bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effects of Cocaine on the Expression of Brain‐Derived Neurotrophic Factor in Female Rat Reward Circuits
Authors: Del Mar Nieves Torres Damaris , Ortíz Angela Silva , Silva Roberto Morales , Domínguez Ursula Gelpí , Rivera Angel Echevarría , Orengo Marian Sepúlveda
Published: April 2020

Cocaine is a highly addictive psychostimulant drug with a high relapse rate. Studies have shown that women have greater subjective responses to cocaine depending on the hormonal phase they are in, making them more susceptible to addiction. Literature shows that sex hormones can modulate the activities of Brain‐Derived Neurotrophic Factor (BDNF). BDNF modulates the growth and survival of neurons in areas such as the nucleus accumbens (NAc) and prefrontal cortex, specifically the pre‐limbic cortex (PL). However, it is still unknown how hormonal stages influence the expression of BDNF in the different phases of cocaine addiction development: self‐administration, extinction and reinstatement. We hypothesized that the expression of BDNF in the NAc and PL varies depending on the hormonal stage, as well as the stage in the development of cocaine addiction. To determine this, female adult rats were subjected to 12 sessions of short‐access self‐administration of cocaine, 16 sessions of extinction and a session of cue‐primed reinstatement. Tissue from the PL and NAc were extracted to compare the expression of BDNF after each phase of cocaine addiction. Preliminary data suggests that BDNF decreases in the NAc during low‐hormonal phases (Diestrus and Metestrus), while it increases during high‐hormonal phases (Estrus and Proestrus). In addition, BDNF protein levels seem to be elevated in the prelimbic cortex after cue‐primed reinstatement, but further analysis is required to confirm this observation. These findings suggest that BDNF expression depends on the phase of cocaine addiction and the estrous cycle in female rats.

Support or Funding Information

Funding was received from the following sources: UPR PRISE Program: #R25GM096955; Start up funds from Ponce Research Institute; Rise Program: NIH‐NIGMS #2R25GM082406; B.R.A.I.N. and M.A.G.I.C. Core: NIM‐HD MD007579.
Liquid‐Liquid Phase Separation by the Egr Transcriptional Coregulator NAB2
Authors: Correia Adele , Couto Rob , Spolidoro Sarina , King Gordo , Cunningham Michaela , Perez Adam , Adams Kenneth
Published: April 2020

NGFI‐A‐binding proteins 1 and 2 (NAB1 and 2) are transcriptional coregulators with roles in a variety of cell types and behaviors. Their role in the peripheral nervous system is best established, where they are expressed in Schwann cells and essential for development and maintenance of peripheral nerve myelination. NAB1 and 2 have also been implicated in the regulation of hindbrain development, fibroblast activation in response to TGF‐β, neuronal and hematopoietic differentiation, and thymus cellularity. In addition, misregulation of NAB1 and 2 has been implicated in disease, including the peripheral neuropathy Charcot‐Marie‐Tooth disease and cancers that develop in the form of solitary fibrous tumors. NAB1/2 mediate their effects through interactions with the Egr family of transcription factors, which they bind via their amino terminal NCD1 (NAB‐conserved domain 1). Upon binding Egr, they either repress or potentiate gene expression is a gene‐specific manner. Two NAB domains have been shown to possess transcriptional repression activity, an internal NCD2 domain and a carboxy terminal CID (CHD4‐interacting domain). However, a NAB domain responsible for transcriptional potentiation has not been determined, nor have molecular mechanisms responsible for regulating NAB1/2 activity. Here we provide evidence that NAB2 possesses the ability to undergo liquid‐liquid phase separation (LLPS) to form liquid droplets in cells, which has recently been characterized as critical event through which some transcriptional activators mediate their effects on gene expression. Transfection of multiple cell lines with a NAB2‐GFP fusion resulted in the formation of several puncta within the nucleus that exhibit fluorescence recovery after photo‐bleaching (FRAP). Assembly into puncta is prevented by either truncation of the NAB2 NCD1 domain or by alanine substitution of two arginine residues within that region (Arg‐97 and Arg‐98), indicating that NAB2 droplet formation is dependent on the amino acid composition within NCD1. Examination of the NAB2 amino acid sequence using PONDR predicted 62.5% disordered sequence and 37.5% ordered sequence. Ongoing experiments are evaluating the necessity of additional regions within NAB2 for LLPS as well as whether NAB2 LLPS is affected by phosphorylation as several sites. These data may provide insight into the mechanisms through which NAB2 is regulated and/or mediates its effects on gene expression.

Support or Funding Information

Bridgewater State University Undergraduate Research Program
ERK‐mediated Phosphorylation of the Egr Transcriptional Coregulator NAB2 at Multiple Sites
Authors: King Gordo , Perez Adam , Spolidoro Sarina , Cunningham Michaela , Corriea Adele , Couto Rob , Adams Kenneth
Published: April 2020

The early growth response (Egr) family of transcription factors comprises five members, Egr1‐4 and Wilms Tumor 1 (WT1), which play roles in various cell behaviors including proliferation, apoptosis, and differentiation in a cell type‐ and stimulus‐specific manner. Roles for Egr1‐3 are particularly well‐documented in the nervous system. Egr1 and 3 contribute to learning and memory through regulation of genes that mediate synaptic plasticity and long‐term potentiation. Egr2 controls hindbrain development through regulation of Hox gene expression and drives peripheral nerve myelination through, at least in part, activation of the gene encoding myelin protein zero in Schwann cells. Egr1 and 2 also contribute to transcriptional network that drives neuronal differentiation of PC12 cells. However, the mechanisms that regulate Egr1‐3 activities in the nervous system are not fully understood. One mechanism of regulation involves protein‐protein interactions with co‐regulators NAB1 and 2 through a conserved R1 domain in Egr1‐3. Most reports demonstrate that NABs repress Egr1‐3 transactivation activity when bound, however, NABs can also enhance transactivation in a target gene‐specific manner. The mechanisms controlling the effect of NABs on Egr activities are not clear. Here, we provide evidence that NAB2 regulation may involve its phosphorylation at several sites by ERK. Using PC12 cells as a model system, our data demonstrate that nerve growth factor (NGF) induces a NAB2 mobility shift in SDS‐PAGE within 10 minutes of treatment. The mobility shift was reversed when lysates from NGF‐treated cells were treated with lambda phosphatase, suggesting the shift is due to phosphorylation. Inhibition of ERK signaling during NGF treatment blocked the mobility shift, while recombinant ERK phosphorylated recombinant NAB2 in an in vitro kinase assay, together indicating that direct phosphorylation of NAB2 by ERK may be responsible for the NAB2 mobility shift in response to NGF. Nine potential ERK phosphorylation sites were identified in NAB2, which were mutated individually to alanine within a NAB2 expression construct to evaluate phosphorylation at those sites based on their mobility in SDS‐PAGE. Of the nine mutants, three exhibited clear changes in NAB2 mobility, which were additive in double and triple mutants for those sites. Ongoing experiments are evaluating the effect of NAB2 phosphorylation on its localization and regulation of Egr1‐3.

Support or Funding Information

Bridgewater State University Undergraduate Research Program
The Effect of Primary Cilia Restoration on Cancer Cells
Authors: Jamal Maha H. , Nauli Surya M.
Published: April 2020

Primary cilia are sensory organelles that exist on the surface of most cells. Since primary cilia play an important role in cell cycle and cilia‐related signaling pathways (e.g. Hedghog and Wnt signaling pathways), cilia have been proposed to be involved in cancer development. In our current study, we characterized the expression of the primary cilia in two prostate cancer (PC3 and DU145), and one lung cancer (NL20‐TA) cell lines by examining cilia formation and measuring cilia length. Moreover, we investigated the correlation between primary cilia and cilia‐related signaling pathways. We found that the frequency and the length of the primary cilia were reduced in PC3 prostate cancer. Primary cilia were absent in DU‐145 prostate cancer and NL20‐TA lung cancer. Furthermore, the β‐catenin of Wnt signaling pathway was elevated in the prostate and lung cancers, suggesting an association between primary cilia and Wnt signaling in cancer cells. We also assessed the ability of rapamycin to restore primary cilium expression in cancer cells. We found that treatment with rapamycin for 8 days increased the percentage of ciliated cells in PC3 prostate cancer and restored primary cilium formation in DU145 prostate cancer and NL20‐TA lung cancer. We next investigated the correlation between primary cilia and Wnt signaling pathways after restoring primary cilia expression in cancer cells. We found that treatment with rapamycin for 8 days reduced the level of β‐catenin in prostate cancer cells. In summary, our data show a potential role of primary cilia in cell proliferation.

Support or Funding Information

SACM
Radiotelemetry and Diffusion Tensor Imaging Revealed that Baroreflex Exhibits Disproportional and Paradoxical Changes During Isoflurane Anesthesia
Authors: Chan Samuel H.H.
Published: April 2020

Using isoflurane as the representative anesthetic agent, the aim of this study was to address the issue of whether general anesthetics induce their depressive actions equally on all aspects of cardiovascular functions and their regulations and whether the degree of depression is directly related to the doses. To mimic the clinical setting during surgery, C57BL/6 mice were instrumented to record by radiotelemetry changes in blood pressure (BP), heart rate (HR) and both arms of the baroreflex from a conscious state to incremental concentrations of isoflurane. We found that there was a significant but disproportional reduction in the cardiovascular parameters when mice were subjected to 1.5% isoflurane anesthesia. Compared with the conscious state, the averaged reduction in BP or HR amounted to 23.5% or 20.5%. On the other hand, the experimental indices for baroreflex‐mediated sympathetic vasomotor tone and cardiac vagal baroreflex manifested a precipitous decline of 90.0% and 63.4%. Paradoxically, on incremental concentrations of isoflurane up to a maximum of 4%, mice exhibited sustained BP and HR, alongside returning of both arms of the baroreflex towards the conscious level. These results were corroborated by diffusion tensor imaging of the medulla oblongata. Tractographic analysis of the baroreflex neural circuits showed a decrease in fraction anisotropy indicative of reduced magnitude of functional connectivity between the nucleus tractus solitarii and rostral ventrolateral medulla or nucleus ambiguus accompanied the augmented baroreflex‐mediate sympathetic vasomotor tone or cardiac vagal baroreflex in response to increased concentrations of isoflurane. We further found that bilateral adrenalectomy exerted minimal effects on the changes in BP, HR and baroreflex in mice subjected to incremental concentrations of isoflurane. It is therefore unlikely that norepinephrine and epinephrine released from the adrenal medulla may account for the disproportional and paradoxical changes in BP, HR and baroreflex under isoflurane anesthesia. These surprise observations offer at least two challenges to the general assumptions on general anesthesia. The effects of isoflurane on cardiovascular functions and their regulations are not exerted proportionally, and both baroreflex‐mediated sympathetic vasomotor tone and cardiac vagal baroreflex exhibit large functional capacity and plasticity in terms of regulating BP and HR.

Support or Funding Information

Supported in part by the Ministry of Science and Technology, Taiwan (MOST‐107‐2320‐B‐182A‐024 and MOST‐108‐2320‐B‐182A‐007).
2,3‐Dichlorophenylpiperazine (DCPP)‐derived Antipsychotics Obstruct Cholesterol Biosynthesis in hiPSC‐derived Neural Precursors and Early Postmitotic Neurons
Authors: Kim John , Neely M. Diana , Kim Hye-Young , Tallman Keri , Porter Ned
Published: April 2020

Antipsychotic prescriptions have steadily expanded in recent years as have their uses for “off‐label” indications such as bipolar affective disorder and major depressive disorder. Aripiprazole (Abilify) and cariprazine (Vraylar) are atypical antipsychotics which share a 2,3‐dichlorophenylpiperazine (2,3‐DCPP) moiety in their chemical structure. Preclinical and clinical studies have shown these small molecules have off‐target inhibitory effects on 7‐dehydrocholesterol reductase (DHCR7) which converts the highly oxidizable lipid 7‐dehydrocholesterol (7‐DHC) to cholesterol. Recent studies have also demonstrated that 2,3‐DCPP alone can potently inhibit DHCR7. The grave consequences of impaired DHCR7 function are demonstrated in Smith‐Lemli‐Opitz syndrome (SLOS), a rare neurodevelopmental disorder in which genetic defects in the enzyme result in the abnormal accumulation of 7‐DHC. Given the developmental deficits seen in SLOS, the use of DHCR7 inhibitors during the first trimester of pregnancy has been suggested to have deleterious outcomes in fetal development. Recognizing the dynamic nature of cellular biochemistry in neural development, we examined the impact of two structurally similar antipsychotics on cholesterol biosynthesis precursor profiles in highly proliferative NPCs and early post‐mitotic neurons differentiated from human‐induced pluripotent stem cells (hiPSCs). We exposed cortical glutamatergic and mesencephalic dopaminergic NPCs and early postmitotic neurons to increasing concentrations (1–1000 nM) of aripiprazole and cariprazine for 48 hours after which we quantified lipid extracts via liquid chromatography‐tandem mass spectrometry (LC‐MS‐MS). Cortical glutamatergic and mesencephalic dopaminergic lineages of both developmental stages exhibited dose‐dependent increases in DHCR7 substrates and corresponding decreases in DHCR7 products. While in early postmitotic neurons both compounds showed similar potency with observable changes in sterol profile at >10 nM exposures, proliferative NPCs demonstrated a particularly high sensitivity to cariprazine with significant effects at 1 nM. These findings are highly significant as patients on cariprazine retain plasma levels of cariprazine and its metabolites above 100 nM for many months. Despite the substantial alterations in sterol profile, no significant cytotoxicity was observed at any dosage. Overall, these observations illustrate the importance of assaying DHCR7 activity as it relates to teratogenicity of novel pharmaceuticals, especially those bearing the 2,3‐DCPP structural unit.

Support or Funding Information

This work was supported by the SyBBURE Searle Program and NIH R01HD064727‐01 (NAP). Cell counts were obtained via the ImageXPress instrument of the Vanderbilt High Throughput Screening (HTS) facility.
Various Physiological Factors for Regulating Activation Speeds in the Delayed Calcium‐Dependent Activation of ANO6/TMEM16F
Authors: Roh Jae Won , Nam Joo Hyun
Published: April 2020

Anoctamin6/TMEM16F (ANO6) is a transmembrane protein that functions as a Ca2+‐activated lipid scramblase and Ca2+‐activated ion channel. Unlike other ion channels in its family, such as ANO1/TMEM16A or ANO2/TMEM16B, ANO6 shows unique characteristics of delayed ion channel activation in the presence of relatively high intracellular Ca2+. This property raises questions regarding its physiological function as an ion channel. Here, we demonstrate that the delayed activation of ANO6 ion channel activity can be modified by different splicing variant types and various intracellular physiological factors, including temperature, Mg‐ATP, and actin cytoskeleton. Human ANO6 has three splicing variants, designated 1, 2, and 5, with variant 1 showing the longest delayed activation, and variants 2 and 5 showing a relatively short delay in whole‐cell patch‐clamp experiments. Conversely, murine ANO6 has only one splicing variant which corresponds to human variant 1 ANO6, thus exhibiting long‐delayed activation. Using a human whole transcriptome database (e.g., TCGA, ISOexpress), we analyzed mRNA levels of each ANO6 splicing variant in human tissues. Interestingly, variant 5 was predominant in all healthy human tissues. This finding implies profound experimental and clinical importance, given that almost all ANO6 studies are performed using murine ANO6. Furthermore, the present study focused on elucidating the different activation mechanisms of ANO6 splicing variants. Similar to variants 2 and 5, variant 1 showed no delayed activation on the excised inside‐out patch‐clamp experiment, implying the existence of unknown cytosolic factors that modulate and delay the activation of ANO6 variant 1. We also discovered that increasing intracellular ATP and stabilizing the actin cytoskeleton extended delayed activation of ANO6 variant 1. Temperature also participates in ANO6 activation. Increasing the temperature to 37□ markedly reduced the delayed activation and increased the Ca2+ sensitivity to physiological levels. Specific mechanisms through which ATP, actin cytoskeleton, and temperature modulate delayed activation remains to be studied further. Collectively, the data obtained in the present study revealed that humans and mice possess different ANO6 splicing variants with different delayed activation kinetics, and many cytosolic factors modulate delayed activation of ANO6 variant 1 which corresponds to murine ANO6. This finding is important because most studies on ANO6 utilize murine ANO6. We posit that physiological and clinical research data should be considered separately when evaluating human and murine ANO6.

Support or Funding Information

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT & Future Planning (MIST) of the Korean government (No. NRF‐2015R1C1A1A02037738).
Characterization of Viraemic Cells in a Murine Model of EHV‐1 Infection
Authors: Awan Aftab , Tulp Orien L. , Einstein George P. , Field Hugh
Published: April 2020

Equine herpes virus (EHV‐1) causes respiratory infection in equine, abortion, paresis, neonatal death, and retinopathy and the virus may become latent following initial infection. Virus entry is via respiratory route, and the virus replicates in ciliated and non‐ciliated epithelial cells of the respiratory tract and in Type 1 and Type 2 pneumocytes in the lung parenchyma. After viral replication in the respiratory system, the virus is disseminated to other parts of body via viraemic cells. The virus crosses the placenta which leads to abortion of live or dead fetuses without premonitory signs. Infected horses show transient immunity after natural or experimental infection and immune responses to EHV‐1, but the immunoprotective status begins to decline after few months of infection. Due to the transient immune response, recovered horses are not protected and thus are prone to subsequent re‐infection. Immunity is not long lived after experimental or natural infection, and as a result the development of an effective vaccine has remained a challenge. In this study viraemic cells were studied in a murine EHV‐1 infection model. Mice were infected intranasally and viraemic cells were studied on days three and five of infection (peak of infection). This study may help to identify the nature of viraemic cells and their role in the transient immune response to infection. Buffy coat cells were removed and stained with a fluorescent antibody test for EHV‐1 antigen, and lung specimens were subjected to transmission electron microscopy. Both techniques confirmed the presence of viraemic cells. These viraemic cells were further stained for EHV‐1 antigen and for CD4 or CD8 biomarkers and results are discussed re: possible identification of viraemic cells in a murine model and their role in transient immune responses.

Support or Funding Information

Supported by Institutional Resources of the University of Cambridge, UK, the University of Health and Humanities, British Virgin Islands, the Einstein Institute, and University of Science, Arts & Technology, Montserrat BWI.
Anomalous AMPK‐Regulated Angiotensin AT1R Expression and SIRT1‐Mediated Mitochondrial Biogenesis at RVLM in Hypertension Programming of Offspring to Maternal High Fructose Exposure
Authors: Chan Julie Y.H. , Chao Yung-Mei
Published: April 2020

Tissue oxidative stress, sympathetic activation and nutrient sensing signals are closely related to adult hypertension of fetal origin, although their interactions in hypertension programming remain unclear. Based on a maternal high‐fructose diet (HFD) model of programmed hypertension, we tested the hypothesis that dysfunction of AMP‐activated protein kinase (AMPK)‐regulated angiotensin type 1 receptor (AT1R) expression and sirtuin1 (SIRT1)‐dependent mitochondrial biogenesis contribute to tissue oxidative stress and sympathoexcitation in programmed hypertension of young offspring. Increased systolic and diastolic blood pressure, plasma norepinephrine level and sympathetic vasomotor activity were exhibited by young HFD offspring. Reactive oxygen species (ROS) level was also elevated in rostral ventrolateral medulla (RVLM) where sympathetic premotor neurons reside, alongside augmented protein expressions of AT1R and pg91phox subunit of NADPH oxidase, decrease in superoxide dismutase 2; and suppression of transcription factors for mitochondrial biogenesis, peroxisome proliferator‐activated receptor g co‐activator a (PGC‐1a) and mitochondrial transcription factor A (TFAM). Maternal HFD also attenuated AMPK phosphorylation and protein expression of SIRT1 in RVLM of young offspring. Oral administration of a HMG‐CoA reductase inhibitor, simvastatin, or an AMPK activator, metformin, to young HFD offspring reversed maternal HFD‐programmed increase in AT1R and decreases in SIRT1, PGC‐1a and TFAM; alleviated ROS production in RVLM, and attenuated sympathoexcitation and hypertension. Together these results suggest that dysfunction of AMPK‐regulated AT1R expression and SIRT1‐mediated mitochondrial biogenesis may contribute to tissue oxidative stress in RVLM, which in turn primes increases of sympathetic vasomotor activity and blood pressure in young offspring programmed by excessive maternal fructose consumption.

Support or Funding Information

This work was supported by research grants from the Chang Gung Medical Foundation to J.Y.H. Chan (CMRPG8C0053‐33 and OMRPG8G0011). Y.M. Chao was supported by a Postdoctoral Fellowship (MOST107‐2811‐B‐182A‐501) from the Ministry of Science and Technology, Taiwan.
Effects of Exogenous Lactate Intake During Exercise on Energy Metabolism and Energy Substrate Utilization in Mice
Authors: Jang Inkwon , Kim Jisu , Kyun Sunghwan , Lim Kiwon
Published: April 2020

Purpose

In the past, lactate has been considered a by‐product of lack of oxygen in the skeletal muscle and accumulates in muscles and blood, which causing acidosis and muscle fatigue. However, lactate is always produce, removed or used regardless of presence of oxygen, and it has been identified as an important metabolite that maintain the constancy of glucose, ATP (adenosine triphosphate) in cells, changing the understanding of lactate. Nevertheless, the effects of exogenous lactate intake have not explained and there is no study of the interrelationship between ingestion time and exercise. Therefore, we aimed to investigate the effects of exogenous lactate intake before exercise on energy metabolism during exercise.

Methods

The male 7‐week‐old ICR mice were randomly divided into 2 groups. Con (vehicle) n = 8, Lac (1 g/kg of sodium lactate) n = 8. After 30 minutes oral administration lactate, during exercise metabolism was measured for 30 minutes at 18m/min and 6° slope. A breathing gas was measured for VO2: oxygen consumption, VCO2: carbon dioxide production, RQ: respiratory quotient, fat oxidation, carbohydrate oxidation, EE: energy expenditure.

Results

Lac was no longer able to exercise for 30 minutes. From the total during exercise metabolism results, there were no significant differences between the two groups. But when the data were divided by 15 minutes, the overall metabolism of Lac tended to changes as the second half progressed. At 16 to 30 minutes, the RQ in Lac tended to increase, while VCO2, carbohydrate oxidation and EE showed significant differences with Con. In addition, the increase in RQ and carbohydrate oxidation caused the fat oxidation to decrease, but there was no significant difference between the two groups.

Conclusion

This study confirmed that exogenous lactate intake 30 minutes before exercise contributes to higher VCO2, carbohydrate oxidation and EE in the second half of the exercise and increase relative intensity. Therefore, our results suggest that lactate intake as an exercise supplement has the potential to increase carbohydrate oxidation and EE relatively, and that it can have a positive effect on improving lifestyle diseases such as diabetes and obesity. However, despite these result, our research shows that acute intakes have increased only in the latter half of exercise and that data have been identified only with open circuit calorimetry system. Furthermore, it could be assessed by the molecular biological approach associated with metabolism in the skeletal muscle.

Support or Funding Information

This work was supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea(NRF‐2019S1A5A8033825)
Insulin‐like Growth Factor 1 Improves Cardiac Function after Acute Myocardial Infarction in a Prediabetic Mouse Model
Authors: Spychala Andre , Heinen Andre , Zimmerhofer Alexandra , Nederlof Rianne , Panjwani Priyadarshini , Gödecke Axel
Published: April 2020

Background

Type 2 diabetes mellitus (T2DM) is a major comorbidity worsening the outcome after acute myocardial infarction (MI). Reduced cardiac glucose uptake may be the underlying mechanism compromising cardiac function even under prediabetic conditions. Insulin‐like growth factor 1 (IGF1) is an anabolic hormone similar to insulin in molecular structure and signaling. IGF1 controls proliferation, differentiation and metabolism of cells. In previous studies we have shown that short‐term IGF1 treatment after MI improved cardiac function and reduced scar size in mouse hearts. Here we investigate whether IGF1 can maintain cardiac function also after MI under conditions of prediabetes.

Methods

Mice were fed standard chow or high fat/high sucrose diet (HFHSD) for 10 weeks to induce obesity and prediabetes. Phenotyping was performed by measurements of blood glucose, insulin levels, glucose (GTT) and insulin tolerance tests (ITT). Mice were subjected to 45 min left anterior descending coronary artery occlusion and 1‐week reperfusion. IGF1 or vehicle were given over 3 days using osmotic mini pumps. Heart function was analyzed echocardiographically before and one week after MI including strain analysis. In addition, a transcriptome analysis was carried out for both the area at risk and the remote area by next‐generation sequencing.

Results

HFHSD led to increased fasting blood glucose levels with up to 200 mg/dl and glucose intolerance. In line with this results, plasma insulin levels were elevated in HFHSD mice with 1.3 ng/L in comparison to 0.4 ng/l in standard chow mice. However, exogenous insulin was able to lower blood glucose in the ITT, indicating prediabetes but no T2DM. Functional analysis by EC indicated a slight improvement of ejection fraction (EF) in HFHSD mice with 71.1% in comparison to 64.0% in the control group. However, cardiac function after MI under prediabetic conditions (EF= 35.3%) worsened to the same extent as in the control group (EF= 34.4%). Treatment of IGF1 after MI preserved cardiac function not only in the control group but also in the prediabetes group, with higher EF (control: 45.5%; IGF1: 48.2%), higher stroke volume and lower end‐systolic volume. Strain analysis revealed that this improvement is rather by an improvement of the remote area than by the area at risk.

Conclusion

Despite a similar signal transduction mediated by IGF1 and insulin, IGF1 was able to preserve cardiac function after MI in a prediabetic mouse model. Thus, an IGF1‐based therapeutic intervention could be a promising approach to improve the worsened cardiac outcome after myocardial infarction even under prediabetic conditions and may also be protective under T2DM.

Support or Funding Information

This work was funded by the German Research Foundation (CRC 1116 “Master Switches in Cardiac Ischemia”, TP A06).
Acute and Chronic Effects of Prostaglandin E2 on NKCC2, NCC and ENaC in Ex Vivo Renal Tubules
Authors: Esteva-Font Cristina , Krogager Toke P. , Hoorn Ewout J. , Fenton Robert A.
Published: April 2020

The major renal prostaglandin, prostaglandin E2 (PGE2), is enzymatically generated from arachidonic acid in a series of steps catalyzed by various enzymes including Cox1/2. PGE2 has modulatory effects on renal Na+ handling, but the molecular targets of these effects in the distal segments of the renal tubule (TAL, DCT, CD) are less well characterized. Here, we assessed acute (30 minutes) and long‐term (24 h) actions of PGE2 (0.1 nM to 10 μM) on various Na+ transport proteins in these segments using tubule suspensions isolated from mouse kidney. In the long‐term, no significant effects could be detected on the abundance of the NaCl cotransporter NCC, the Na‐K‐Cl cotransporter NKCC2 or the epithelial Na+ channel ENaC (αENaC). Altered phosphorylation of NCC (T58) and NKCC2 (T105) or proteolytic cleavage of αENaC was not observed. Similar results were obtained in the presence of Cox1/2 inhibition and no sex differences were observed. Acutely, no effects on NKCC2 or αENaC were observed, but phosphorylation of NCC was decreased. A similar decrease in NCC phosphorylation was detected in NCC‐expressing MDCK cells. Data mining of online kidney RNAseq databases suggested that prostanoid receptor EP4 was expressed in the DCT, and ongoing studies are centered on determining if the actions of PGE2 on NCC are via this receptor.

Support or Funding Information

This project has received funding from The European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska‐Curie grant agreement no 754513, The Aarhus University Research Foundation and NNF Laureate Research Grant ‐ Novo Nordisk Fonden
Structural analysis of determinants for high‐affinity binding of G proteins to muscarinic and histaminic receptors
Authors: Buenemann Moritz , Jelinek Volker
Published: April 2020

GPCRs typically couple to a preferred G protein family. The question is, how receptors recognize their cognate G protein and how this selectivity is mechanistically achieved. Due to the enormous progress in resolving structures of GPCR ‐ G protein complexes, there is overwhelming evidence that the C‐terminus of Ga subunits binds into the cavity of GPCRs that is opened upon outward movement of helix 5 and 6 and thereby is intimately involved in the mechanism responsible for G protein activation. However, other structures of the G protein must contribute as well, as switching C‐termini results only partially in a corresponding switch of the G protein selectivity. Based on M3 receptors we previously found a correlation of the stability of the ternary complex under nucleotide‐free conditions with the binding affinity to the receptor and the potency of receptors to activate G proteins (Ilyaskina et al. PNAS, 2018).

The muscarinic M3receptor and the histaminic H1 receptor were chosen as a model system to investigate possible structures of the G protein because they are able to bind to two different G protein classes with different affinities. Chimeric Gqo and Goq subunits were created, in which several parts of the G protein were exchanged with the respective ones of the other G protein class. The relative binding affinities to the receptors were then compared with the native Gq and Go subunits by using a FRET based assay performed in single permeabilized HEK293T cells. Our results show that the binding affinities of certain Gqo and Goq chimeric subunits to M3 and H1 receptors were similar to the native G proteins, carrying the same backbone. For other chimeras, the binding affinities were significantly changed, which could be attributed primarily to alterations in the dissociation kinetics. Specifically, the high affinity receptor binding properties of Gq proteins could be transferred onto Go‐chimeras by exchanging structures at the N‐ or the C‐terminus or in the middle of the G protein. Furthermore, the combination of several chimeric structures showed even stronger effects. In contrast, exchanging similar regions using Gαq as the backbone it was much more difficult to impair the high affinity of chimeric Gq proteins.

According to these results, the very end of the C‐terminal α5 helix of the G protein seems not to be the crucial part in generating the high‐affinity binding to M3 and H1 receptors. However, it is still important, as in combination with other exchanged structures, the binding affinity was significantly altered. Furthermore, the N‐terminal region of this α5 helix might be more important than previously thought, as it had a pronounced impact on the binding affinity in our experiments.
Squalene Monooxygenase at the Nexus between Cholesterol Homeostasis and Proteostasis
Authors: Chua Ngee Kiat , Howe Vicky , Jatana Nidhi , Hart-Smith Gene , Scott Nicola A. , Thukral Lipi , Brown Andrew J.
Published: April 2020

Cholesterol homeostasis is governed by cholesterol uptake, synthesis and efflux. Cellular cholesterol synthesis relies on numerous enzymes. One of these enzymes is squalene monooxygenase (SM), an endoplasmic reticulum localized rate‐limiting enzyme in the cholesterol synthesis pathway, previously implicated as another target for cholesterol‐lowering and recently gained increasing attention in cancer metabolism. Excess cholesterol stimulates the proteasomal degradation of SM, a classic example of negative feedback regulation to lower cholesterol production. The cholesterol‐accelerated degradation of SM requires the first 100 amino acids of SM (SM N100), which represents the cholesterol‐responsive degron of SM. How the SM N100 cholesterol‐responsive degron operates to fine‐tune SM levels is not well‐understood. Here, we dissected degron features of SM N100, revealing key mechanistic insights into degron features and protein factors mediating the degradation of this degron. We identified (1) a 12‐amino acid amphipathic helix (Gln62 – Leu73) that allows SM N100 to sense membrane cholesterol levels, (2) serine ubiquitination as a non‐canonical mode of ubiquitination to trigger protein degradation, and (3) valosin‐containing protein/p97 as a protein that facilitates the removal of the hydrophobic SM N100 degron from the endoplasmic reticulum membrane. Our findings highlight concerted actions of cholesterol‐sensing and protein degradation mechanisms in regulating SM protein abundance. We also discuss our observations in the context of degron features and sterol‐sensing mechanisms reported by other studies – defining general themes that make proteins amenable to destruction in response to fluctuating lipid levels.

Support or Funding Information

This work was supported by the National Health and Medical Research Council Grant 1060515, Australian Research Council Grant DP170101178, and a Gold Star award from University of New South Wales Sydney (to A. J. B). N.J. was supported by a Department of Biotechnology, Government of India, Research associateship. L.T. was supported by DST‐INSPIRE Faculty Grant LSBM‐45. G.H.S. was supported by the Australian Research Council Grant DP170100108. N.K.C. was funded by a University International Postgraduate Award (UIPA) from UNSW Sydney.
Muscle Fiber Atrophy With Single‐Limb Immobilization is Associated With a Significant Decrease in Muscle‐Derived α‐Klotho of Male Mice
Authors: Parker Emily , Khayrullin Andrew , Mendhe Bharati , Yu Kanglun , Hamrick Mark
Published: April 2020

Loss of muscle mass associated with aging, known as sarcopenia, is attributed to an increased risk of fractures and falls among the elderly population. The mechanisms underlying the development and progression of sarcopenia are, however, not well‐understood. Recent studies suggest that the anti‐aging factor α‐Klotho (α‐KL) plays a role in muscle regeneration, though its relevance for muscle disuse atrophy and atrophy has not been examined. This study aimed to investigate the response of α‐KL and inflammatory factors after immobilization. Twelve‐month C57/Bl6 mice were subjected to two weeks of hindlimb immobilization, consisting of wrapping surgical tape from the pad of the foot, connecting to cast padding and winding several times around the hindlimb. A layer of vetwrap was put on top followed by multiple layers of tape for protection with a drop of super glue applied on the top of the foot. This immobilization technique induced muscle atrophy through significant reduction of relative quadriceps weight (p<0.005) and a significant reduction in fiber size (p<0.005) in the immobilized legs of wild type mice. A significant increase in body weight was seen in the male mice (N=9, p=0.006), while no difference was seen in the females. PCR investigation showed that immobilized muscles had significantly increased TNF‐alpha expression (p<0.05) by a mean change of 67.37%. α‐KL ELISA analysis of muscle lysate revealed that muscle α‐KL was significantly decreased by a mean change of 44.23% in immobilized male muscles compared to controls (p<0.05), while female muscles showed the same trend but was not significant (p=0.2). RT2 profiling of myogenesis and myopathy in mouse skeletal muscles revealed that ten genes were upregulated (Dysf, Dmpk, Prkag3, Akt1, Musk, Myf6, Acvr2b, Prkab2, Hdac5, Myh1) and six were downregulated (Igfbp3, Tnnt1, Myat, Crayb, Trim63, Mb). Transcription factor Sp1, which regulates α‐KL expression, is also involved in regulating three of these genes (Tnnt1, Dmpk, Acvr2b, Cryab). Our data reveal that immobilization simulated disuse atrophy resulting in increased TNF‐α expression which lead to decreased α‐KL in muscle cells in male mice. This indicates that perhaps treatment with α‐KL could alleviate atrophy seen with acute muscle injury.

Support or Funding Information

This research was funded by the National Institutes of Aging (NIA P01 AG036675).
Neuroimmune Contributions to T‐lymphocyte‐Driven Inflammation in a Preclinical Model of Post‐traumatic Stress Disorder (PTSD)
Authors: Elkhatib Safwan K. , Moshfegh Cassandra M. , Watson Gabrielle F. , Case Adam J.
Published: April 2020

Post‐traumatic stress disorder (PTSD) is a devastating psychological disorder with an idiopathic etiology. Interestingly, patients with PTSD have a >3 fold risk of developing comorbid autoimmune diseases compared to matched controls, yet, the exact mechanisms underlying this risk remain unclear. A hallmark of PTSD is increased sympathetic tone, and previous work from our lab has shown that immune cells, primarily T‐lymphocytes, display an autoreactive phenotype when exposed to elevated levels of norepinephrine (NE). Therefore, we hypothesized that the sympathoexcitation observed in PTSD may be causally linked to T‐lymphocyte‐driven inflammation and the predisposition to autoimmune diseases. To test this hypothesis, we adapted a preclinical mouse model of PTSD known as social defeat stress. After validating the PTSD‐like behavioral phenotype of our trauma‐exposed mice, we investigated the neuroimmune interactions of T‐lymphocytes in the spleen that may govern the increased predilection for inflammatory diseases in PTSD patients. The spleen is a primary site of adaptive immune system activation, and is innervated exclusively by the sympathetic efferent splenic nerve. Trauma‐exposed animals exhibited a 2.5 fold increase in splenic catecholamines and 4.1 fold increase in splenic tyrosine hydroxylase (TH) content (p<0.05) compared to controls. Additionally, purified splenic T‐lymphocytes from socially‐defeated mice showed elevations in the pro‐inflammatory cytokines interleukin 6 (IL‐6, 1.84 fold increase, p<0.0001) and interleukin 17A (IL‐17A, 5.5 fold increase, p=0.0032). Importantly, TH content was tightly correlated with T‐lymphocyte IL‐6 and IL‐17A expression (R=0.43, p=0.0001 and R=0.87, p<0.0001, respectively), suggesting a neuroimmune mechanism of pro‐inflammation. To test this, we performed targeted splenic denervation to deduce the contribution of the splenic nerve to T‐lymphocyte‐driven inflammation after psychological trauma. Denervation reduced splenic NE content by 80% (p<0.0001), and persisted to at least 30 days after denervation. While splenic denervation did not alter the PTSD‐like behavioral changes after social defeat, denervation ameliorated the characteristic increase in gene expression of splenic T‐lymphocyte IL‐6 and IL‐17A after social defeat stress. Overall, our data suggest that psychological trauma results in altered inflammation through sympathetic modulation of splenic T‐lymphocytes. Further elucidation of this neuroimmune interaction will provide possible new biomarkers or therapeutic strategies to reduce the inflammatory‐driven morbidity and mortality of PTSD patients.

Support or Funding Information

NIH R00HL123471, T32NS105594
The Stress of Success: An Online Module to Help First‐Year D.M.D. Students Cope with the Impostor Phenomenon
Authors: Metz Cynthia J. , Ballard Erin , Metz Michael J.
Published: April 2020

Professors often spend a great deal of time counseling poorly‐performing students, which limits the time dedicated to high achievers. This may be problematic, as psychiatrists have determined that successful students may be susceptible to feelings of inadequacy despite positive external evidence. Dr. Pauline Clance coined the term “Impostor Phenomenon (IP)” to describe these traits. It was desired to create an online module as an intervention to educate first‐year D.M.D. students about IP and provide six coping mechanisms. After viewing the video at the beginning of the semester, students completed the Clance IP Scale to determine the prevalence of IP thoughts. This was repeated at the end of the semester to determine the impact of the online training module. There was a decrease in post‐semester scores from 63.44 (SD 14.92) to 59.12 (SD 14.56, n=103, 86% response rate, paired‐samples t‐test, p < 0.001). The percentage of students reporting intense impostor experiences decreased from 13.6% to 4.9%. Females exhibited statistically significant higher scores than males on both the pre‐ and post‐semester IP surveys. However, there was no detected significant impact of age or race on results. The most common coping strategies reported by students were a reduction in time spent on non‐essential tasks and the use of scheduling to prevent procrastination. Ten students also reported seeking professional help through mental health services for their IP thoughts. These results suggest that an online training module can improve awareness of the Impostor Phenomenon and help high‐achieving students to cope with their stress and feelings of inadequacy.
The HNF‐1a variant induces endogenous glucose production and Ketonuria in female mice
Authors: Sebastian Manuel , Morriseau Taylor , Dolinsky Vernon , Doucette Christine
Published: April 2020

BACKGROUND

Type 2 diabetes (T2D) diagnoses in the Anishininiiwuk (Oji‐Cree) linguistic group of central Canada are among the highest in the world. A genetic variant in the transcription factor hepatic nuclear factor‐1a (HNF‐1a), known as HNF‐1a G319S is present in this population which has been strongly associated with earlier onset of T2D. Historically, T2D was rare in Anishininiiwuk communities provided an active lifestyle required for land‐based food procurement, and potentially with prolonged periods of fasting. As a result of colonization, this lifestyle was abruptly altered leading to a decrease in hunter‐gatherer societies. In the liver, HNF‐1a plays an important role in glucose homeostasis and suppression of hepatic steatosis, but it is unclear if the G319S variant affects liver function. We
hypothesize that the G319S variant promotes greater hepatic fuel production and confers a metabolic advantage in the fasted state.

METHODS

CRISPR/Cas9 gene editing was used to knock‐in the HNF‐1a G319S G>A 955 single nucleotide substitution into C57BL6 mice. These mice express the G319S variant at endogenous levels and in appropriate tissues. Mice fed chow diet ad libitum were sacrificed at 6 months of age, and liver tissues were collected for gene expression analysis, glycogen content measurements, and for hematoxylin and eosin staining. In addition, pyruvate tolerance tests were performed, and urine ketone levels measured in mice fasted for 12 hours.

RESULTS

Differences in hepatic steatosis and glycogen content were not observed between the wildtype (G/G), heterozygous (G/S), and homozygous (S/S) HNF‐1a mice. Female mice with two copies of the variant S‐allele (S/S) displayed elevated endogenous glucose production on a pyruvate tolerance test, as well as elevated ketones in the urine.

MAIN FINDINGS/CONCLUSIONS

Independent of hepatic steatosis, female homozygous (S/S) HNF‐1a mice show greater endogenous glucose production, and higher ketone levels in their urine, indicative of a greater capacity to generate and mobilize endogenous fuels.

Support or Funding Information

This work was supported by a CIHR project grant awarded to CAD (PJT‐159633).