bluuebunny/crossref_metadata_2025 huggingface dataset:
Does the Mu‐Delta Opioid Receptor Heterodimer Repress Akt Kinase to Reduce Opioid Anti‐Nociception?
Authors: Hurtado Kevin , Keresztes Attila , Barker Natalie , Langlais Paul , Streicher John M.
Published: April 2020

The mu opioid receptor (MOR) and delta opioid receptor (DOR) have been shown to interact in the regulation of pain, opioid side effects, and similar physiology. However, the mechanism of this interaction is still unknown. Limited in vitro and in vivo experiments have suggested that these receptors heterodimerize (MDOR) to regulate pain and opioid response. However, in vivo studies of this receptor complex are very challenging due to the lack of selective tools to study MDOR activity. We thus designed a first‐in‐class MDOR‐selective antagonist named D24M. Using this tool, we found striking evidence suggesting that the MDOR acts as a negative feedback loop, blocking analgesia while enhancing side effects. We performed phosphoproteomic analysis of D24M and/or oxymorphone‐treated CD‐1 mouse brainstems, and found a network of signaling molecules possibly implicated in MDOR function. Among these altered molecules, we found evidence that the kinase Akt was repressed by oxymorphone treatment and restored by D24M co‐treatment, suggesting that Akt repression may be downstream of the MDOR in vivo, and responsible for the blockade of opioid analgesia by this heterodimer. We tested this hypothesis in male and female CD‐1 mice by intracerebroventricular injection of combinations of oxymorphone, D24M, and the selective Akt inhibitor Akt‐i. We found that D24M increased Oxymorphone‐induced anti‐nociception compared to Oxymorphone alone in the tail flick assay, as in our previous studies. We also found that Akt‐i co‐treatment blocked the increase in anti‐nociception caused by D24M in male, but not female mice. These results suggest that Akt may be repressed in a sex‐specific manner by the MDOR to repress opioid anti‐nociception as part of an anti‐opioid negative feedback loop. These results also suggest that MDOR‐selective antagonists could be novel therapeutics to enhance opioid efficacy while decreasing side effects.

Support or Funding Information

Funding Information

These studies were funded by NIH R21DA044509 and UG3DA047717 to JMS. The authors have no relevant conflicts of interest to declare.
Alpha adrenergic receptor regulation of blood flow during passive leg movement: The impact of age.
Authors: La Salle D. Taylor , Cerbie James , Alpenglow Jeremy , Jarret Catherine , Craig Jesse C. , Broxterman Ryan M. , Richardson Russell S. , Trinity Joel D.
Published: April 2020

Passive leg movement (PLM) evokes a robust and transient increase in limb blood flow (LBF). The magnitude of this response is attenuated with aging and this reduction has been largely attributed to diminished nitric oxide (NO) bioavailability. Importantly, aging is also associated with heightened sympathetic activity and altered vasomotor tone, which may contribute to the age‐related reduction in PLM. Therefore, this study sought to determine the contribution of α‐adrenergic signaling to the PLM‐induced LBF response in aging. The central hypothesis is that aging is associated with sympathetically‐mediated restraint of the LBF during PLM. Following intravenous infusion of the β‐blocker propranolol, five young (24±6 yr) and five old (68±4 yr) healthy male subjects underwent PLM during intra‐arterial infusion of saline (CON; control), phenylephrine (PE; α1‐adrenergic receptor agonist) and phentolamine (PHEN; nonspecific α‐adrenergic receptor antagonist). LBF through the common femoral artery was determined second‐by‐second by Doppler ultrasound. Compared with CON, PE did not reduce baseline LBF in the young (CON: 251±11; PE: 193±18 ml/min, p=0.23) or old (CON: 265±18; PE: 239±36 ml/min, p=0.74); however, PHEN increased baseline LBF in both the young (PHEN: 725 ± 63 ml/min, p<0.01 vs. CON) and the old (PHEN: 680 ± 122 ml/min, p<0.01 vs. CON). During PLM in the young, PE reduced the peak change in LBF (ΔPeakLBF) (CON: 595±67; PE: 393±46 ml/min, p=0.038) while PHEN increased the ΔPeakLBF (827 ± 89 ml/min, p=0.02 vs. CON). In contrast, in the old, no changes in ΔPeakLBF in response to PE or PHEN were evident (CON: 342±17; PE: 311±22, PHEN: 378±65 ml/min, p=0.37 for the treatment effect). The magnitude of α‐adrenergic receptor contribution (PHEN ΔPeakLBF – PE ΔPeakLBF) was different between young and old (young: 434±97; old: 67±75 ml/min, p=0.02). The overall hyperemic response, assessed by the LBF area under the curve (LBFAUC) during PLM, was not different when comparing CON (127 ± 23 ml/min) to PE (67 ± 19 ml/min, p=0.22) nor PHEN (210 ± 31 ml/min, p=0.095) in the young; however, LBFAUC was significantly different between PE and PHEN (p=0.02). Neither PE nor PHEN altered the LBFAUC in the old (CON: 82 ± 17, PE: 65 ± 9, PHEN: 127 ± 32 ml/min, p=0.29 for the treatment effect). Although both groups exhibited robust increases in baseline LBF after adrenergic receptor inhibition, only the young demonstrated alterations to the PLM response following stimulation and inhibition of adrenergic receptors. In line with previous findings, we observed sympathetic restraint of resting LBF in both young and old individuals. However, the age related reduction in ΔPeakLBF and LBFAUC during PLM in old individuals does not appear to be associated with augmented sympathetic regulation of vasomotor tone.
Baroreflex Resetting of Sympathetic Action Potential Subpopulations During Fatiguing Isometric Handgrip and Post‐Exercise Circulatory Occlusion
Authors: Klassen Stephen A. , Badrov Mark B. , Moir M. Erin , Shoemaker J. Kevin
Published: April 2020

The arterial baroreflex exerts heterogeneous control over the varying‐sized action potential (AP) subpopulations active within human muscle sympathetic nerve activity (MSNA) under baseline conditions. How baroreflex control of sympathetic AP discharge becomes reset to facilitate sympathoexcitation during physical exercise remains to be investigated. Therefore, we quantified the baroreflex gain for each sympathetic AP cluster (i.e., reflecting different c‐fibres) within the MSNA filtered neurogram (microneurography and continuous wavelet transform) during baseline conditions (BSL), the last 2‐min of a 5‐min fatiguing isometric handgrip (20% of max; IHG), and during post‐exercise circulatory occlusion (PECO) in six healthy young participants (3 females, 23–31 years). Baroreflex gain was measured as the slope of the linear function between AP probability (%) versus diastolic blood pressure (DBP; Finometer). We classified AP clusters based on peak‐to‐peak amplitude using the following allocations: small (clusters 1–2), medium (clusters 3–8) and large (clusters 9–15). During BSL, medium AP clusters (e.g., cluster 3: −9.4 ± 2.7 %/mmHg) expressed the greatest baroreflex gains while small (e.g., cluster 1: −1.9 ± 0.4 %/mmHg) and large (e.g., cluster 9: −2.4 ± 0.9 %/mmHg) clusters expressed lesser gains (both P < 0.05 vs. cluster 3). Compared to BSL, baroreflex functions for most AP clusters during IHG and PECO were reset upwards to greater firing probabilities (both Condition‐by‐Cluster Interactions: P < 0.05) and rightwards to higher DBP (BSL: 69 ± 4, IHG: 81 ± 3, PECO: 79 ± 2 mmHg; both P < 0.05 vs. BSL). However, compared to BSL, the gains of the AP cluster baroreflex functions remained unchanged with IHG (e.g., cluster 1: −1.6 ± 0.4, cluster 3: −8.2 ± 2.9, cluster 9: −4.3 ± 1.8 %/mmHg, Main Effect: P > 0.05). Nonetheless, AP cluster gains were augmented above BSL during PECO (e.g., cluster 1: −2.6 ± 1.0, cluster 3: −16.0 ± 4.4, cluster 9: −5.5 ± 1.8 %/mmHg; Main Effect: P < 0.05). IHG (e.g., cluster 15: −1.6 ± 0.5 %/mmHg) and PECO (e.g., cluster 15: −0.6 ± 0.3 %/mmHg) recruited a subpopulation of previously silent larger and faster conducting APs that expressed weak baroreflex gains. These data suggest that during fatiguing isometric handgrip exercise the interaction between the central command feature and the skeletal muscle metaboreflex resets baroreflex control of discrete AP subpopulations to higher blood pressures and greater levels of discharge. In addition to resetting the baroreflex functions, independent activation of the muscle metaboreflex augments the strength of baroreflex control over subpopulations of previously active APs.

Support or Funding Information

Supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada.
Does Teaching an Etymological Dissection Tool Aid Student Understanding and Retention of Terminology?
Authors: Mohr Madison , Olive Elizabeth , Barremkala Mallikarjuna , Browne Berkley , Venuti Judith
Published: April 2020

Based on the premise that teaching a person a useful skill is ultimately more beneficial than filling a temporary need, we developed a tool for medical students to learn new terminology in embryology. This tool, the etymological dissection approach (EDA), dissects new words using their etymological origins. The goal is to provide students with a tool they can apply, not only for the improved understanding of embryological terminology, but medical terminology in general.

After IRB approval (IRB# 1201615‐1), data was obtained from class of 2021 focus groups (Phase I), which guided the development of content, timing, and mode of delivery of an EDA resource for the incoming class of 2023. The data recommended newly admitted students be provided with a pre‐matriculation worksheet and a “hands on” workshop early in their first semester to walk them step‐by‐step through the EDA.

In Phase II, we created and delivered a pre‐matriculation EDA worksheet that provided an introduction to word parts and a list of commonly encountered word roots, suffixes and prefixes. This was provided to all incoming students via the Student Academic Success Website. Students were also invited to complete a survey that asked their background in embryology, primary and secondary languages, etc. At the end of the first week of classes, a hands‐on workshop was delivered consisting of EDA exercises outlining the 5 steps for “dissecting” each new term: Connect, Deduce, Confirm, Understand, and Integrate. The exercises were also provided to students via the Academic Success Website, whether they attended the workshop or not. Students were encouraged to use the tool and, at the end of their first semester, complete a survey.

Data from the pre‐survey revealed students had little to no prior embryology knowledge (2.323 std. dev. +/− 0.989; 1 = no knowledge; 5 = extremely knowledgeable); only 7% have taken an embryology course and 15% a developmental biology course. While 55% of students reported they were moderate to fluent in Spanish, they reported little to no familiarity with Greek (99%) and or Latin (93%). Interestingly, students reported they sometimes (2.327 std. dev. +/− 1.203; 1 = never; 5 = always) attempt to understand terminology using a technique similar to the EDA, perhaps because 17% have taken a medical terminology course. Those students that reviewed the EDA worksheet prior to the workshop, found it to be moderately useful (3.051 std. dev. +/− 0.972; 1 = not at all useful; 5 = extremely useful).

The goal of the post‐survey is to determine if students attended the workshop, found it useful, used the EDA during the semester, and if the EDA technique was efficacious in guiding their understanding and retention of terminology. It is hoped that the EDA will add to a medical student’s skillset and provide a tool that can be used throughout a career requiring life‐long learning.
A Novel Distal Convoluted Tubule‐Specific Tamoxifen‐Inducible Cre‐Recombinase Driven by the NaCl Cotransporter Gene
Authors: Cornelius Ryan J. , Sharma Avika , Su Xiao-Tong , McMahon Andrew P. , Ellison David H. , McCormick James A.
Published: April 2020

The use of knockout and transgenic mouse models coupled with Cre‐lox technologies has revolutionized research in kidney transport physiology by allowing site‐specific genetic recombination in individual nephron segments. Although several groups have tried to generate a distal convoluted tubule (DCT)‐specific mouse Cre‐recombinase driven by the thiazide‐sensitive NaCl cotransporter (NCC) promoter, this goal has remained elusive. The DCT is a major site for the regulation of Na+ and K+ transport. The segment consists of two distinct segments the early (DCT1) and late (DCT2) DCT. The only previously recognized mouse model available allowing targeted gene modification in the DCT is the DCT1‐specific mouse with Cre‐recombinase under control of the Pvalb gene encoding parvalbumin. The model, however, has limitations including activity in neurons that prevent comprehensive characterization of transport pathways in the DCT. Recently, a novel mouse line expressing estrogen‐inducible DCT‐specific Cre was generated and donated to Jackson Laboratory. CRISPR/Cas9 was used to introduce Cre‐ERT2 into the 3' UTR near the stop codon of the Slc12a3 gene encoding NCC (NCC‐Cre‐ERT2 mice). Here, we crossed NCC‐Cre‐ERT2 mice with YFP floxed mice to test whether the Cre expression would mimic that of NCC, and to determine whether the construct is ‘leaky’. Without tamoxifen, approximately 6% of NCC positive cells expressed YFP, indicating minimal leakiness. After five days of tamoxifen injection, mice showed YFP expression in almost all NCC positive cells and there was complete overlap of YFP expression in NCC positive cells. Western blotting showed lower NCC abundance in NCC‐Cre‐ERT2 males compared to controls, but female NCC‐Cre‐ERT2 mice NCC abundance was not significantly different from the abundance in control littermates. Despite the difference in NCC abundance in males, abundance of the active phosphorylated form of NCC did not differ. Furthermore, functional analysis of NCC showed no effects on NCC activity in NCC‐Cre‐ERT2 mice. Plasma K+ and Mg2+ concentrations, and thiazide‐sensitive Na+ and K+ excretion did not differ in NCC‐Cre‐ERT2 mice compared to controls. Thus, the NCC‐Cre‐ERT2 mice have high recombination efficiency, minimal leakiness, and complete fidelity in cell‐specificity. Our data show that Cre expression is entirely localized to the DCT and the genetic modification has very mild or no effect on NCC expression and renal function. The NCC‐Cre‐ERT2 mice are the first mice generated with Cre recombinase activity along the entire DCT, and will be a powerful tool to study DCT function.

Support or Funding Information

NIH grants DK098141 and DK117903 to JAM. NIH grant DK51496 and VA grant 1I01BX002228‐01A1 to DHE.
Renal Distal Segment‐Specific BMAL1 Regulates Blood Pressure in the Setting of Potassium Depletion
Authors: Crislip Gene Ryan , Douma Lauren G. , Cheng Kit-Yan Ryan , Barral Dominique , Lynch I. Jeanette , Wingo Charles S. , Gumz Michelle L.
Published: April 2020

Introduction

Circadian clock factors in the kidney influence systemic blood pressure control. BMAL1 is a core mammalian circadian gene responsible for tissue‐specific transcription regulation. Previously, we generated renal distal segment BMAL1 knockout mice (dsBMAL1 KO) that exhibit lower blood pressure compared to control mice (CNTL) in males but not females. Additionally, male dsBMAL1 KO retain less sodium following potassium depletion compared to CNTL, a difference not seen in females.

Goal

The goal of this study was to determine if dysregulation of the renin‐angiotensin‐aldosterone system (RAAS) contributes to the sex‐specific solute handling response to potassium depletion and to test the hypothesis that male dsBMAL1 KO display lower blood pressure than CNTL adapted to a potassium deficient diet.

Methods

Our mouse model was generated using floxed exon 8 BMAL1 mice crossed with kidney‐specific cadherin Cre+ mice to generate dsBMAL1 KO. Floxed Cre‐littermates were used as CNTL. Arterial blood samples from 14–18 week old male and female mice were collected at noon for basal and 5 days post potassium depletion for serum measurements (0% K, 0.2% Na; N=4–8). Additional male mice were implanted with telemeter devices for 24‐hour monitoring of blood pressure before and during administration of a potassium depleted diet over 5 days (N=7–8). Two‐way ANOVA was used to compare diet and genotype effects within each sex.

Results

Serum angiotensin II levels were unchanged following potassium depletion regardless of genotype or sex. As expected, serum aldosterone levels were lower following potassium depletion compared to baseline in males and females (M: P<0.0001; F: p<0.01), however, there was no difference between genotypes (M: P=0.5; F: P=0.8). Interestingly, potassium depleted males had higher serum aldosterone at midnight compared to noon (P<0.001), however, there was no time difference in females (P=0.8). Following potassium depletion in males, systolic blood pressure significantly decreased in both genotypes (P<0.0001), but a greater decrease over the treatment period was observed in dsBMAL1 compared to CNTL (CNTL: 125±2.4 to 121±1.4; KO: 123±1.7 to 116±1.7 mmHg; PINTERACTION<0.05).

Conclusions

BMAL1 in renal distal segments contributes to blood pressure regulation. Potassium depletion decreases blood pressure in this experimental setting with male dsBMAL1 KO demonstrating a greater drop in pressure than CNTL. These data suggest RAAS does not contribute to genotype‐dependent differences seen in blood pressure following potassium depletion.

Support or Funding Information

NIH/NIDDK, 1R01DK109570‐01A1; 19POST34450134 AHA Postdoc Fellowship; 5T32HL083810‐10 Postdoc Training Grant, Gatorade Trust through the UF Department of Medicine, North Florida/South Georgia Veterans Health Systems
Palmitic acid induced lncRNA PARAIL regulates inflammation in human monocytes and macrophages
Authors: Tanwar Vinay Singh , Reddy Marpadga A. , Das Sadhan , Amaram Vishnu , Ganguly Rituparna , Stapleton Kenneth , Lanting Linda , Natarajan Rama
Published: April 2020

Elevated circulating levels of long chain saturated fatty acids such as Palmitic acid (PA) enhances inflammation, metabolic disorders like diabetes and associated complications. Accumulated evidence shows that long non‐coding RNAs (lncRNAs) play key roles in the regulation of inflammation, however, their role in PA‐induced inflammation in monocytes/macrophages is not well known. Strand‐specific RNA‐seq was performed to identify PA‐induced lncRNAs in CD14+ human monocytes. The RNA‐seq results showed that 2624 transcripts including lncRNAs were differentially expressed in PA treated CD14+ human monocytes. Interestingly, PA significantly induced genes associated with inflammatory phenotype. Here we studied a PA‐induced lncRNA RP11‐37B2 located adjacent to RIPK2, which we designated as
PA‐
regulated
anti‐
inflammatory
lncRNA (PARAIL). PARAIL was induced by PA in a time and dose dependent manner with optimal induction by 200 μM PA at 24 h in THP1 monocytes differentiated into macrophages. Analysis of publicly available datasets showed that PARAIL is also upregulated by lipopolysaccharide (LPS) and bacterial infection in human monocytes and macrophages. PARAIL promoter showed binding motif for NF‐kB, and the NF‐κB inhibitor BAY 11‐7082 significantly inhibited PA‐induced expression of PARAIL. Furthermore, PARAIL knockdown with GapmeR antisense oligonucleotides increased pro‐inflammatory cytokines and suppressed anti‐inflammatory chemokines in THP‐1 macrophages. In addition, mouse orthologous Parail was down regulated in peritoneal macrophages from diabetic db/db mice and bone marrow derived macrophages from Apoe−/− mice, a model of accelerated atherosclerosis. Together, these results show that PA‐induces lncRNA PARAIL in human monocytes via NF‐kB activation, and its knockdown elicits pro‐inflammatory phenotype in macrophages. These results suggest that upregulation of PARAIL in the short term by PA and LPS can mediate feedback mechanisms to protect from fatty acid‐induced inflammation associated with metabolic disorders, and this protection is lost during downregulation of PARAIL in chronic disease.

Support or Funding Information

This research work was funded by National Institutes of Health grant NIH R01 DK 065073 to RN.
Cobalt Nanoparticles Cause Major Platelet Aggregation In Vitro While Chromium Nanoparticles Induce Platelet Lysis In Vitro.
Authors: Pasieka Paweł Melchior , Marycz Krzysztof , Radomski Marek , Santos-Martinez Maria Jose , Pękala Przemysław Andrzej , Tomaszewska Iwona , Tomaszewski Krzysztof Andrzej
Published: April 2020

Introduction

The usage of artificial joint prostheses has recently gained significant popularity, with roughly two million total hip, knee and spinal arthroplasties performed annually worldwide. However, the presence of prostheses in organism is associated with significant release of nanoparticles (NPs). When in the bloodstream they can affect various blood components, such as platelets. Despite that the problem of NPs’ influence on platelets function has not been thoroughly analyzed. Therefore the aim of our study was to analyze the impact of Cr and Co particles on thrombocytes in vitro with usage of novel quartz crystal microbalance with dissipation (QCM‐D) methodology.

Materials and methods

The light transmission aggregometry, flow cytometry and QCM‐D were utilized to assess the ability of NPs to cause activation and aggregation of platelets. Transmission electron microscopy (TEM), scanning electron microscopy and optical and immunofluorescence microscopy were used to confirm the outcomes given by QCM‐D.

Results

Compared to the controls under flow there was a significant change of frequency and dissipation in platelet‐rich plasma incubated with Co 28nm, CoO 50nm, Co2O3 50nm, Co3O4 30–50nm, Cr 35–45nm, Cr2O3 60nm NPs (at concentrations of 5, 2.5, 1, 0.5 μg/mL). The NPs induced platelet aggregation even at the lowest concentrations. Other utilized modalities confirmed aggregation of thrombocytes. Furthermore, TEM showed that Cr NPs induce swelling and lysis.

Conclusion

Our study indicates that both Cr and Co NPs influence thrombocytes function in vitro. Also two different underlying mechanisms of platelet aggregation as Cr NPs cause swelling and lysis while Co particles induce typical aggregation. We conclude that any physician should take into consideration monitoring level of Cr and Co NPs of patients having Co‐Cr prosthesis.
Tissue‐specific Humanized Models Expressing Human UGT1A1
Authors: Mennillo Elvira , Yang Xiaojing , Weber André , Chen Shujuan , Maruo Yoshihiro , Tukey Robert H.
Published: April 2020

The human UDP‐glucuronosyltransferases (UGTs) represent an important family of drug‐metabolizing enzymes. Among the nine UGT1A proteins, UGT1A1 has been the most extensively studied. UGT1A1 is the sole enzyme responsible for the glucuronidation of bilirubin and is involved in the conjugation of endogenous and many exogenous substances, including pharmaceutical drugs. Although the liver is considered a major organ for detoxification, intestinal UGT1A1 is also an important contributor for drug clearance. In order to investigate the role of both intestinal and hepatic human UGT1A1, we generated humanized strains that express only the UGT1A1 gene in either liver (hAlb1A1) or small intestine (hVil1A1). The two models were characterized by demonstrating hepatic and intestinal tissue specific expression of human UGT1A1. Body weight (BW) and total serum bilirubin (TSB) levels were monitored during different developmental stage. Both humanized models exhibited similar BW and normal TSB values in the adult stages. Differences between the two animal models were observed in the neonatal stage where hVil1A1 pups presented significantly lower TSB levels than hAlb1A1 mice, confirming that the intestinal expression of UGT1A1 is crucial in preventing bilirubin toxicity during development. Of importance is the finding that we have new humanized UGT1A1 mouse models to examine tissue‐specific glucuronidation. Given the importance of the intestinal tract in xenobiotic activation, metabolism and detoxification, we subsequently have isolated intestinal crypt organoids from these same animal models. The use of these in vivo models and their in vitro complementary primary cell models provide important tools for studying intestinal and hepatic toxicity of drugs and environmental toxicants that are metabolized by UGT1A1.

Support or Funding Information

Supported by National Institutes of Health grants GM126074 and ES010337
Rates of Synthesis of Individual Proteins in Muscles of Old Mice: A Heavy Water Approach.
Authors: Owen Euan , Hammond Dean E. , Staunton Caroline , Franco Catarina , Prescott Mark , Brooks Susan V. , Jackson Malcolm J. , Lau Edward , Beynon Rob J. , McArdle Anne
Published: April 2020

Age‐associated loss of muscle mass and function is a major factor in the development of frailty, but the mechanisms responsible for this loss are unknown. Unquestionably, muscle atrophy is the consequence of changes in the balance between protein synthesis and breakdown. However, the nature and extent of these changes in old age is unclear, with some studies reporting reduced basal muscle protein synthetic rate in the aged compared with younger people and others suggesting no difference. Whole organism heavy water ([2H2]O) dynamic labelling of proteins, followed by LC‐MSMS, provides a significant improvement in analysis of protein turnover, allowing the rates of synthesis and degradation of both slow and fast turnover muscle proteins to be accurately modelled. The aim of this study was to use a [2H2]O labelling approach to determine the synthesis rates of individual proteins in the gastrocnemius muscles of adult and old mice.

Ten adult and ten old male mice were primed with an intraperitoneal bolus injection of saline, prepared with 99% [2H2]O and subsequently, provided with 8% (v/v) [2H2]O in the drinking water, for up to 60 days. The sampling interval was distributed to obtain sufficient data points to cover both high and low turnover proteins. Muscle proteins were extracted and digested using standard procedures for analysis on a high‐resolution mass spectrometer (Thermo Q‐Exactive) and the raw data files were processed to allow protein identification using Crux‐tide (
McIlwain et al, 2014). The software RIAna (:
https://github.com/Molecular‐Proteomics/riana) was then used to track time dependent incorporation of [2H] into tryptic peptides, yielding synthesis rates of proteins to be determined.

Muscle mass was significantly reduced in the old mice and preliminary data demonstrated little change in the rate of synthesis of a large proportion of identified muscle proteins between adult and old mice. Several individual proteins showed a marked decrease in protein synthesis rates in muscles of old compared with adult mice, particularly structural proteins. Other proteins, particularly redox‐responsive proteins demonstrated an increased synthesis in muscles of old mice. Proteomic analysis of unlabelled muscle protein pools and RNASeq data will be integrated with the turnover data to further interpret these findings.

This study was supported by the University of Liverpool, MRC and NIA (AG051442).
A Striking Bone Phenotype in the Familial Danish Dementia (FDD) Tg+ Mouse
Authors: Suryadevara Vidyani , Krehbiel Connor J. , Vidal Ruben , Willis Monte S.
Published: April 2020

Familial Danish Dementia (FDD) is an autosomal dominant neurodegenerative disease caused by a 10‐nucleotide insertion mutation in the BRI(2) gene (aka ITM2B) resulting in a frame‐shift. The resulting Danish amyloid precursor protein (ADanPP) is then cleaved an amyloidogenic peptide that deposits in the brain and cerebral vessels. FDD in patients results in a range of clinical defects, including cataracts, hearing defects, cerebellar ataxia, slurred speech, gait disturbances, psychosis, and dementia. With emerging evidence that human bone defects are associated with neurodegenerative disease, we phenotyped bone in 12‐month FDD Tg+ mice. FDD Tg+ mice exhibit abnormal grooming by 9 months and have Ab plaques and behavior deficits by 12 months, paralleling mechanisms in Alzheimer disease. At 12 months of age, femurs from FDD Tg+ mice were assessed by mCT (SkyScan 1172, 9 mm resolution) in the distal metaphysis (1 mm) and mid‐diaphysis regions for trabecular and cortical areas, respectively. Both male and female femurs exhibited significantly reduced bone volumes (44%) vs. age‐, strain‐matched wildtype mice; male FDD Tg+ femurs showed a 24% reduction in bone mineral density.

Trabecular bone

Females FDD Tg+ femurs had a 24% reduction in the trabecular thickness (vs. controls), whereas male FDD Tg+ femurs exhibited a 24% increase in the trabecular separation and 40% reduction in the trabecular number (vs. controls).

Cortical bone

Female FDD Tg+ femurs had a 15% reduction in bone marrow area and 8.6% endocortical thickness compared to controls. Male FDD Tg+ femurs had reduced total cross‐sectional area and endocortical thickness by about 8%, a 15% reduction in the marrow area, along with a reduced periosteal thickness and increased cortical thickness vs. controls. These results indicate that FDD Tg+ mice have significant bone phenotypes in parallel with previously observed brain defects, suggesting therapeutic interventions may want to consider how neurodegeneration might impact other critical organs in the body such as bone.

Support or Funding Information

Lilly Foundation/Indiana Center for Musculoskeletal Health/ IU School of Medicine, Physician Scientist Initiative, Scientific Research Initiative
Mapping Circuit Connections of Brainstem Respiratory Regions with Retrogradely Transporting Viral Vectors in Adult Transgenic Rats
Authors: Sheikhbahaei Shahriar , Gamarra Julio , Chopra Varuna , Zhang Ruli , Smith Jeffrey C.
Published: April 2020

In mammals, breathing is a constantly regulated key motor behavior that is generated by a rhythmic neural process in the brainstem. The preBötzinger complex (preBötC), a medullary region critical for generating the breathing rhythm, has been proposed to have circuit connections ipsilaterally and contralaterally to functionally distinct neighboring medullary regions, such as the respiratory Bötzinger complex (BötC), the rostral and caudal Ventral Respiratory Groups (rVRG/cVRG), and retrotrapezoid nucleus (RTN) for coordination of the rhythmic activity of multiple respiratory neuronal populations. Because breathing is integrated with other volitional and physiological behaviors, it is proposed that brainstem respiratory circuits, especially within the preBötC, act as critical nodes for control of breathing, and may have connections to neurons in other regions throughout the brain involved in motor control and behavioral integration such as motor cortex for volitional control of breathing or other aspects of motor integration. However, the axonal projections of preBötC neurons throughout the brain, and the input connections from other brain regions have not been mapped. In the present study, we therefore performed brain‐wide mapping employing adult transgenic rats expressing Cre recombinase under control of the glutamic acid decarboxylase 1 (GAD1) promoter to specifically target GABAergic inhibitory neurons. To map input connections to the preBötC, we used combinations of Cre‐dependent and Cre‐independent retrogradely transporting viral vectors (rAAV2‐retro) driving expression of different fluorescent proteins to allow connections of GABAergic neurons to be differentiated from connections by other neuron types. This approach allows for targeted mapping of local circuit neurons and their axonal projections as well as retrograde labeling of neurons with long‐range input connections. With this approach, we have obtained evidence of retrograde GABAergic and non‐GABAergic neuronal labeling at multiple levels of the brainstem (including BötC, rVRG, RTN, and pontine Kölliker‐Fuse/Parabrachial nuclei) and numerous suprabrainstem regions including cortical and subcortical regions. Our results indicate that various constructs of rAAV2‐retro in combination with specific Cre transgenic lines are effective tools for brain‐wide mapping of input connections to circuits in functionally defined respiratory regions, including providing genetic access to specific populations of projection neurons.

Support or Funding Information

IRP of NIH, NINDS
SPARC: Correlation between Patient Characteristics in Gastrointestinal Evaluation using Principal Component Analysis
Authors: Dabiri Yaghoub , Gregersen Hans , Kassab Ghassan
Published: April 2020

Introduction

anorectal function is complex. Assessment of anorectal physiology and diagnostics for anorectal disorders face problems due to inconsistent results with different technologies and huge inter‐subject variability. This limits development of therapeutic strategies. Devices used to evaluate anorectal function include Fecobionics, EndoFLIP, Anorectal manometry and Ultrasound. The goal of this study was to find correlation in patient characteristics based on data obtained with these measurements.

Methods

the four tests mentioned above were used to evaluate anorectal function in normal subjects and patients with fecal incontinence (FI) or constipation. The data were cleaned by replacing unknown values with the average of measurements, and they were standardized. In total there were 90 measurements and 35 features. The collected data included sex, age (60.0±14.3 years), weight (59.0±9.0 kg), height (1.6±0.1m), FI score (16.8±12.8 with normal values =< 5), constipation score (6.5±4.4 with normal values =< 8), anorectal maximum pressure (46.9±10.1 mmHg), resting pressure (61±25.8 mmHg), expel time (46.3±63.2s), and absence or presentation of rectoanal inhibitory reflex (RAIR), etc. Principal component analysis (PCA) was used to find the contribution of each reduced feature in variance of data and the correlation between features, utilizing singular value decomposition. We used Python and its libraries.

Results and Discussion

the variance in data was mostly explained using 25 PCs. The percentage of cumulative variance explained by up to the 1th, 25th and 35th PC was 15%, 98% and 100%, respectively. The PCs provided insights about the relation between features. For example, according to the first PC the maximum pressure, resting pressure, maximum tolerance and age were positively correlated. Also, weight, absence of RAIR and constipation score were positively correlated. However, the latter group of parameters were negatively correlated to the former group. Because the number of patients used in this study were limited results should be interpreted with caution. However, more data are currently being collected, which will be included in our analysis. We plan to extend our analysis to cluster patient data using machine learning.

Conclusions

in this study we analyzed data gathered in anorectal evaluation using PCA. Data obtained provided insights about relation between characteristics of the patients. Analysis of data obtained in anorectal evaluation will help to better understand factors involved in anorectal and other gastrointestinal disorders, which could lead to better preventive, diagnostic and therapeutic strategies.

Support or Funding Information

NIH SPARC funding

Acknowledgements

NIH SPARC funding.
Tregs Fail to Expand in Pregnancy after Recovery from Acute Kidney Injury in Sprague Dawley Rats
Authors: Gillis Ellen E. , Sullivan Jennifer C.
Published: April 2020

Recent clinical studies report that women with a history of acute kidney injury (AKI) have adverse maternal and fetal outcomes in pregnancy, despite having normal renal function prior to conception. Pregnancy is a unique state of immunosuppression, with immunosuppressive cells, such as regulatory T cells (Tregs), increasing to combat the immune response to the allogenic fetus. Tregs are also known to play a critical role in recovery after AKI, and may serve as a critical link between AKI and poor pregnancy outcomes. Our laboratory has developed an animal model of pregnancy after recovery from AKI to further explore the mechanisms by which AKI predisposes women to poor pregnancy outcomes. In the current study, we hypothesized that the Tregs would be decreased during pregnancy after recovery from AKI. Renal ischemia reperfusion (IR) injury was used as our experimental model of AKI. Briefly, female Sprague Dawley rats (12 wks of age, n=4) were randomized to receive either 45 minutes of warm, bilateral renal IR injury or sham surgery. Rats were then allowed 30 days to recover, and renal recovery was confirmed by plasma creatinine and urinary protein excretion. Rats were then randomized to either be mated or serve as an age‐matched virgin control. Pregnancy was confirmed by vaginal cytology, with presence of sperm on the slide indicative of day 1 of pregnancy. Pregnancies were terminated on pregnancy day 20, and blood and kidneys were collected for flow cytometric analysis of T cells. Total CD3+ cells increased in sham and IR pregnant dams compared to virgin controls in the kidney (Table
, Effect of pregnancy, p=0.02, 2‐way ANOVA). Pro‐inflammatory Th17 cells also increased in pregnancy in both IR and sham dams compared to virgin controls, as anticipated (Table
, Effect of pregnancy, p=0.01 kidney, p=0.03 blood). Interestingly, Tregs only increased in sham pregnant dams (Table
, Interaction effect p<0.001 kidney, p=0.004 blood). Tregs in IR dams and virgin controls were similar to the virgin sham rats. These data suggest that Tregs fail to expand during pregnancy after AKI, potentially contributing to the increased rate of fetal demise and poor pregnancy outcomes observed in this model. Ongoing studies in the laboratory are further exploring the role of Tregs in pregnancy in this model, testing the hypothesis that expanding Tregs in pregnancy will improve fetal and maternal outcomes after AKI.

Renal CD3 (% renal cells)

Renal Tregs (%CD3+CD4+ cells)

Renal Th17 (%CD3+CD4+ cells)

Blood CD3 (% renal cells)

Blood Tregs (%CD3+CD4+ cells)

Blood Th17 (%CD3+CD4+ cells)

Sham Virgin

0.9±0.2

4.7±0.7

1.9±0.2

27±2

5.7±0.3

5.0±0.3

Sham Pregnant

4.3±0.5

12.6±0.3

8.5±0.6

28±2

13.4±1.2

9.3±0.7

IR Virgin

1.1±0.4

5.0±0.3

2.1±0.2

26±4

6.6±0.4

5.3±0.1

IR Pregnant

4.0±0.3

5.2±0.7

8.7±0.5

27±3

6.8±0.5

9.0±0.6
Anatomical reorganization within the hand and forelimb of Perodicticus potto
Authors: Boettcher Marissa Lauren , Dickinson Edwin , Leonard Kaitlyn , Herrel Anthony , Hartstone-Rose Adam
Published: April 2020

Historical behavioral accounts note extraordinarily high grip strength in Perodicticus potto. Anatomical analysis of the potto’s hand and forelimb reveal several specializations that may facilitate this enhanced gripping ability, including a specialized vascular bundle (the retia mirabilia) within the wrist, a hyper‐abducted pollex, and a highly reduced second digit. The myological correlates of grip strength (i.e., the contractile potential of the hand and wrist musculature) of P. potto, however, have yet to be quantitatively evaluated. The current study contrasts the myology of the hand and forearm muscles within P. potto to a series of closely‐related strepsirrhines (Nycticebus coucang, Otolemur crassicaudatus, Hapalemur griseus, and Eulemur mongoz), as well as comparing the forearm musculature to a broader (n= 41) primate sample. Despite reports of the potto’s high grip strength, our findings demonstrate relatively below‐average force capacity (relatively low physiological cross‐sectional area) within the digital flexors of this species, which suggests that the gripping ability of this species may be less extraordinary than previously hypothesized. In addition to this observation, we report a number of unusual myological features within the potto, including the largest brachioradialis (an elbow flexor) of any taxon within our sample. Even more surprisingly, we identified an independent extensor indicis within P. potto, despite the vestigial nature of the second digit, and the absence of this muscle within ~25% of other taxa within our sample ‐ all of which possessed a more substantial second digit than the potto. These data underscore the curious nature of the potto’s antebrachial musculature, but suggest that adaptations within the potto’s forelimb may be more related to maximizing endurance grip potential than towards increasing myological force.

Support or Funding Information

National Science Foundation (IOS‐15‐57125 and BCS‐14‐40599)
Developing a FRET‐based Platform of Monitoring GPCR‐induced p38 Activation Illuminates the Foundational Basis of Spatiotemporal Regulation in Proinflammatory Signaling
Authors: Burton Jeremy Caleb , Okáľová Jennifer , Grimsey Neil Justin
Published: April 2020

Mitogen‐activated protein kinase (MAPK) p38 is a critical component in pro‐inflammatory signaling associated with chronic obstructive pulmonary disorder (COPD) and asthma, and is considered a potential therapeutic target for proinflammatory disease. Unfortunately, direct targeting of p38 activity has proven unsuccessful in modulating inflammation, due to the physiologically ubiquitous necessity of p38 activity in tissues. G‐protein coupled receptor (GPCR) stimulation has been shown to mediate p38 autophosphorylation via interaction with TGF‐Beta Activated Kinase 1 Binding Protein 1 (TAB1) independently of typical p38 activation by MKK3/6. We predict that inhibition of p38‐TAB1 interaction will reduce pathological pro‐inflammatory signaling without affecting critical physiological p38 activation. We predicted that the spatiotemporal regulation of this under‐characterized pathway of p38 signaling is critical for downstream signaling responses associated with proinflammatory disease. In this work we have developed a Forster’s Resonance Energy Transfer (FRET) system of subcellular localized biosensors capable of detecting P38 activity. Through exploration of two GPCRs known to activate atypical p38 signaling (PAR1 and EP2) we demonstrate differential signaling kinetics of cytosolic and plasma membrane p38 activation in HeLa cells co‐transfected with the receptor and subcellular‐directed biosensor. The variances in subcellular p38 activation are the subject of current investigations. We have established a basis for further expansion of this system to include acute detection of nuclear and endosomal p38 activation, and will adapt this platform for small scale screening of p38 inhibitor compounds in stable cell lines expressing subcellular localized biosensors. Combined, these studies provide key foundational insight into the complex interplay and relationships between GPCRs and downstream effectors on vascular stability, inflammation, and angiogenesis, potentially leading to the discovery of novel therapeutic approaches to addressing chronic inflammatory disease.
Krill Oil Addition to 8‐weeks of High‐fat Diet Enhances mTORSer2448 Phosphorylation in Rat Heart
Authors: Butcko Andrew , Andraka John , Sharma Naveen
Published: April 2020

Krill oil (KO) derived from shrimp‐like crustaceans, has been marketed as a substantial source of dietary long chain omega‐3 (LC n‐3) polyunsaturated fatty acids, particularly rich in eicosapentaenoic acid (EPA; 20:5 n‐3) and docosahexaenoic acid (DHA; 22:6 n‐3). Consumption of EPA and DHA has been linked to the reduction of cardiovascular disorders including coronary heart disease and myocardial infarction, however the cellular mechanism for these cardioprotective benefits is unclear. The mechanistic target of rapamycin (mTOR) is a Ser/Thr kinase that acts a nutrient sensor, subsequently controlling various key cellular processes including cell growth, cell survival, and protein synthesis. Diets high in saturated fat content have been associated with myocardial dysregulation through alteration of the mTOR signaling pathway, suggesting that interventions that ameliorate mTOR signaling may counter these deleterious effects in the heart. The goal of the current study is to determine the effect of krill oil supplementation to a high saturated fat diet on mTOR signaling in cardiac muscle. Male Sprague‐Dawley rats (approximately 12 months‐old), were assigned by mass to one of three isocaloric dietary groups for 8 weeks: control diet group (CON; 17% saturated fat, 54% carbohydrate, 29% protein; n=8), a high saturated fat group (HF; 42% saturated, 43% carbohydrate, 15% protein; n=12), and a high fat group with krill oil supplementation (KO; 34% saturated fat, 8% krill oil, 43% carbohydrate, 15% protein; n=12). All rats were given ad libitum access to food and water. Body weights and food consumption was recorded weekly. After 8 weeks of dietary intervention and an overnight fast, blood and hearts were collected from anesthetized rats during a terminal surgery. Western immunoblot analysis was used to determine phosphorylation levels of key mTOR signaling proteins including mTORSer2448, p70S6KThr389, 4E‐BP1Thr37/46, eIF4ESer209, and eIF2αSer51. There was no difference in body mass between dietary groups after 8 weeks of treatment. There was a significant increase (p<0.05) in phosphorylation of mTORSer2448 in the KO group when compared to the HF and CON groups. Phosphorylation of downstream targets of mTOR was not different between groups. Eight weeks of KO supplementation can enhance phosphorylation of mTORSer2448, however the cardioprotective impact of this remains unknown. Further research is needed to examine how KO consumption can affect other key downstream mTOR‐related targets associated with improved cardiovascular health.

Support or Funding Information

Research was supported by intramural grants from the College of Health Professions and the Office of Research and Graduate Studies at Central Michigan University.
Overexpression, Purification, and Preliminary Crystallographic Analysis of the Archaeosine Synthase GAT‐QueC
Authors: Susanto Albert , Honberger Matthew , Murphy Ryan , Bayooz Susan , Yi Stuart , Swairjo Manal A.
Published: April 2020

Archaeosine (G+) is a highly modified nucleosides found exclusively at position‐15 in the dihydrouridine loops (D‐loop) of archaeal tRNAs where it contributes to tRNA structural stability. In a late step in G+ biosynthesis, the precursor nucleoside 7‐cyano‐7‐deazaguanine (preQ0) is inserted into tRNA by the enzyme tRNA‐guanine transglycosylase (arcTGT) before conversion to G+ by archaeosine synthases that vary among archaeal phyla: ArcS in Euryarchaeota, and QueF‐like or GAT‐QueC in Chrenoarchaeota. GAT‐QueC is a two‐domain enzyme harboring an N‐terminal glutamine amidotransferase (GAT) domain, and a C‐terminal domain homologous to QueC, the enzyme that produces preQ0. Consistent with its 2‐domain structure, GAT‐QueC functions in vivo as both a preQ0 synthase and an amidinotransferase, generating G+‐modified tRNA. However, it remains unknown whether the GAT domain acts on preQ0 before or after its insertion into tRNA by arcTGT. Here we describe overexpression, purification and preliminary biochemical and crystallographic characterization of GAT‐QueC from Sulfolobus tokodaii (StGAT‐QueC). Analytical gel filtration chromatography and electrophoretic mobility shift analysis reveal that StGAT‐QueC is a monomeric protein with molecular weight of 55 kDa and no affinity for tRNA, suggesting that the enzyme acts on free preQ0, not preQ0‐modified tRNA. High throughput crystallization of the apoenzyme (and in the presence of preQ0) using the vapor diffusion method yielded crystals that grew in 21 days from solutions containing methyl pentanediol and sodium acetate, and diffracted to 5.8 Å without cryo protection. The crystals belong to cubic space group F23, exhibit unit cell length=292 Å and contain 4 protein molecules per asymmetric unit.

Support or Funding Information

NIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).