bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inactive 5‐HT7 Receptors Associate with Inactive and Active Gs Heterotrimers
Authors: Jang Wonjo , Lambert Nevin A. 
Published: April 2020

G protein‐coupled receptors (GPCRs) interact cooperatively with agonists and heterotrimeric G proteins to form ternary complexes, leading to activation of G proteins. Following exchange of GDP for GTP the complex is thought to rapidly disassemble as nucleotide‐bound G proteins adopt conformations that have low affinity for GPCRs, and heterotrimers dissociate into Gα and Gβγ subunits. Unlike most GPCRs, serotonin 5‐HT7 receptors are known to preassemble with GDP‐bound Gs heterotrimers prior to agonist binding. Here we show that these receptors also interact with GTP‐bound Gs heterotrimers. We used bioluminescence resonance energy transfer (BRET) and luciferase complementation assays to assess the effects of ligands and nucleotides on 5‐HT7‐Gs coupling. We find that stimulation with agonist (5‐HT) in intact cells promotes net dissociation of 5‐HT7‐Gs complexes, whereas inverse agonists (e.g. methiothepin) produce net association. To determine how nucleotide binding by Gs affects interactions with receptors, we replaced endogenous nucleotides with the non‐hydrolyzable analogs GTPγS or GDPβS in permeabilized, apyrase‐treated HEK293 cells. Unexpectedly, ligand‐mediated effects on 5‐HT7‐Gs complexes were largely maintained in the various nucleotide conditions. Agonist‐induced 5‐HT7‐Gs dissociation still occurred when only GDPβS was present, indicating that dissociation does not require Gs activation. Similarly, inverse agonist‐induced 5‐HT7‐Gs association still occurred when only GTPγS was present, suggesting that activated Gs heterotrimers retain affinity for inactive 5‐HT7 receptors. Moreover, we found that inverse agonist‐bound 5‐HT7 sequestered free Gβγ in the presence of GTPγS, suggesting that inactive 5‐HT7 receptors stabilized active Gs heterotrimers. These results reveal unconventional coupling of inactive 5‐HT7 receptors to both inactive and active Gs heterotrimers. This represents a significant departure from the standard allosteric model of GPCR‐G protein coupling, wherein G proteins interact primarily with active GPCRs, and GTP‐bound Gα subunits dissociate from both receptors and Gβγ subunits.Support or Funding InformationSupported by GM130142 (to N.A.L.)
Mutation of Eftud2 in Mouse Neural Crest Cells Models MFDM and Reveals Contribution of P53 in Abnormal Craniofacial Development
Authors: Beauchamp Marie-Claude , Djedid Anissa , Bareke Eric , Aber Rachel , Majewski Jacek , Majewska Loydie Jerome 
Published: April 2020

Haploinsufficiency of EFTUD2 is associated with MFDM (mandibulofacial dysostosis with microcephaly), but the etiology of this syndrome remains unknown. Our goal is to determine the tissue and temporal requirement for Eftud2 during craniofacial development. We generated a mouse with a conditional mutation of exon 2 of Eftud2 (Eftud2 flox) using the CRISPR/Cas9 editing system. We used the Wnt1‐Cre2 transgenic line to delete exon 2 of Eftud2 specifically in the neural crest cells. Embryos carrying one mutated allele of Eftud2 were normal. However, when both alleles were mutated, all embryos displayed hypoplasia of the midbrain and pharyngeal arches at E9.5. By E11.5, most of these embryos showed an open neural tube and all live embryos exhibited exencephaly at E14.5. At this stage, only 3 out of 12 embryos were still alive. Cartilage preparations revealed an absence of cartilage in the head, reduced/or absence of Meckel’s cartilage, and abnormal inner ear development. Crosses with the Rosa26RlacZ mice reporter line revealed a reduced number of neural crest cells in the pharyngeal region at E9.5 and E10.5, but not at E9.0. We next performed RNA sequencing analysis from the head of Eftud2 neural crest cells mutant embryos at E9.0, before any phenotype was observed. The P53 pathway was the most significantly upregulated in the mutant embryos, compared to their wild‐type littermates. Overexpression of P53 target genes identified in the RNAseq (Ccng1, Phlad3 and Trp53inp1) was validated by qRT‐PCR. Additionally, 373 genes showed increased skipped exons in the mutant embryos. Among them, only 2 genes were also overexpressed, including Mdm2, a major regulator of P53. We found that mutant embryos had significantly increased levels of the Mdm2 transcript lacking exon 3, which was previously reported to produce a truncated MDM2 protein, allowing stabilization of P53. Finally, we evaluated the contribution of P53 in craniofacial malformation of Eftud2 mutants by treating pregnant females with daily i.p. injection of pifithrinα, a P53 inhibitor, from E6.5 to E8.5. 37% of pifithrinα‐treated mutant embryos had normal midbrain development at E9.5. Also, the area perimeter of the first pharyngeal arch of pifithrinα‐treated mutant embryos was significantly higher than the vehicle‐treated ones. Altogether, these data suggest that Eftud2 specifically regulates Mdm2 splicing and leads to neural crest cell death due to overactivation of the P53 pathway. We propose that cranial neural crest cell death is responsible for craniofacial malformations in MFDM patients.Support or Funding InformationCanadian Institutes of Health Research
CTRP3 Prevents Macrophage Activation
Authors: Musick Adam 
Published: April 2020

Sepsis is a life‐threatening medical emergency that is caused by the body’s extreme response to an infection. The initial infection results in an overproduction of cytokines which recruits additional immune cells. Adipose tissue is an active endocrine organ which secrets several anti‐inflammatory mediators, collectively called adipokines which could contribute to the control of the body’s response to a septic event. Our previous work has identified that the circulating levels of the anti‐inflammatory adipokine called C1q TNF Related Protein 3 (CTRP3) is significantly reduced in response to sepsis. We have also demonstrated that CTRP3 binds directly to circulating macrophages. Therefore, the purpose of this project was to examine the potential anti‐inflammatory effects of CTRP3 on macrophages.METHODSThioglycolate‐induced peritoneal macrophages were isolated from CTRP3 overexpressing transgenic (Tg) and wild‐type (WT) mice. The isolated macrophages were then treated with lipopolysaccharides (LPS). Morphological changes were analyzed via bright‐field microscopy and the transcription levels of the inflammatory cytokines tumor necrosis factor alpha (TNF) and interleukin‐6 (IL6) were measured by Real‐Time PCR.RESULTSMorphological qualitative analysis indicated that LPS reduced macrophage adhesion and the effect was more pronounced in the Tg macrophages. Gene expression analysis demonstrated that LPS stimulation significantly increased IL6 and TNF levels in all samples (~4000‐fold and 300‐fold respectively). However, LPS‐induced increase in IL6 expression was significantly attenuated in Tg macrophages (fold elevation compared to vehicle treated: WT 273±92; Tg 78±68). There was no difference in LPS‐induced TNF expression in Tg compared to the WT macrophages.CONCLUSIONCombined these data indicated that CTRP3 attenuates the ability of LPS to activate the pro‐inflammatory functions of the macrophages. Future studies are needed to confirm these findings and to determine the unique mechanism for the CTRP3‐induced alterations to macrophages. Regardless, CTRP3 is a unique contributor to macrophage’s response to inflammatory signals.Support or Funding Information1R15DK114740
Women In STEM‐ Intrinsic and Extrinsic Factors That Impact Retention and Yield
Authors: Benore Marilee Ann 
Published: April 2020

Statistics gleaned from international sources paint a dismal picture of the number of women choosing, then leaving, STEM. The lack of women in the STEM workforce has become of significant concern as studies document the inequity, negative economic impact and loss of research when a disturbingly large number of capable individuals are discouraged or lost. The situation is not unique to the US; the percentage of women in STEM varies globally by field, nation and culture.Yet, in spite of the problems, many women persist and thrive. Understanding factors that impact persistence, resilience and self‐efficacy of successful women in STEM is necessary to determine how to encourage and retain women. Examining factors and experiences, especially those that are subtle or often unrealized, can be determined by utilizing techniques more commonly used by anthropologists and historians. Interviews and oral histories have emerged as excellent methods to identify personal experiences, as the narrative data can be more thoroughly analyzed, provides the opportunity to probe a topic deeply, and can be archived as videos for mentoring. By interviewing and surveying successful STEM women in Europe and the USA, specific factors were observed that may be valuable in retaining women and encouraging girls in STEM.Support or Funding InformationUniversity of Michigan
Pre‐hypertensive molecular alterations in the paraventricular nucleus of the hypothalamus during angiotensin‐II hypertension
Authors: Jeong Jin Kwon , Simonyan Hayk , Young Colin N. 
Published: April 2020

The paraventricular nucleus of the hypothalamus (PVN) plays a critical role in the development of hypertension in response to elevated levels of angiotensin‐II (Ang‐II). Upstream circumventricular organs, regions lacking a blood‐brain‐barrier, sense circulating Ang‐II and in turn influence endocrine and autonomic control via efferent projections to the PVN. Despite progress in unraveling the function of the PVN in hypertension development, the underlying signaling mechanisms remain unclear. Notably, the majority of investigations have examined alterations in the PVN during established hypertension, which limits our understanding of temporal changes that may occur prior to elevations in arterial blood pressure. Based on this, we investigated key molecular targets in the PVN during the pre‐hypertensive phase of Ang‐II hypertension. C57Bl/6J male mice were implanted with subcutaneous osmotic minipumps for chronic delivery of low dose Ang‐II (600 ng/kg/min). Brains were harvested for immunohistochemistry analysis of target molecules (n=3–4/group) at baseline (day 0) and pre‐hypertensive (days 3, 5, 7) timepoints. Histological analysis of synaptophysin, a marker for synaptic terminal innervation, indicated a progressive increase in immunoreactivity that peaked by ~10% within the first week of Ang‐II infusion (day 7 fold change: 1.1± 0.03 density/area, p<0.05); indicative of increased synaptic input to the PVN prior to elevations in arterial blood pressure. Previous findings have demonstrated that circulating arginine vasopressin protein (AVP) is elevated during chronic Ang‐II hypertension. Consistent with this, AVP immunoreactivity in the PVN was elevated as early as day 3 of Ang‐II infusion (day 3 fold change: 1.7 ± 0.09 density/area, p<0.05), and this response was sustained for up to 7 days (day 5 fold change: 1.4 ± 0.02; day 7 fold change: 1.3 ± 0.03 density/area, both p<0.05). Interestingly, we also observed synaptic end AVP immunoreactivity in the circumventricular subfornical organ very early during Ang‐II infusion (e.g. day 3 fold change: 1.2 ± 0.02 density/area, p<0.05), suggesting a possible feedback loop between the PVN and subfornical organ in AVP regulation. Unlike AVP, PVN oxytocin immunoreactivity was not changed during Ang‐II hypertension (e.g. day 7 fold change: 1.01 ± 0.08 density/area, p>0.05). Collectively, these findings indicate early molecular changes within the PVN prior to the development of Ang‐II hypertension including: 1) Gradual synaptic innervation of the PVN during the first week of Ang‐II infusion; 2) Very early Ang‐II associated elevations in PVN AVP expression, but not oxytocin; and 3) Potential alterations in the innervation of hypothalamic AVP fibers to the circumventricular subfornical organ in response to Ang‐II.Support or Funding Information19CDA34630010 (to Jin Kwon Jeong) and 1R01HL141393 (to Colin N. Young)
Efficacy of Student‐Directed Learning in a Gross Anatomy Course for Undergraduate and Graduate Students
Authors: Garner Sarah 
Published: April 2020

IntroductionWith a decrease in the number of contact hours available to teach gross anatomy and no corresponding decrease in the amount of course material, instructors must come up with innovative ways to teach this information in less time. Contact hours involving active learning promote increased retention rates. Thus, many institutions are moving away from lecture‐based courses and towards active learning classroom sessions. Providing pre‐recorded lecture videos facilitates student‐directed learning outside of the classroom so that students can utilize class time and interaction with instructors in a productive, active manner. While the use of flipped classrooms has been widely studied in anatomy, students’ opinions of the efficacy in‐class activities has not been closely examined. The goal of this project is to determine if the implementation of pre‐recorded lecture videos increases conceptualization rather than rote memorization and which in‐class active learning techniques are well‐received by gross anatomy students.MethodsGraduate and undergraduate students enrolled in a gross anatomy course will be provided with pre‐recorded lectures and corresponding lecture documents prior to entering the class. Once in class, students will participate in a variety of active in‐class activities including individual and group activities such as quizzes, clinically oriented questions and case studies, and osteology activities. Students will be invited to participate in surveys and interviews regarding course content, efficacy of pre‐recorded lecture videos and in‐class activities, methods of studying, and methods of assessment. Surveys and interviews will be utilized throughout the semester including the beginning of the semester, the end of each block of course material, and at the end of the course.ResultsIt is anticipated that the students will feel uncomfortable with self‐directed learning at the beginning of the semester, due to unfamiliarity and a feeling of increased work. However, it is also anticipated that the subsequent surveys and interviews will show an increased appreciation for the pre‐recorded lecture videos and an increased focus on conceptualization, with a decreased focus on memorization.ConclusionWhile pre‐recorded lecture videos may be more time consuming for instructors to create and increase the amount of work outside of class expected from students, the videos will improve conceptual knowledge of material and the quality of in‐class contact hours. Students will find group work most effective and prefer group work and clinically oriented questions to individual quizzes. Furthermore, students will feel more prepared for examinations and enjoy classroom time more with the interactive in‐class activities as opposed to traditional lectures. Therefore, the utilization of pre‐recorded lecture videos as an adjunct tool in anatomy education should be considered.
Chronic Binge Alcohol‐induced Hyperalgesia in SIV‐infected Rhesus Macaques
Authors: Foret Brittany , Coleman Larry , McGarrah Heather , Simon Liz , Molina Patricia 
Published: April 2020

Chronic pain affects more than half of people living with HIV (PLWH), and the current pain management strategies, such as opioids, capsaicin, and gabapentin, remain inefficient in PLWH. Moreover, the pathogenesis of HIV‐associated pain remains widely unknown, hindering implementation of evidence‐based treatments. Alcohol and pain have a bidirectional relationship, where people consume alcohol to relieve pain, and unhealthy alcohol uses leads to chronic pain. Approximately 30% of PLWH develop alcohol use disorder; therefore, it is important to understand the effects of alcohol on pain in this population. Using a relevant preclinical model of HIV‐infection, the aim of the study was to determine if chronic binge alcohol (CBA)‐administration causes hyperalgesia in SIV‐infected macaques. We hypothesized that CBA‐administration causes hyperalgesia in rhesus macaques prior to infection, and these findings will be exacerbated during peak viremia in SIV infection. Rhesus macaques, four to six years old were administered CBA or isovolumetric water (VEH) through an intragastric catheter for 30 minutes, five days a week, with blood alcohol concentrations reaching 50–60 mM. Three months after the start of CBA administration, macaques were inoculated with SIVMac251 and 2.5 months later initiated on antiretroviral therapy. To assess thermal hyperalgesia, tail withdrawal test was used. Conscious macaques were restrained in chairs and their tails are placed in water baths at 40, 50, and 55C, and the latency to remove their tail (in seconds) determined. The test was performed three months after the initiation of CBA administration (24 hours after a CBA administration session), and repeated 2.5 months after SIV‐infection. Molecular analysis for proteins involved in pain circuitry will be determined in the spinal cord and periaqueductal gray. Our results indicate that three months of CBA administration causes hyperalgesia in rhesus macaques. Whether SIV infection exacerbates hyperalgesia, and if the molecular measures correlates to behavioral measures warrants further investigation. These data provide mechanistic insight into how unhealthy alcohol use affects hyperalgesia in HIV infection, and will provide a scientific basis for pain management strategies in this population.Support or Funding InformationNIH/NIAAA P60 AA009803 and 5T32AA007577‐19
Knockout of Per1 Exacerbates the Hypertensive Phenotype of the Dahl Salt Sensitive Rat
Authors: Spires Denisha R. , Zietara Adrian , Levchenko Vladislav , Gumz Michelle L. , Staruschenko Alexander 
Published: April 2020

The dynamic between blood pressure, its circadian rhythm, and the circadian clock genes has been considered as a factor in the subsequent renal injury that accompanies hypertension. Various circadian clock proteins such as BMAL1 and PERIOD (PER) function in a marriage of positive and negative feedback loops that control transcriptional regulation. Of particular interest is PER1 which naturally acts in the negative loop of the cycle as a repressor, but recent findings show that it may have additional roles as a transcription activator. PER1 has a role in renal sodium handling as a target of aldosterone, and it has been linked to the expression of the α‐subunit of the epithelial sodium channel (α‐ENaC). Investigators have found that knockout of PER1 in 129/sv mice causes a decrease in mean arterial pressure (MAP); however, when the gene is knocked out in the C57BL/6J mouse on a 4% NaCl diet (high salt, HS) with deoxycorticosterone pivalate (DOCP) treatment, there is a significant increase in MAP with animals displaying a “non‐dipping” pattern of MAP. The wildtype mice that were on HS with DOCP treatment had a 11.4 ± 1.9% decrease in MAP from night to day whereas the knockout mice with the same conditions had only a 5.7 ± 1.4% decrease. To test the contribution of PER1 in the development of salt‐induced hypertension, we created the Per1 knockout rat on the Dahl Salt‐Sensitive rat background (SS; knock out SSPer1−/−). Eight to 9‐week old SS and SSPer1−/− male rats were implanted with DSI telemetry units to measure blood pressure on a 4% NaCl diet (high salt, HS) for 3 weeks. MAP in SSPer1−/− rats was significantly higher compared to wild type SS rats (191 ± 7 vs. 168 ± 4 mmHg, P = 0.031; respectively) after 3 weeks of HS. We also revealed a significant increase in albuminuria in SSPer1−/− rats on HS compared to SS rats on HS (0.10 ± 0.01 vs. 0.05 ± 0.01 Alb/Cre Clearance ratio, P = 0.027; respectively). Knockout of Per1 in SS rats on HS also resulted in a significant increase in cholesterol (200.0 ± 11.8 vs. 135.0 ± 9.7 mg/Dl, P = 0.003; respectively) and alkaline phosphatase (37.5 ± 3.6 vs. 77.6 ± 3.3 U/L, P = <0.001; respectively). Furthermore, SSPer1−/− rats after 3 weeks of HS diet had significant decreases in creatinine clearance compared to SS rats (65 ± 6 vs. 127 ± 24 mL/hour, P = 0.036; respectively). Here, we show that lack of PER1 causes a dramatization of the salt‐sensitive phenotype of the SS rat with a decline in renal function. These data propose that the role PER1 plays in sodium homeostasis in the kidney is pivotal for proper blood pressure regulation.Support or Funding InformationNational Institutes of Health Grants R35 HL135749 (AS) and R01 DK109570 (MLG) and by the American Heart Association 18PRE34030127 (DS)
Age‐Related Loss of Phrenic Motor Neurons: Reduced Myogenic Influence?
Authors: Wilfred Ann M. , Fogarty Matthew J. , Mantilla Carlos B. , Sieck Gary C. 
Published: April 2020

There is an age‐associated loss of phrenic motor neurons (PhMNs) and withdrawal of pre‐synaptic terminals at neuromuscular junctions (NMJs) in 24‐month old Fischer 344 (F344) rats. Previously, we showed that age‐related PhMN loss is concomitant with neuromuscular transmission failure and diaphragm muscle (DIAm) sarcopenia (an age‐related atrophy of muscle fibers and a decrease in specific force). It is unknown if pre‐synaptic terminal withdrawal precedes PhMN loss, which would implicate a reduction in a trophic influence emanating from muscle fibers (myogenic). We hypothesized that age‐related PhMN loss results from pre‐synaptic terminal withdrawal and reduced myogenic influence, which occurs predominantly at type IIx and/or IIb DIAm fibers (innervated by larger PhMNs). F344 rats were obtained from the National Institutes of Aging. Experimental ages were chosen to represent 100% and 50% survival at 6 months and 24 months‐old, respectively. PhMNs were first retrogradely labeled by intrapleural injection of cholera toxin‐subunit B (CTB), which is dependent on binding to pre‐synaptic terminal gangliosides. Thus, with pre‐synaptic terminal withdrawal, PhMNs would not be labeled. Subsequently (3 days after CTB), PhMNs were retrogradely labeled by sectioning the phrenic nerve and dipping the proximal end into a Rhodamine dextran (Rho) solution – this labeling is not dependent on pre‐synaptic terminals. CTB and Rho labeled PhMNs were visualized by confocal microscopy. Only optical slices containing the nucleus of Rho‐ and/or CTB‐labeled PhMNs were counted. We analyzed the differences in labeling of PhMNs by CTB and Rho in both age groups. In labeled PhMNs, short and long axis measurements were used to determine somal surface areas by assuming a prolate spheroid shape. In 6‐month old rats, there was no significant difference in the number of PhMNs labeled by CTB or Rho. In 24‐month old rats, the total number of labeled PhMNs was reduced compared to the 6‐month old rats, which primarily affected larger PhMNs. Furthermore, at 24‐months, fewer PhMNs were labeled with CTB compared to Rho and those labeled with Rho had larger somal surface areas. These results support our hypothesis that the age‐related loss of larger PhMNs results from pre‐synaptic terminal withdrawal and reduced myogenic influence.Support or Funding InformationSupport: NIH R01‐AG044615American Physiological Society