Singh Nilmani , Reyes-Ordonez Adriana , Compagnone Michael , Moreno Jesus , Yao Lucy , Chen Jie
Published: April 2020

The human genome encodes over 250 PH domain containing proteins, all of which share an invariable structure. PH domains are presumed to mediate protein‐lipid interactions specifically to phosphoinositides (PIPs), and yet only a small fraction of PH domain‐containing proteins have been reported to bind PIPs with specificity. Most reported protein‐lipid interaction studies employ methods that either require purified proteins or utilize non‐physiological presentation of lipids, and rely on isolated PH domains rather than full‐length proteins. To circumvent these problems, we recently developed a total internal reflection fluorescence (TIRF) microscopy‐based single‐molecule pulldown (SiMPull) assay, called lipid‐SiMPull, to study protein‐lipid interactions using GFP‐tagged full‐length proteins in whole cell lysates and lipid vesicles. Here, we applied the lipid‐SiMPull assay to probe the lipid binding properties of 67 human PH domain proteins. We found that >50% of those full‐length proteins bound at least one of the phospholipids, with 44% of them displaying various levels of selectivity towards PIPs. About two‐thirds of those proteins had not been previously reported to bind PIPs with any specificity. Additionally, we examined lipid binding by 17 isolated PH domains and found that most of them did not behave like their full‐length counterparts. One of the proteins, XPLN (ARHGEF3), was found to bind specifically PI‐4,5‐P2 in lipid‐SiMPull. Further characterization revealed that PI‐4,5‐P2 binding was necessary for XPLN membrane association, activation of RhoA, and stimulation of stress fiber formation. Taken together, our findings suggest that specific PIP recognition by PH domain proteins is more prevalent than previously believed, and that full‐length proteins display lipid binding properties distinct from the isolated PH domains.

Support or Funding Information

NIH NIGMS R01GM089771
Coordinated development of the mitochondrial reticulum in skeletal muscle
Authors: Kim Yuho , Lindberg Eric , Bleck Christopher K.E. , Glancy Brian
Published: April 2020

Skeletal muscle cells form a mitochondrial network, or reticulum, in a fiber‐type specific manner with grid‐like and fiber‐perpendicular mitochondrial network configurations found in oxidative and glycolytic fibers, respectively. However, it is not well characterized how the networks form during postnatal muscle development. Using focused ion beam scanning electron microscopy (FIB‐SEM) and machine learning based 3D image segmentation and analysis, we have sought to understand the 3D mitochondrial development in oxidative (soleus) and glycolytic (gastrocnemius) muscles of mice at different postnatal days 1 (P1), 14 (P14), and 42 (P42). Both oxidative and glycolytic fibers show tortuous, parallel mitochondrial formations at P1. At P14, oxidative fibers have more elongated, fiber‐parallel mitochondria, while glycolytic fibers have a more grid‐like mitochondrial reticulum. To better understand the intra‐ and inter‐organelle interactions of the mitochondrial reticulum during development, we additionally assessed intermitochondrial junctions (IMJ) and mitochondrial interactions with lipid droplets (LD). During early maturation (P1‐P14), many IMJ in oxidative fibers are arranged in both diagonal (~40%) and parallel (~40%) directions, yet matured oxidative fibers have almost half (~50%) of IMJ in the perpendicular direction. In contrast, after having ~50% of IMJ along the parallel axis at P1, glycolytic fibers gradually change IMJ orientations more into both perpendicular and diagonal axis and IMJ are located in all directions at P42. Although mitochondria in glycolytic fibers maintain similar sizes of the IMJ contact sites between P1 and P14 and have smaller areas at P42, oxidative fibers have ~2‐fold larger IMJ areas at P14 as compared to early (P1) and matured (P42) fibers. Meanwhile, both oxidative and glycolytic fibers at P1 have many, big LD that sharply decrease in size during the course of postnatal development. In the matured muscles, oxidative fibers have few, tiny LD and glycolytic fibers have no LD. Both fibers maintain similar LD contract areas per mitochondrion across P1‐P42. Notably, mitochondria directly in contact with LD have volumes ~2–3‐fold larger than non‐LD‐contacted mitochondria at all time points. In addition, the LD‐contacted mitochondria have ~2‐fold bigger IMJ areas as compared to non‐contacted one, with the largest differences observed at P14. In conclusion, development of the muscle mitochondrial reticulum occurs in coordination with altered interactions among mitochondria and with LD. However, future studies are warranted to study possible interactive roles of calcium handling structures such as sarcoplasmic reticulum and t‐tubules.

Support or Funding Information

This study is supported by intramural programs within the National Heart, Lung, and Blood Institute and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (grant 1ZIAHL006221‐02).
Medical Faculty’s Perception of the Use of Altmetrics
Authors: Brown Kirsten , Byram Jessica , Lazarus Michelle , Wilson Adam
Published: April 2020

In recent years, Academia is increasingly engaging with online scholarly tools (e.g., Mendeley) and social media platforms (e.g., Twitter), providing potentially impactful alternative pathways for research dissemination and engagement. These platforms flatten access to knowledge with online platforms fostering easier access by both peers and the broader community alike. Termed altmetrics, or “alternative metrics”, these platforms carry with them novel methods for tracking scholarly impact and reach whose potential is not yet fully understood. These non‐traditional Altmetrics capture quantitative data indicative of social media shares, clicks and plays. The use of altmetrics as indicators of scholarly/societal impact is garnering increased attention over the last decade. However, the degree to which faculty demographics, career stage, professional roles, and areas of research focus influence perceptions and use of altmetrics within academia is yet to be characterized. This study examined the perceptions of medical faculty regarding their use of altmetrics, exploring how factors, such as career focus and social media use, influence these opinions.

Medical faculty responded to a Qualtrics survey distributed through social media platforms and organizational listservs. The survey collected demographic information, and data on social media usage, the likelihood of disseminating altmetric outcomes in academic documents (e.g., CV), and perceptions on the value/utility of altmetrics.

Of the 48 respondents, 23 were male and most were from North America or Oceania (81%). All age groups and career levels were represented. While only 17% indicated their institution endorses altmetrics, slightly more than half (54%) indicated altmetrics were ‘moderately’ to ‘extremely valuable’. A Pearson’s correlation revealed that frequent social media usage was strongly and positively correlated with favorable perceptions of altmetrics (r=0.512, p<0.001). Neither age range, career level, nor years of experience explained one’s level of social media usage (p>0.402) or one’s favorable perceptions of altmetrics (p>0.200). Irrespective of respondents’ primary professional roles, areas of research focus, or whether their institution endorsed altmetrics, no significant differences (p>0.05) in social media usage or altmetric perceptions scores were detected.

This study suggests that more favorable perceptions of altmetrics are associated with higher social media use. Even though institutions may not endorse altmetrics for promotion and tenure purposes, many medical faculty still regard them to be valuable. These results also indicate that favorable perceptions of altmetrics traverse career level, age ranges, and years of experiences, supporting a more universal appreciation for these non‐traditional evaluations of impact. Future work will explore, through qualitative inductive interviews, motivations for those engaging (or not) in social media platforms and altmetrics.
ROLE OF TRPV4 CHANNEL SIGNALING IN SHEAR STRESS RESPONSES
Authors: Aria Kevin M.
Published: April 2020

Shear stress along the vascular intima promotes blood flow and preserves vascular wall structure, providing vital protection against progressive cardiovascular disease. Indeed, obstructive cardiovascular disease states such as atherosclerosis are associated with early shear stress disruption that leads to endothelial dysfunction and vascular remodeling. A key feature of shear stress action is a rapid and sustained increase in dynamic (spatially and temporally) Ca2+ signals within the endothelium, which promotes vasodilation and opposes inflammation. Although specific mechanisms remain unclear, recent studies have implicated mechanosensitive TRPV4 cation channels in shear stress mediated Ca2+ entry. We hypothesize that shear stress‐induced endothelial Ca2+ signals arise from graded recruitment of TRPV4 Ca2+ transients. We performed Ca2+ imaging studies on arteries isolated from wild type and genetically altered TRPV4 knockout mice. Carotid arteries were isolated, opened, and mounted in specialized flow chambers. The chambers were connected to a peristaltic pump and placed on the stage of a microscope adapted for high‐speed confocal imaging (488 nm ex, 510 nm emission; 8 frames/s). Endothelial Ca2+ dynamics were recorded at increasing flow rates (shear stress 0, 12, 52, 75, and 102 dynes/cm2). Image analysis was performed using ImageJ and custom software (LC_Pro) designed to discern distinct profiles of signal frequency, amplitude, duration and spatial spread. Artery segments were also subjected to immunostaining to assess relative expression and distribution of TRPV4 channels. We found that the wild type and TRPV4 knockouts both responded to shear stress. The TRPV4 knockout exhibited a reduced Ca2+ event frequency and shorter event durations compared to wild type controls in response to physiologic elevations in shear stress. Future experiments will focus on expounding upon the flow response, particularly the importance of TRPV4 channels in the time‐course of Ca2+ signal patterning changes under chronic low flow conditions.

Support or Funding Information

Sponsored by Mark Taylor and David Weber, Ph.D., Department of Physiology/Cell Biology, University of South Alabama College of Medicine, Mobile, AL. Summer Medical Student Research Program
Dysbiosis in hypertension modulates kidney function through TLR4‐mediated gut barrier disruption
Authors: Majumder Suravi , Pushpakumar Sathnur , Sen Utpal
Published: April 2020

Inflammation is a major mediator in the development and progression of hypertension‐induced chronic kidney disease (CKD). Recent studies demonstrated that altered gut microbiota contributes to blood pressure (BP) dysregulation and kidney dysfunction in hypertension. Both animal and clinical studies in hypertension revealed decreased gut microbial richness and diversity. Further, a plethora of evidence suggests that gut barrier impairment allows bacteria genes and their products to enter circulation and affect physiological function in different organs. Although the relationship between Angiotensin II‐induced (Ang II) hypertension and TLR4‐mediated inflammatory response has been established, the contribution of TLR4 in the gut‐kidney axis to modulate kidney function is unclear. We therefore sought to determine the possible link between TLR4 and the gut‐kidney axis in hypertension‐induced renal injury and dysfunction. Mice with normal TLR4 (C3H/HeOuJ, HeOuJ) and mice lacking functional TLR4 (C3H/HeJ, HeJ) were treated without or with Ang‐II (1000 ng/kg b.w.) for 4 weeks. Increased BP and reduced renal function (GFR) were recorded in HeOuJ mice following Ang II treatment. HeJ mice were partially resistant to Ang II‐induced hypertension. 16S rRNA sequencing analysis of gut microbiome revealed differentially altered microbial richness and diversity in HeOuJ mice compared to HeJ mice during hypertension. Further, HeOuJ mice showed gut barrier disruption to Ang II as indicated by decreased tight junction proteins (ZO‐1, Occludin, Claudin‐1) and increased gut permeability (FITC dextran leakage into the bloodstream) compared to HeJ mice. Increased bacterial component translocation was observed (16S rRNA) in hypertensive HeOuJ kidney compared to other groups. In response to Ang‐II, gut and kidney inflammation (IL1b, IL6, TNFa) was markedly increased in HeOuJ mice but blunted in HeJ mice. Taken together, our data demonstrates that gut dysbiosis during hypertension triggers TLR4 activation and gut barrier disruption leading to renal inflammation and dysfunction and mice lacking normal TLR4 are protected from dysbiosis/TLR4 mediated renal injury.

Support or Funding Information

DK116591, DK104653, 15SDG25840013
Prolonged Fasting Increases DNA Methylation in Northern Elephant Seal Pups
Authors: Gibson Emily , Torres-Velarde Julia Maria , Vazquez-Medina Jose Pablo , Crocker Daniel
Published: April 2020

Northern elephant seals (NES) are naturally exposed to extreme conditions, including prolonged food and water deprivation (fasting). NES pups initially nurse for a month before they are weaned and fast for two months. During this fasting period NES pups lose about 25% of their body mass, while maintaining biochemical homeostasis and supporting muscle development. The environment, especially stressful environmental conditions, can modify the methylation status of DNA, consequently regulating gene expression. We compared global DNA methylation between early (1–2 weeks) and late (7–8 weeks) fasting NES pups. DNA was extracted from white blood cells collected from fasting NES pups. Global DNA methylation was measured using a MethylFlash™ Global DNA Methylation (5‐mC) Kit. Prolonged fasting significantly increased global DNA methylation (p = 0.0463) in NES pups, suggesting a decrease in transcription activity. We are in the process of identifying changes in specific genes involved in growth, hypoxia tolerance, and metabolism between early and late fasting pups by analyzing the methylation status of CpG islands in putative promoter region sequences. Our initial results suggest that DNA methylation is an important regulator of gene expression during natural, prolonged food deprivation.
CTRP3 and Alcoholic Liver Disease in Female Mice
Authors: Root Callie Leah
Published: April 2020

Introduction

C1q TNF Related Protein 3 (CTRP3), is a cytokine that is primarily secreted from adipose tissue, also known as an adipokine. Our previous research has shown that CTRP3 prevents alcoholic fatty liver disease (ALD) in male mice. However, even when accounting for confounding factors such as absolute and relative alcohol intake females are more sensitive to the effects of ethanol compared to male mice. Therefore, the goal of this project was to determine whether CTRP3 prevented ALD in female mice or determine whether the beneficial effects of CTRP3 are sex specific.

Methods

Female wild type (WT) and CTRP3 transgenic over expressing (Tg) mice were fed an ethanol containing liquid diet (5% v/v) for 6 weeks. Daily weight and food intake measurements were taken and external heat‐pads were placed under a portion of the cage to facilite thermoregulation. Hepatic steatosis was determined by total triglyceride quantification and lipid droplet quantitation in liver sections. Data were analyzed by repeated measures ANOVA, t‐test, or Log‐rank (Mantel‐Cox) test as appropriate.

Results

There was no difference between WT and Tg mice in food intake or body weight. There was no difference in survival between WT and Tg mice, however, Tg mice trended towards a reduced rate of survival compared with WT mice (78% in WT versus 44% in Tg, p=0.13). Stereological analysis indicated no difference in the percent of lipid liver volume between the two groups (WT 7.2±3.6 vs Tg 5.1±4.1%). This finding was consistent with no difference in total hepatic triglyceride accumulation observed between WT and Tg mice (12.7±4.4 vs. 13.1±6.8 mg triglycerides/gram liver protein).

Conclusion

Combined these data indicate that unlike previous studies with male mice, CTRP3 is not protective against alcohol‐induced hepatic steatosis. Combined, these data indicate that the adipokines such as CTRP3 contribute to physiology in a sex‐specific manner.
Comparison of academic performance in undergraduate Anatomy and Physiology using in‐person versus asynchronous on‐line laboratory instruction
Authors: Peterson Jonathan M. , Brown Patrick
Published: April 2020

OBJECTIVE

To compare the effectiveness of asynchronous on‐line laboratory instruction with in‐person instruction on learning in undergraduate Anatomy and Physiology.

METHODS

A retrospective analysis was conducted on student academic performance on the first semester of a 2‐semester Anatomy and Physiology course. All students were enrolled in an asynchronous on‐line Anatomy and Physiology lecture course and had the option of enrolling in either an in‐person or asynchronous on‐line laboratory. Assessment of learning for all students was obtained via course management software. Specific assessments included 3 lecture examinations, 2 laboratory examinations, and 12 specific content quizzes for lecture and laboratory respectively (24 quizzes total). All examinations were proctored, whereas students were not proctored for quizzes and were encouraged to use their textbooks and notes. Student engagement was assessed based on number of independent course log‐ins, number of completed tasks, and total time spent.

RESULTS

Overall the students who enrolled in the in‐person laboratory instruction had mean examination scores ca. 10% higher than the on‐line laboratory students that was greatly dependent on topic area. Specifically, there was no difference between the two groups in the first lecture exam (regional anatomy, cellular anatomy, and histology), with a 9% difference in the second exam (musculoskeletal system), and a 13% difference in the third lecture exam (Nervous system). There was no difference between the two sets of students in the measures of student engagement or in the lecture quiz scores. There was a 22% decrease in the mean laboratory quiz grade for students enrolled in the on‐line laboratory. There was a 14% and 22% difference in the mean scores for the 2 laboratory exams. Further, the reduction in mean laboratory examination score was still significant when normalized to the student’s individual mean lecture examination score.

CONCLUSIONS

The use of asynchronous on‐line laboratory instead of in‐person laboratory instruction can significantly negatively impact student academic performance.

Support or Funding Information

NIDDK R15DK114740
Sympathetic Neurotransmitters Decrease Muscle Spindle Afferent Response to Muscle Stretch
Authors: Salazar Alexandra , Harnisch Arthur , Valdespino Steven , Wilkinson Katherine A.
Published: April 2020

Proprioception is the ability to sense body position and motion in space. The primary proprioceptors are the Group Ia and II muscle spindle afferents that innervate the muscle spindle and comprise the sensory arm of the muscle stretch reflex. In‐vivo animal studies have shown that increased sympathetic outflow decreases dynamic and static stretch sensitivity of muscle spindle afferents. However, the direct effect of sympathetic neurotransmitters on muscle spindle afferent stretch sensitivity is not well understood. We hypothesized that the addition of sympathetic neurotransmitters would cause a decrease in responsiveness of muscle spindle afferents via direct action in the muscle spindle. To test this hypothesis, we used an in‐vitro mouse muscle nerve preparation to record muscle spindle afferent firing rates during stretch. The muscle was stretched once every three minutes to 5% resting length for 4 s. Muscle spindle afferent firing rates were determined during the plateau phase of stretch and compared before and after drug addition. Instantaneous firing frequency during a static muscle stretch was measured at different time intervals: beginning of stretch (0.5 seconds into the hold phase or Initial Static Time (IST)) and end of stretch (3.5 seconds into the hold phase or Final Static Time (FST)). All values are expressed as a percentage of the average of 6 pre‐drug baseline responses (BL) and compared to no drug control mice stretched for the equivalent time. Exposure to 100μM norepinephrine (n=6, IST=54.18% BL, FST=37.70% BL) significantly decreased the response to stretch as compared to no drug control animals (n=12, IST=90.82% BL, FST=91.50% BL; p<0.05 One‐way ANOVA w Tukey’s post‐hoc to control). The addition of epinephrine decreased muscle spindle afferent firing, however only significantly at the lower dose of 30μM (n=6, IST=76.90% BL, FST=73.20% BL; p<0.05). The α1 adrenergic receptor agonist, phenylephrine, was used to test the hypothesis that the decrease seen following norepinephrine was due to binding at the α1 adrenergic receptor. No statistically significant decrease was found following phenylephrine administration, however, the sample size used was small (n=3, IST=96.70% BL, FST=96.98% BL). These results show direct effects of sympathetic neurotransmitters on muscle spindle afferent stretch sensitivity and support the hypothesis that sympathetic innervation of the muscle spindle plays an important role in modulating stretch responsiveness. Future studies will use optogenetic techniques to stimulate the sympathetic neurons to determine whether physiologically relevant release of sympathetic neurotransmitters controls muscle spindle afferent sensitivity.

Support or Funding Information

This work was supported by NIH SC3 GM127195 (KAW) and NIH R25 GM071381 (AS and SV).
Endotoxic hepatotoxicity in rats is exacerbated by tacrolimus and diminished by cyclosporine or sirolimus: modulation by androgenic hormones
Authors: Elzokm Shrouk S. , El-Deeb Nevine M. , Abdel Moneim Rahab A. , Fouda Mohamed A. , El-Mas Mahmoud M.
Published: April 2020

Liver toxicity contributes to poor prognosis and increased mortality rate in septic and endotoxic patients. Given the discrepant hepatic influences of immunosuppressant therapies, biochemical and histopathological studies were performed to determine whether endotoxic hepatotoxicity is variably altered by calcineurin‐dependent (cyclosporine and tacrolimus) and independent immunosuppressant (sirolimus) in rats. The data showed that 6‐hr treatment of rats with lipopolysaccharide (LPS, 3 mg/kg i.p.) significantly increased serum levels of serum glutamic pyruvic transaminase (sGPT) and glutamic oxaloacetic transaminase (sGOT). Morphologically, livers of endotoxic rats showed evidence of portal tract inflammation, neutrophil infiltration, central vein congestion, and focal lytic necrosis. Except for mild portal inflammation and congestion, these hepatic anomalies were largely compromised in endotoxic rats pretreated 2 hr earlier with cyclosporine (10 mg/kg i.p.) or sirolimus (1 mg/kg i.p.). By contrast, the prior exposure to tacrolimus (2 mg/kg i.p.) accentuated hepatotoxic manifestations of endotoxemia as indicated by the significant elevations in serum sGPT and sGOT beyond endotoxic levels and loss of regular cord arrangement of hepatocytes, apparent bile secretion within bile canaliculi, hemorrhagic areas, cytoplasmic vacuolization, and marked congestion. We also show that functional androgenic pathways seem necessary for the demonstration of hepatotoxic manifestations caused by LPS or its combination with tacrolimus because such effects were remarkably attenuated in rats with surgical (castration) or pharmacologic (flutamide, androgen receptor blocker) testosterone depletion. Our data indicate that immunosuppressants exhibit diverse, calcineurin‐independent, testosterone‐mediated effects on endotoxic hepatotoxicity in male rats.
Effect of Prenatal Glucocorticoid Exposure on Circadian Rhythm Gene Expression in the Brains of Adult Rat Offspring
Authors: Murray Alyssa , Khurana Sandhya , Lalonde Christine , Tharmalingam Sujeenthar , Nguyen Phong , Tai T.C.
Published: April 2020

Circadian clocks developed in organisms as a way of estimating the time of day. In mammals, the circadian clock is based on molecular oscillators that operate through transcriptional‐translational feedback loops. While normal circadian signaling relies on a ‘master clock’, in the suprachiasmatic nucleus (SCN), to synchronize peripheral oscillators to environmental light, glucocorticoid receptor (GR) signaling has the ability to reset the phase of peripheral clocks. GR is responsive to cortisol and is central to the regulation of genes involved in stress response, immune regulation and metabolism. Phase shifts allow peripheral clocks to become uncoupled from the master clock. The SCN does not express GR and this ensures it stays tied to the light‐dark cycle. It has been shown that maternal exposure to glucocorticoids (GCs) can lead to modification of hypothalamic‐pituitary‐adrenal (HPA) function and impact stress‐related behaviours via GR activation. While exploring the effects of prenatal GC exposure on rat offspring, our lab previously demonstrated altered circadian rhythm signaling in the adrenal glands using whole transcriptomic profiling. Pregnant WKY rats were given daily subcutaneous injections of 0.1 mg/kg/day DEX (Dexamethasone, a synthetic GC), or a saline vehicle throughout the third semester, and offspring sacrificed at 18 weeks of age for tissue collection. Brain regions were isolated through a micropunch technique and RNA extracted. Results from the current study show that prenatal GC exposure affects circadian rhythm gene expression in the molecular oscillators of the amygdala, hippocampus, prefrontal cortex, paraventricular nucleus as well as the main oscillator in the SCN. Transcripts of circadian rhythm genes in these tissues were measured through RT‐qPCR and demonstrated both sex and tissue specific alteration in expression of circadian genes including Bmal1, Clock, Npas2, as well as the Per and Cry genes. This widespread dysregulation of the circadian rhythm system points to a mechanism whereby the dysfunction seen previously in the adrenal gland arises from a programmed dysregulation of the entire circadian system beginning at the master clock in the SCN.

Support or Funding Information

Supported by grants from CIHR and NOSMFA Research Development Fund.
Structural and functional analysis of the role of the chaperonin CCT in mTOR complex assembly
Authors: Willardson Barry M. , Cuellar Jorge , Ludlam Grant , Tensmeyer Nicole C. , Aoba Takuma , Dhavale Madhura , Santiago César , Bueno-Carrasco Teresa , Mann Michael J. , Plimpton Rebecca L. , Makaju Aman , Franklin Sarah
Published: April 2020

The mechanistic target of rapamycin (mTOR) kinase forms two multi‐protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, β‐propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 Å) cryo‐EM structure of the human mLST8‐CCT intermediate isolated directly from cells shows mLST8 in a near‐native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N‐ and C‐termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar β‐propeller proteins.

Support or Funding Information

This research was supported by the grant BFU2016‐75984 (AEI/FEDER, EU) and the Madrid Regional Government (grant S2013/MIT2807) to JMV as well as the US National Institutes of Health grant EY012287 to BMW and fellowships from the Brigham Young University Simmons Center for Cancer Research to WGL, NCT, TA and MD.
Endothelial Senescence in Regenerative Lung Growth after Pneumonectomy
Authors: Mammoto Akiko , Hendee Kathryn , Mammoto Tadanori
Published: April 2020

Impairment of lung regeneration and repair in aging animals contributes to the pathogenesis of aging‐associated diseases, including chronic obstructive pulmonary disease (COPD). Angiogenesis – the formation of new blood capillaries ‐ plays a key role in organ regeneration. We have reported that inhibition of angiogenesis attenuates lung growth after unilateral pneumonectomy (PNX) and that post‐PNX regenerative lung growth is inhibited in aged mice. While accumulation of senescent cells in aged adults is linked to the age‐related decline in regenerative ability, senescent cells also play important roles in injury repair and tissue remodeling. Here we investigate the role of endothelial cell (EC) senescence in regenerative lung growth after PNX in young vs. aged mice. The levels of the senescence marker, p16INK4A and a major angiogenic factor, angiopoietin2 (Ang2) transiently increase in ECs isolated from 2‐months (2M) old post‐PNX mouse lungs compared to those from sham‐operated control mice and decrease at the later stage. The baseline levels of p16INK4A are high in 24M old mouse lung ECs but post‐PNX transient induction of p16INK4A and Ang2 is inhibited in 24M old mouse lung ECs. Depletion of senescent cells from 2M old p16‐3MR mice by treating with ganciclovir suppresses post‐PNX lung growth. When ECs isolated from 2M old post‐PNX mouse lungs are mixed in the gel and implanted on the mouse lungs, vascular network formation is stimulated in the gel, while these effects are inhibited in the gel mixed with 24M old post‐PNX mouse lung ECs. These results suggest that post‐PNX transient induction of EC senescence is required for vascular morphogenesis after PNX. Deregulation of this mechanism may contribute to age‐dependent inhibition of regenerative lung growth. Understanding this mechanism will lead to the development of efficient strategy for aging‐associated lung diseases.

Support or Funding Information

NIH R21AG054830, NIH R21AG062893, NIH R01HL142578, AHA 18TPA34170129
Effects of Perinatal Inhibition of Gonadal Steroid Hormones on Behavioral Changes in Rats
Authors: Hammel Jessica Jean , Niepoetter Paige , Viernow Coral , Sundaram Ram , Gopalan Chaya
Published: April 2020

Sex differentiation of a rat brain occurs during the perinatal period (four days prior to and four days after birth). The undifferentiated brain becomes a female brain unless it is exposed to testosterone (T) which gets metabolized to 17β‐estradiol (E2) and dihydrotestosterone (DHT). While aromatase converts T to E2, 5α‐reductase converts T to DHT. 17β‐estradiol exerts its effects by binding to an estrogen receptor (ER) whereas DHT via an androgen receptor (AR). We tested the significance of DHT and E2 during the organizational phase in the differentiation of the rat brain. During the last four days of pregnancy, seven timed‐pregnant rats received either the vehicle (5% ethanol and 95% sesame oil), exemestane (Aromasin; 4mg/kg/ml), or flutamide (20 mg/kg/ml) subcutaneously. Pups from each group continued to receive their specific treatments during the first four days after birth. From postnatal day 65, these animals were subjected to open field (OF), spatial working memory, and sexual motivation (SM) tests. Male rats receiving flutamide exhibited a significant increase in exploratory behavior. Additionally, male rats receiving exemestane exhibited significantly greater interest in the estrus rat compared to the control group. In conclusion, under AR blockade, where E2 becomes the main gonadal hormone, improvements in exploratory behavior in male rats were observed. Moreover, when exemestane was used to block E2 conversion, and DHT becomes the main gonadal hormone, increased SM in male rats was observed. Thus, the presence of both E2 and DHT during organizational phase appears to be essential for normal socio‐sexual and exploratory behaviors.
Distinct Bacterial Populations Colonizing Plastic Debris in Coastal Waters of Southern California
Authors: Leask Allison , Barral Ana Maria , Simmons Rachel E.
Published: April 2020

Microbes that attach to plastic form the so‐called plastisphere, a unique ecosystem of microbes that may impact the ocean and its inhabitants. While the microbial populations present on microplastics, tiny fragments resulting of physical and chemical breakdown of plastic debris, have been described in multiple studies, much less is known about the initial stages of bacterial colonization. This project is part of a study to evaluate the presence and diversity of bacteria colonizing floating plastic in coastal waters using metagenomic DNA sequencing and live culture experiments. We particularly focus on colonies isolated from two different sampling locations using different culture media and some of their physiological characteristics.

The objective of this research is to identify bacteria colonizing ocean plastic using 16S rDNA barcoding from cultures isolated from exposed plastic. In addition, some physiological characteristics of the bacteria, such as antibiotic production and plastic degradation, is explored in parallel.

Three types of floating plastic (High and low density polyethylene and polypropylene) were exposed to beach water at Doheny State Beach (Dana Point, CA) and near the Scripps Institution of Oceanography pier (La Jolla, CA). Samples were collected at different time points, in a range up to 2 months. Colonies were grown on Salt Water Agar (SWA) or Marine Agar, as well as ChromAgar Vibrio (CAV) media and characterized using 16S amplicon sequencing. Fourier Transform Infrared Spectroscopy (FTIR) was used to analyze the breakdown of plastic over time and scanning electron microscopy (SEM) to visualize the biofilms attached to plastic on day 40.

No clear pattern of colonization was observed over a month period. With longer incubation times, an increase in unknown/uncultured bacteria species was noted. Antibiotic production was observed in several colonies of the genus Phaeobacter. Signs of chemical breakdown were observed as early as after 21 days of incubation.

Support or Funding Information

Supported by NU internal grants as well as the NSF HSI’s new to NSF award #1832545
Halophile Inoculation Significantly Improves Growth of Alfalfa Under Saline Conditions
Authors: Miller Ashley , Knowles Alisa , Nielsen Brent , Hill Jonathon
Published: April 2020

Saline soils limit crop production, and salinity is increasing in farmland in many parts of the world. A halophilic bacterial species has been isolated from salt‐tolerant plants, and when used as an inoculum stimulates growth of alfalfa in salty soil. The hypothesis is that the microbial inoculant induces gene expression changes in the plant, resulting in the increased growth of the plant in salt. To test this, RNA was isolated from inoculated and uninoculated alfalfa plants grown under saline conditions, with additional controls in the absence of salt. RNAseq analysis of these four treatments led to identification of differentially expressed genes (DEG). Several of these DEGs are being examined in more detail using quantitative reverse‐transcriptase PCR. This will lead to the identification of major plant gene expression changes induced by the bacterial inoculum under saline conditions.

Support or Funding Information

Our work is being funded by a grant from the Roger and Victoria Sant Educational Endowment for a Sustainable Environment, a John Topham and Susan Redd Butler BYU Faculty Research Award (The Charles Redd Center for Western Studies), and the Department of Microbiology and Molecular Biology, Brigham Young University.
The Role of Ceramide Synthases Response to the Heavy Metal Cadmium Chloride
Authors: Peck Margaret Eleanor , Staab Trisha , Chan Jason , Moats Alicen G. , Kalil Azia
Published: April 2020

Cadmium is an extremely toxic heavy metal that is hazardous to human health. In the United States, the primary source of cadmium exposure is in the food supply. Ingestion results in a buildup of cadmium in the body, specifically in the kidneys, which can lead to organ damage and cancer. This toxin has been known to induce stress in organisms by causing an accumulation of reactive oxidative species (ROS). To counter stress, cells respond with a variety of signaling pathways. We are interested in studying the sphingolipid pathway, which is known to mediate stress responses, but its role in defending against cadmium toxicity is unclear. We hypothesized that ceramide synthase, a sphingolipid metabolism enzyme, is required for survival to cadmium stress and decreased accumulation of ROS in animals. To test this, we utilized the model organism Caenorhabditis elegans. Ceramide synthase converts sphingosine to ceramide in the cells. We examined animals with mutations in ceramide synthase (hyl‐2 mutants) by treating worms to acute and chronic cadmium exposure and examining changes in survival, reproduction, and locomotion. We present data showing that cadmium does not affect animal reproduction, but it does affect survival upon exposure. Cadmium exposure resulted in decreased survival, however, and this result was less prominent in organisms possessing hyl‐2/ceramide synthase. We also found that the hyl‐2 gene is vital for oxidative stress response mechanisms. In addition, we present data on the effects of cadmium exposure on gene expression of sphingolipid metabolism enzymes using qPCR. These results are important because stress plays a role in aging, and if we better understand how stress is regulated via sphingolipids, it could become a potential pharmaceutical target to slow the degradative aging process.

Support or Funding Information

NIH‐ 2R15AG052933
Role of conserved serine hydrolases in controlling acetaldehyde toxicity in yeast
Authors: Barton Jennifer Marie , Johnson R. Jeremy
Published: April 2020

Acetaldehyde is a toxic by‐product of ethanol consumption that can lead to cancer through to its damaging effect on DNA. Among the genes hypothesized to control acetaldehyde toxicity are a family of conserved serine hydrolases from Saccharomyces cerevisiae, known as FSH1, FSH2, and FSH3. Our goal was to determine the relative role and redundancy of these three serine hydrolases in controlling acetaldehyde toxicity. Their relative redundancy was investigated by combining growth assays with combinatorial knock‐out strains and by measuring the relative substrate specificity of each individually purified hydrolase against esters of similar structures and properties to acetaldehyde. The growth of single deletion strains of FSH1, FSH2, and FSH3 in the presence of various unsaturated aldehydes and ketones did not show substantial differences between the various toxins tested. This suggested that the three homologs likely play compensatory roles. Double and triple knockouts are being constructed to dissect their potentially overlapping roles. For measuring their substrate specificity, FSH1 and FSH2 were expressed heterologously and purified to homogeneity with the purification of FSH3 ongoing. Overall, FSH1 and FSH2 were active against a variety of overlapping esters with higher average catalytic activity for FSH1. Together, we plan to build a map of the complex regulation and overlapping activities of these three serine hydrolase homologues in regulating acetaldehyde toxicity in yeast.
A Metabolomic Analysis of the Hispanic Paradox
Authors: Patterson Jeffrey , Bresette William , Liu Jingping , Eghlimi Ryan , Atlas Sarah , Gu Haiwei
Published: April 2020

INTRODUCTION

Obesity has increased in prevalence among Latinos in the United States over the last several decades and continues to trend upward. The current obesity rate for this population is 34.4%, which is significantly higher than that of the general American population (28.7%). However, increased body fat and excessive weight are not necessarily associated with an increase in premature mortality.

METHODS

In this cross‐sectional analysis, we used a metabolomic analysis to look at the mechanisms behind the Hispanic Paradox. To do this, we examined the role dietary intake, body mass index (BMI; kg/m2), and generational level (GL) played on serum metabolomic fingerprints in 70 Mexican Americans (26 male, 44 female). Metabolomic phenotypes of plasma were measured using liquid chromatography tandem‐mass spectrometry (LC‐MS).

RESULTS

There was no significant metabolic separation among BMI groups (normal weight = BMI < 25; overweight = 25 £ BMI < 30; obese = BMI ³ 30), suggesting that the metabolome remains unchanged despite an increase in BMI. When stratified for caloric intake (< 2200 kcal/d vs > 2200 kcal/d), a log‐transformed OPLS‐DA model showed clear separation in men, while females remained relatively unchanged. Only one metabolite, pipecolinic acid (P = .009; fold change = 3.43), was significantly greater among obese women relative to their non‐obese counterparts, whereas no metabolites were significantly different among men with different BMI status. A log‐transformed PLS‐DA model found similar results between GLs 1, 2, and 3–5 Mexican Americans, potentially signifying the resistance to alteration of the female metabolome. A significant metabolite between the female generation levels was N‐acetylethanolamine, an anti‐inflammatory, analgesic, and satiety producing compound. Additionally, a pathway analysis of significant metabolites between men and women revealed significant alterations in branched‐chain amino acid (BCAA) and aromatic amino acid (AAA) profiles and mechanisms, both prevalent in obesity and type 2 diabetes (T2DM).

CONCLUSION

While Mexican American males demonstrated variance in metabolic profiles when examined for caloric intake and generational level, the metabolome of female participants showed resilience and largely remained unaffected. Further research is needed to better understand how significant pathways associated with comorbidities may not affect the relatively consistent metabolome of Mexican Americans.