bluuebunny/crossref_metadata_2025 huggingface dataset:
Understanding Osteogenesis Through Penis Bone Evolution
Authors: Schultz Nick , Chipalkatti Naina , Dean Matthew D
Published: November 2023

The exaggerated morphology, phylogenetic distribution, and potential reproductive function of the mammalian penis bone, baculum, have made it the subject of studies for centuries. Nevertheless, a detailed understanding of the evolution of the baculum is lacking. Our recent research indicates that the baculum has independently evolved 9 times and been lost at least 10 times, while the structure that it develops in, the phallus, has a single origin. This fascinating evolutionary trend implies that pre‐cursor cells within the penis of mammals can either modify conserved developmental pathways, or have evolved novel genetic networks to gain or lose the potential to develop a baculum. Here we distinguish these two hypotheses by studying osteogenesis in femurs and genitalia of three mammal species, mice, ferrets, and rabbits. These species represent two independent gains of the baculum (mouse, ferret), and a species without a baculum (rabbit), while the femurs of all three species have a single origin. Specifically, we will isolate pre‐cursor cells from the developing penis and femur to create a transcriptional atlas of osteogenesis across the three species. Since these bones commonly develop through endochondral ossification the transcription factor Sox9 will be used as a marker for osteogenesis. Sox9 positive cells will be identified through immunochemistry, isolated using FACS sorting, and then processed for RNA sequencing. Sequencing results will be compared both between species, and between bones to provide insight not only into the genetic regulation of baculum development, but will also enrich our understanding of osteogenic regulation.

Support or Funding Information

This work was supported by the National Institutes of Health grant #GM098536
Effect of Cyclophosphamide on the Rat Urinary Bladder and the Possible Protective Role of Thymoquinone
Authors: Sabry Hassan Sherif M. , Al‐Eyd Ghaith , Thomann Charity , Ahmad Zulfiqar
Published: November 2023

Background

Cyclophosphamide (CP) is a chemotherapeutic agent used for treating many neoplastic disease. Its application is limited now‐a‐days due to its multiple side effects including hemorrhagic cystitis. Thymoquinone (TQ) is one of the active ingredients of Nigella sativa seeds, known for its healing potentials.

Aim of study

The current study has been carried out to examine the protective effect of 2 different doses of TQ against CP‐induced oxidative injury in the urinary bladder in rat. In addition to the possible mechanism through which TQ produces its protective effect.

Methods

5 groups of 40 rats were used in this study. An untreated control group receiving no treatment, a sham control group where TQ (10 mg/kg/day) was administered using gastric gavage, in a single oral dose for 10 consecutive days. A toxicity group where CP‐induced toxicity was induced via 100 mg/kg, intraperitoneal for 2 days. In addition to 2 protection groups, both receiving CP at a dose of 100 mg/kg, intraperitoneal for 2 days but receiving 2 different doses of TQ, at concentrations of 10 mg/kg/day for 10 days and 100 mg/kg/day for 5 days.

Results

CP caused alteration of the urinary bladder histology in rat, with significant oxidative stress (ROS), as revealed by the reduction in the catalase (CA) and glutathione reductase (GR) in blood, in addition to increased lipid peroxidation to compensate for the ROS as revealed by the increase in the malondialdehyde levels (MDA) in blood. CP also induced cell death, as revealed by the activation of caspase‐3 in blood. Post treatment with TQ (at a dose of 100mg/kg/day for 5 consecutive days), however reduced CP‐induced ROS and apoptosis.

Conclusion

Concomitant use of TQ with CP could ameliorate the CP‐induced toxicity on the rat urinary bladder via its antioxidant and antiapoptotic effects.

Support or Funding Information

Self Fund
Development of a Porcine Model to Study the Aortic Plexus and Innervation to the Internal Urethral Sphincter: Implications for Preventing Retrograde Ejaculation
Authors: Beveridge Tyler S. , Groh Adam M. , Moore Colin W. , El‐Warrak Alexander , Johnson Marjorie , Power Nicholas , Allman Brian L.
Published: November 2023

INTRODUCTION

The sympathetic nerves and ganglia overlying the abdominal aorta in humans are symmetrically organized into a bilateral network called the aortic plexus that must be spared during retroperitoneal surgery in order to maintain antegrade ejaculation. It is well established that complete resection of the aortic plexus causes retrograde ejaculation (RE) that results from de‐innervation of the internal urethral sphincter (IUS). At present, however, it remains uncertain which specific nerves of the aortic plexus control the IUS, and thus, which nerves are important for ejaculation.

To ultimately determine which nerves are imperative to maintain ejaculation, the present study aimed to first establish the pig as a comparable model of the human aortic plexus in both structure and function. It was our working hypothesis that the pig exhibits comparable anatomy in terms of gross organization, number of infrarenal splanchnic nerves and constituent ganglia, and that stimulation of the left and right hypogastric nerves (homologous to the human superior hypogastric plexus) would evoke contraction of the IUS.

METHODS

The porcine aortic plexus was examined in 18 boars by gross dissection followed by a histological examination to differentiate ganglia from nerve. Innervation of the IUS was examined using a lesion‐and‐test method, in which stimulation with a bipolar electrode was used to excite the hypogastric nerves distal to the caudal mesenteric ganglia before and after transection (n=5).

RESULTS

As predicted, the anatomy of the porcine aortic plexus appeared comparable to humans, exhibiting two bilateral cords that were supplied by two or more infrarenal lumbar splanchnic nerves. Right and left caudal mesenteric and spermatic ganglia were invariably observed adjacent to their respective arteries, and given their topography within the plexus, they appear homologous to the prehypogastric, inferior mesenteric and spermatic ganglia in humans, respectively. At the junction of the upper infrarenal lumbar splanchnic nerves, two additional ganglia were observed within the cords of the porcine aortic plexus (prevalenceright = 4/8; prevalenceleft = 3/8); these structures have not been reported in humans. Electrostimulation of each hypogastric nerve evoked bladder neck contraction (ΔPright = 6.2 ± 2.2 mmHg, ΔPleft = 4.5 ± 2.2 mmHg), which was abolished following transection.

CONCLUSIONS

Collectively, our results confirm that the pig represents an effective preclinical model for studying not only the structure and normal function of the aortic plexus, but also the implications of unintended surgical trauma on the delicate nerves controlling ejaculation.

Support or Funding Information

This work is supported by CIHR
Cell‐matrix interactions in temporomandibular joint homeostasis
Authors: Reed David A
Published: November 2023

Mechanobiological signaling is a critical regulator of tissue homeostasis in the temporomandibular joint (TMJ), and is achieved through chondrocyte receptor mediated interactions with the pericellular microenvironment. Mechanical and/or inflammatory stress is hypothesized to disrupt these interactions and promote cartilage degeneration. However, defining select and specific cell‐matrix derived signals in the context of TMJ degenerative pathophysiology has yet to be achieved. Here we evaluate the interaction of type VI collagen, an abundant pericellular matrix molecule that affects the material properties of the chondrocyte microenvironment, with nerve/glial antigen 2 (NG2), a transmembrane proteoglycan that binds with type VI collagen. In other cell types, proteolysis of the NG2 ectodomain internalizes an intracellular domain, influencing transcriptional activity in the cell through a PDZ binding motif. Here we test the hypothesis that mechanical and/or inflammatory stress in the TMJ will be associated a spatiotemporal dysregulation of NG2‐type VI collagen interactions and high levels of internalized NG2. In vivo, degeneration was induced in the TMJ of male c57 mice using unilateral partial discectomy. In vitro, mandibular condylar cartilage explants were exposed to a 10 N, 1 Hz compressive load for five days in a compression bioreactor environment. NG2‐type VI collagen colocalization was calculated from immunofluorescence confocal microscopy, and confirmed with proximity ligation assay. In vivo, non‐surgical controls yielded NG2‐type VI collagen colocalization in articular, prechondroblastic and chondroblastic cells (n = 3). High levels of internalized NG2 were observed in hypertrophic cells. Following degeneration, colocalization values decreased (n = 3; p < 0.05) and internalized NG2 levels increased in articular cells (n=3). In vitro, exposure to mechanical stress yielded similar results, with high levels of internalized NG2 in articular and hypertrophic chondrocytes. These data illustrate that NG2 colocalizes with pericellular type VI collagen in mandibular chondrocytes, and that mechanical and/or inflammatory stress affects colocalization and NG2 internalization. Future work will define the functionality of internalized NG2 in the pathophysiology of degenerative temporomandibular disorders.

Support or Funding Information

The Department of Oral Biology, UIC College of Dentistry
Age‐Based Degenerative Joint Disease of Intracapsular Anatomy of the Knee: A Cadaveric Study
Authors: Immonen Jessica , Siefring Chris , Sanders Luke
Published: November 2023

The objective of this study is to determine specific degenerative changes to intracapsular anatomy of the knee joint with aging. The Center for Disease Control states that approximately 14% of Americans 25–65 years old and 33.6% of Americans 65+ experience symptoms of osteoarthritis (OA) and/or degenerative joint disease. The knee joint heavily contributes to OA‐related deaths and hospitalizations in the United States and these values are likely underestimated. Metamorphic analyses to cadaveric intracapsular anatomy of the knee were performed using Image Pro software on three age populations: < 70 years old, 70–79 years old and greater than or equal to 80 years old. The articular cartilage of the medial tibial plateau in the greater than or equal to 80 years old specimens showed a 1.7‐fold increase in surface area degeneration (mm2) compared to 70–79 years old specimens (P<0.05). The articular cartilage of the lateral tibial plateau experienced a 15% and 26% approximate loss in surface area when comparing the < 70 years old age group to the 70–79 years old age group and the greater than or equal to 80 years old age group, respectively. The medial meniscus experienced degenerative changes with aging. These values were expressed as decreases in tibial plateau surface area coverage. Specimens < 70 years old had an average of 49% coverage of the medial tibial plateau whereas the 70–79 years old and the greater than or equal to 80 years old age groups had approximately 38% and 36% coverage, respectively. No major degeneration with aging was seen on the lateral meniscus. 100% of the specimens contained either the anterior meniscofemoral ligament or the posterior meniscofemoral ligament. This additional support to the lateral meniscus could reduce its degeneration as well as the degeneration of underlying lateral articular cartilage. The anterior cruciate ligament (ACL) experienced substantial degenerative thinning with aging. The 70–79 years old specimens had a 1.2‐fold decrease in average ACL circumference (mm) compared to the < 70 years old specimens (P<0.001). The greater than or equal to 80 years old specimens had a 1.24‐fold decrease in ACL circumference compared to the < 70 years old specimens (P<0.001). Weakness of the tensor fascia lata muscle, excessive foot pronation during gait, obesity and skeletal pathology such as shortening or fracture of the femur all may contribute to the increased lateral degeneration with aging on the tibial plateau compared to the medial respects. Contrarily, the medial respects of the joint may experience accelerated degeneration of the meniscus and articular cartilage due to normal compressive forces during gait and obesity. ACL thinning during aging may be leading to substantial articular cartilage degeneration given the cruciate ligaments and the musculature of the knee joint are the primary restraints that combat shearing forces at the knee joint.

Support or Funding Information

Internal Grant from RMUoHP for Protocol No. 150430‐04
Non‐ablative radiofrequency enhance collagen content on the injured Achilles tendon after one year in rats
Authors: Akamatsu Flávia Emi , Itezerote Ana Maria , Saleh Samir , Hojaij Flávio , Andrade Mauro , Teodoro Walcy
Published: November 2023

Degenerative changes are observed in tendons from people over 35 years of age. Also, Tissue repair in the geriatric patients occurs more slowly than in young adult. The mechanism and effects of radiofrequency (RF) treatment have not yet been described in animal model one year after tendon injury. We investigated the action of RF on the healing process one year after induced experimental lesions of the Achilles tendon in rats. Five adult male Wistar rats weighing between 250 and 300 g were surgically subjected to bilateral partial transverse sectioning of the Achilles tendon. After one year the right tendon was treated with RF (RFT), monopolar RF (Tonederm™) adjusted to 650 kHz and 2 W for two minutes for five days/one week, whereas the left tendon served as control (CT). The tendons were weighed and processed for hydroxyproline content. Statistical analysis used Student t test (α= 5%). We observed a significant increase in hydroxyproline content in treated tendons. Radiofrequency enhances collagen synthesis, suggesting that it can be useful for treatment of chronic tendon lesions.
Effects of High‐Fat Diet and Age on the Blood Lipidome and Circulating Endocannabinoids of Female C57BL/6 Mice
Authors: Pati Sumitra , Krishna Saritha , Lee Jung Hwa , de La Serre Claire , Harn Donald , Wagner John , Filipov Nikolay , Cummings Brian
Published: November 2023

Obesity is a growing public health concern that increases the risk of inflammatory and metabolic disorders. While the mechanisms linking obesity and metabolic disorders remain unknown, several studies suggest that alterations in lipid‐mediated metabolism play a significant role. The growing literature demonstrating the complexity of the plasma lipidome and its dysregulation within the context of several diseases has led to studies aimed at identifying lipid markers of obesity and/or type 2 diabetes. We assessed the effects of dietary fat and aging at the level of the lipidome using shotgun lipidomics with electrospray ionization‐mass spectrometry (ESI‐MS) and did so in females. Blood lipid profiles of female C57BL/6 mice following high‐fat diet (HFD) and low‐fat diet (LFD) consumption for short (6 weeks), long (22 weeks), and prolonged (36 weeks) periods were analyzed to evaluate the effects of HFD feeding. To compare lipid alterations with metabolic and liver regulation, we also examined glucose tolerance, insulin sensitivity, endocannabinoid levels, plasma esterase activity, and liver homeostasis. Multivariate analysis of blood lipidomic data indicated differences in the lipid profiles between HFD‐ and LFD‐fed mice with the most notable differences following 6 weeks of consumption. Age‐related alterations demonstrated the most striking effect on the lipidome where the most variation was observed between 6 week and 22–36 week treatment groups, irrespective of dietary treatment. Both HFD‐consumption and age altered the relative abundances of lipid species belonging to phospholipid, fatty acyl, and glycerolipid classes. Interestingly, HFD feeding also affected endocannabinoid levels in an age‐dependent manner. Plasma esterase activity was increased by HFD feeding at all time points, with the most pronounced effect following 6 weeks of consumption. Liver mRNA levels of three important metabolic regulators, PPARα, PPARγ, and CD36, were all affected by HFD consumption. These findings indicate an interaction between dietary fat consumption and aging with widespread effects on the blood lipidome, which may provide a basis for identification of female‐specific obesity‐ and age‐related lipid biomarkers.

Support or Funding Information

This project was supported in part with funds from the University of Georgia's Obesity Initiative (http://obesity.ovpr.uga.edu/).
Method Validation for the Analysis of Perfluorinated Compounds in Rice
Authors: Choi Seogyeong , Jeong Yu‐Jin , Chun Su‐Hyun , Lee Seul Gi , Kim Junghoan , Kho Younglim , Lee Kwang‐Won
Published: November 2023

Perfluorinated compounds (PFCs) are ubiquitous in environment, and occur in food, water, soil and air. Despite the concern over widespread distribution PFCs, little is known about the presence of PFCs in rice which the most preferred staple in Asia. We analyzed 19 species of PFCs in rice by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). PFCs were extracted from samples by homogenized, ion‐pairing extraction and liquid‐liquid extraction. We validated precision, accuracy, limit of detection (LOD) and linearity. As a result, precision less than or equal to 20% and accuracy within 80 to 120%. LOD depends on the compound from 0.01 to 0.1 ng/g. Linearity was observed with correlation coefficients higher than 0.999. Therefore, these results confirm the accuracy of the analysis method for the determination of the PFCs in rice.
High‐fat diet increases Clostridium clusters XIVa in obese rodents
Authors: Jiao Na , Baker Susan S , Nugent Colleen A. , Tsompana Maria , Guan Lingyu , Wang Yong , Buck Michael J , Genco Robert J , Baker Robert D , Zhu Ruixin , Zhu Lixin
Published: November 2023

Background

A number of studies have associated obesity with altered gut microbiota, although results are discordant regarding compositional changes in the gut microbiota of obese animals. Herein we aim to obtain an unbiased evaluation of structural and functional changes of the gut microbiota of diet‐induced obese rodents using a meta‐analysis.

Methods

Our analysis included nine 16S sequencing datasets generated with high‐fat‐diet (HFD)‐induced obese mouse or rat models. The raw sequencing data of all nine studies were processed with QIIME to obtain gut microbiota compositions. Biological functions were predicted and annotated with KEGG pathways using PICRUSt.

Results

No significant difference was observed for alpha diversity and Bacteroidetes : Firmicutes (B/F) ratio between obese and lean rodents. Bacteroidia, Clostridia, Bacilli, Erysipelotrichi were dominant classes in obese rodents but gut microbiota compositions varied among studies. Meta‐analysis of the nine datasets identified 12 differential taxa and 67 differential pathways between obese and lean animals. In obese rodents, we observed increased abundance of Dorea, Oscillospira, and an unassigned genus of Clostridiales. These three genera belong to Clostridium clusters XIVa, known for polysaccharide fermentation. Enterococcus and Lactococcus also increased in obese animals which could release bile salt hydrolase (BSH) that convert conjugated bile acids into free bile acids through deconjugation. Compared to lean animals, differential functional pathways of the gut microbiome in obese rodents included enriched pyruvate metabolism, pentose phosphate pathway, fatty acid biosynthesis, glycerolipid metabolism and membrane transporter pathways. These pathways converge in the function of carbohydrate metabolism, biosynthesis of lipid and energy transportation.

Conclusion

HFD‐induced obesity alters the structure and function of murine gut microbiota. Altered production of short‐chain fatty acid, lipopolysaccharides, and bile acids by gut microbiota could impact lipid and glucose metabolism, insulin resistance and systemic inflammatory status of the obese rodents.
Pectin and Its Hydrolysates Stimulate Intestinal Mucin Secretion
Authors: Hino Shingo , Morita Tatsuya
Published: November 2023

Objectives

Soluble dietary fibers (SDF) increase small intestinal mucin secretion by increasing the number of goblet cells in proportion to the viscosity of the SDF ingested. Our recent study showed in rats that low methoxyl pectin (LPC) enhanced mucin secretion irrespective of goblet cell number in the small intestine. Mucin secretory effects of LPC were also confirmed in vitro using HT‐29MTX cells, suggesting that pectin directly stimulates mucin secretion from the individual goblet cells. The present study aimed to characterize the mucin secretory effect of pectin among SDF and to clarify active components of pectin related to mucin secretion.

Materials & Methods

HT‐29MTX cells were cultured with 6.25–100 mg /L of the respective SDF including pectin for 24 h. The culture media were gathered and subjected to MUC5AC determination. RNA was extracted from the cells and subjected to gene expression analysis for MUC5AC. Pectin hydrolysates were obtained from citrus pectin using pectinase (polygalacturonase) and fractionated by diethylaminoethanol cellulose column. Mucin secretory effects of the hydrolysates were also examined.

Results & Findings

Only pectin stimulated mucin secretion from HT‐29MTX cells. Mucin secretory effects of pectin were observed irrespective of molecular size, their origins and degree of methoxylation. Pectin hydrolysates also stimulated mucin secretion accompanied by the increase in MUC5AC expression, but oligo‐galacturonic acids did not. The minimal effective concentrations of pectin hydrolysates were 100‐fold lower than those of pectin. Sugar composition analysis showed that pectin hydrolysates contained higher concentrations of rhamnose and neutral sugars compared with the pectin. From these results, it is plausible to assume that hairy region (rhamnogalacturonan carrying side chains) rather than smooth region (homogalacturonan) in the pectin molecule is responsible for mucin secretory effects.

Conclusion

Among the various SDF tested, only pectin and pectin hydrolysates have exerted significant increases in mucin release from the cells. Clearly, they act as mucin secretagogue.
Protective Effect of Propranolol and Nadolol on PTSD‐like Behavior in Rats
Authors: Zaidi Safiyya , Liu Hesong , Valdez Daniel , Kochi Camila , Atrooz Fatin , Salvi Ankita , Bond Richard , Salim Samina
Published: November 2023

Traumatic experiences are reported to cause Post Traumatic Stress Disorder (PTSD). Benzodiazepines and Selective Serotonin Re‐Uptake Inhibitors are considered as standard treatments. Although clinically useful, chronic use causes drastic side effects. Thus, there remains a necessity for improving treatment and finding preventive pharmacological interventions. While promising evidence emerged with administration of b‐adrenergic receptor antagonist Propranolol and PTSD relief, methodological limitations of the studies and efficacy issues dampened the excitement. We believe it is premature to completely eliminate the role of b‐antagonists in PTSD. Using the social defeat paradigm, we examined the effect of two b‐adrenergic receptor antagonists Propranolol and Nadolol on PTSD‐like behavior in Sprague Dawley rats. Propranolol was administered in drinking water at ~18 mg/rat/day for 36 days. Nadalol was provided in rat chow (50 g/rat/day). Social defeat (SD) was performed for 7 consecutive days. In SD, the Sprague Dawley rat was introduced into the home cage of a larger, aggressive male Long Evans (LE) rat. The LE rat perceived the Sprague Dawley rat as an intruder, leading to an attack on the rat. The Sprague Dawley rat was forced into a supine postion repeatedly, inducing depression‐like behavior. We used four rat groups; SD group, SD+ Propranolol, SD+ Nadalol, and naïve control. We performed depression‐like behavior tests 24 hours after SD or control exposures, including forced swim test (FST) and social interaction (SIT). In FST, the rat was placed into a cylindrical water‐filled container for five minutes. The total time spent immobile was recorded. Higher immobility time in the water indicates depression‐like behavior. SD rats had significantly more immobility time as compared to control rats in FST. SD rats treated with Propranolol or Nadolol did not exhibit high immobility in FST. SIT measures animal sociability. The Sprage Dawley rats were placed into an apparatus containing two chambers; an empty chamber and a chamber housing a novel rat. SD rats exhibited higher depression‐like behavior (less interaction time with the novel rat as compared to an empty cup) compared to controls. SD rats pretreated with Propranolol or Nadolol did not exhibit low social interaction when compared to SD rats alone. These results suggest that depression‐like behavior induced by SD was prevented when rats were pre‐treated with Propranolol and Nadolol.

Support or Funding Information

Grant Support: 2R15MH093918‐02
Combination of Gestational Sub‐Therapeutic Prozac with Tryptophan Depletion Sex Dependently Worsens Social Behavior in Adolescence
Authors: Garbarino Valentina Rose , Edwards Marshall Thomas , Gutierrez Ednia , Santos Taylor , Javors Martin , Daws Lynette C. , Gould Georgianna Grimshaw
Published: November 2023

Depression is a serious mood disorder that can require antidepressant use during pregnancy. Safety concerns have been raised about the effects of selective‐serotonin reuptake inhibiting (SSRI) antidepressants on fetal brain development. One of the most common SSRIs prescribed during pregnancy is fluoxetine (Prozac). Fluoxetine blocks serotonin (5‐HT) reuptake by the 5‐HT transporter (SERT). 5‐HT is vital for shaping neural circuitry during development, and aberrant fetal 5‐HT levels can increase the risk of autism risk, which is diagnosed with the presence of social behavior deficits and repetitive behaviors. Placental transfer studies show fetal 5‐HT availability may be highly dependent upon maternal availability of its precursor, tryptophan (TRP). Hence, elucidation of the relationship between maternal and fetal TRP and 5‐HT demand and availability is crucial to understanding their role in autism susceptibility. Using a mouse model of maternal SSRI exposure with dietary TRP manipulations during pregnancy, we measured the social interaction and social novelty preference (3 Chamber Test for Social Interaction and Novelty Preference), as well as the repetitive behavior (Marble Burying Test) of adolescent offspring. We hypothesized a daily sub‐therapeutic dose (1 mg/kg/day) of fluoxetine, would impair offspring sociability with relevance to autistic behaviors. In prior studies higher fluoxetine doses increased 5‐HT turnover. Hence we predicted that if a low dose SSRI was combined with dietary TRP deficiency, offspring could develop persistent sociability deficits. Our preliminary findings partially support this hypothesis, as either prenatal fluoxetine exposure or TRP depletion alone, or in combination, impaired sociability preference, in a sex dependent and sociability test phase dependent manner. Treated males seem to lose interaction preference, spending less time with a social interaction stimulus, but maintain normal preference for social novelty based on dwelling time in chambers. Females differentially demonstrate normal social interaction preference, but show a loss of preference for social novelty with fluoxetine treatment in measures of time spent in chamber and time sniffing social or novel stimuli. Additionally, in a measure of repetitive behavior, females exposed to TRP depletion or the combined treatment buried more marbles than males. Our early results suggest that females may be more sensitive to in utero exposure to 5‐HT altering factors, and that males and females may compensate for their developmentally altering effects by different mechanisms.

Support or Funding Information

Research supported by NICHD: HD081261. V. Garbarino is supported by a Biology of Aging Training Grant: T32 AG021890.
Adenylyl Cyclase Isoform Specific Effects in Lipid Raft and Non‐Lipid Raft Membrane Domains Regulate cAMP Compartmentation in Human Airway Smooth Muscle Cells
Authors: Agarwal Shailesh R. , Miyashiro Kathryn , Latt Htun , Ostrom Rennolds S. , Harvey Robert D.
Published: November 2023

The formation of distinct macromolecular signaling complexes allows different G‐protein coupled receptors to produce diverse functional responses, even while sharing a common second messenger such as cAMP. In human airway smooth muscle (HASM) cells, segregation of specific receptors into different membrane microdomains is thought to critically aid in generating compartmentalized cAMP responses. Whereas, E type prostaglandin receptors (EPRs) have been shown to be expressed in non‐lipid raft domains of the plasma membrane, β‐Adrenergic receptors (βARs) are predominantly expressed in caveolar lipid rafts. In the present study, we tested the hypothesis that adenylyl cyclase type 2 (AC2) preferentially couples to EPRs in a non‐lipid raft domain, while adenylyl cyclase type 6 (AC6) selectively couples to βARs in lipid rafts. To do this, we examined the effect of overexpressing AC2 and AC6 on cAMP responses detected using genetically‐encoded FRET‐based biosensors targeted to lipid raft and non‐lipid raft domains of the plasma membrane, as well as the bulk cytosolic compartment in HASM cells. This approach has the advantage of measuring the kinetics of cAMP production in living cells without the use of PDE inhibitors. Overexpression of AC2 enhanced the cAMP response to EPR activation associated with non‐lipid raft domains, without significantly affecting responses detected elsewhere. AC2 overexpression also had no effect on cAMP responses to βAR activation detected in any subcellular location. These data confirm the hypothesis that AC2 couples exclusively with EPRs in a non‐lipid raft membrane compartment. Overexpression of AC6, on the other hand, actually decreased the response to βAR stimulation associated with lipid rafts, without significantly affecting responses elsewhere. AC6 overexpression also had no effect on the responses to EPR activation detected anywhere in the cell. The ability of AC6 overexpression to inhibit βAR production of cAMP in lipid raft domains was reversed by inhibition of PDE4 activity with rolipram. It was also reversed by overexpression of Ht31 peptide, which disrupts the interaction of protein kinase A with A‐kinase anchoring proteins (AKAPs). These results suggests that AC6 overexpression upregulates and/or recruits PKA and PDE4 activity, which then reduces βAR production of cAMP associated specifically with lipid raft domains.

Support or Funding Information

Support by: NIH R01 GM107094
Region specific proteomic analysis of murine brain after chronic nicotine or menthol
Authors: Mulcahy Matthew J , Wang Jonathan H , Lester Henry A
Published: November 2023

Nicotinic acetylcholine receptors (nAChRs) are pentameric cation channels that are expressed in the mammalian central nervous system, the peripheral nervous system, and the neuromuscular junction. Eleven neuronal nAChR subunits have been identified in mammals (α2‐7, α9‐10, β2‐4). Chronic administration of nicotine has been shown to increase total α4 and β2 subunit expression in specific brain regions. Menthol, a common cigarette flavoring, has also been shown to upregulate α4 and β2 subunit expression in the cortex, hippocampus, and striatum independent of nicotine co‐administration. These data highlight the potential use of changes in β2 nAChR subunit expression as a marker for neuronal nicotine exposure when administered alone and when administered with additional bioactive compounds such as menthol. We investigated the effect of nicotine and of menthol in several murine brain regions on changes in total β2 subunit protein expression using western blot analysis. Absence of β2 immunoreactivity was confirmed in both β2 knockout and α4 knockout mice. To assess the effects of nicotine, male C57bl/6 mice were separated into two treatment groups: control (saline) and nicotine (2mg/kg/hr). Vehicle (ethanol) and menthol alone (2mg/kg/hr) were compared separately. All treatments were administered for 10–12 days. Nicotine‐induced β2 immunoreactivity was significantly upregulated in isolated brain regions, including the cortex, hippocampus, midbrain, striatum, and thalamus. No change in immunoreactivity was observed in the habenula of mice treated chronically with nicotine. In response to chronic menthol treatment, a significant increase was only observed in the hypothalamus. These data indicate that nicotine and menthol differentially effect expression of nAChR subtypes in various regions of the brain. A comprehensive analysis of changes in β2 nAChR subunit expression in various brain regions will facilitate our understanding of the effects of nicotine exposure in the mammalian CNS. Additionally, the validation of a reliable marker for nicotine and menthol exposure in regions associated with various aspects of addiction and dependence would be invaluable in the pursuit of better cessation therapeutics. Using β2 expression as a marker of nicotine exposure will enable a better understanding of possible effects of nicotine and menthol.

Support or Funding Information

This work is supported by NIH DA036061.
VU590 inhibits Kir1.1 and Kir7.1 potassium channels through distinct molecular mechanisms
Authors: Kharade Sujay V. , Figueroa Eric E. , Denton Jerod S.
Published: November 2023

Inward rectifier potassium (Kir) channels play key roles in diverse physiological processes and may represent unexploited drug targets for cardiovascular, metabolic, and neurological diseases. However, the lack of potent and selective small‐molecule tool compounds has slowed efforts to explore the integrative physiology and therapeutic value of most Kir channels. In an effort to begin evaluating the druggability of the emerging diuretic target Kir1.1 (ROMK, KCNJ1), we performed a high‐throughput screen of approximately 225,000 compounds for inhibitors of Kir1.1. One compound, termed VU590, was found to inhibit Kir1.1 with an 50% inhibition concentration (IC50) of approximately 0.2 μM, have no appreciable effect on Kir2.1 or Kir4.1, and inhibit Kir7.1 with an IC50 of approximately 8 μM (Lewis et al., 2009). VU590 has since been used to uncover new roles of Kir7.1 in regulation of uterine muscle excitability (McCloskey et al., 2014) and melanocortin signaling in the brain (Ghamari‐Langroudi et al, 2015). Here, we set out to understand the molecular mechanisms by which VU590 inhibits Kir1.1 and Kir7.1. Voltage clamp measurements revealed that block of both Kir1.1 and Kir7.1 is voltage dependent and reduced by increasing the electrochemical driving force for K+, suggesting the VU590 binding site in both channels is located in the pore. Mutation of Asn 171 to Asp (N171D) or Gln (N171Q) in Kir1.1 led to a ~75‐fold and ~4‐fold loss of VU590 potency, respectively. We therefore hypothesized that the negatively charged residue at the equivalent position in Kir7.1 (E149) accounted for the lower potency of VU590 toward Kir7.1. However, the E149Q mutation reduced, whereas the E149D mutation enhanced block of Kir7.1 by VU590. These data suggest that the ligand‐channel interface is fundamentally different between the two channels. By swapping equivalent residues between Kir1.1 and Kir7.1 pore we found that the Kir7.1‐T153C mutation enhanced block of the channel by ~10‐fold, prompting us to examine the mechanism further. Results from comparative homology modeling, mutagenesis, and medicinal chemistry suggest that T153 acts as an energetic barrier to low‐affinity ligands attempting to access a deeper‐pore binding site. This study provides new insights into the molecular pharmacology and structure of Kir1.1 and Kir7.1.

Support or Funding Information

Supported by R01DK082884 (JSD)
Anagyrine Desensitization of Peripheral Neuronal Nicotinic Acetylcholine Receptors
Authors: Green Ben , Welch Kevin , Lee Stephen
Published: November 2023

Members of the genusLupinus cause crooked calf syndrome in the Western United States. Thissyndrome consists of multiple congenital contracture‐type deformities, and isdue to the inhibition of fetal movement by plant alkaloids found in teratogeniclupines. Anagyrine, a teratogenic quinolizidine alkaloid, has been proposed tobe metabolized to a piperidine alkaloid that inhibits fetal movement, theputative mechanism behind crooked calf syndrome. In this research using SH‐SY5Ycells expressing autonomic nicotinic acetylcholine receptors (nAChR) and TE‐671cells expressing fetal muscle‐type nAChR we tested an alternative hypothesisthat anagyrine directly desensitizes fetal muscle‐type nAChR. Unexpectedly, anagyrine was a more potent desensitizer of nAChR expressed by SH‐SY5Y cellswith a fifty percent desensitization concentration (DC50) value of 0.02 μM in SH‐SY5Y cells compared to a DC50 value of 14.7 μM inTE‐671 cells. By comparison, dimethylphenylpiperazinium (DMPP) a nAChR agonistselective for α3β4 nAChR expressed by SHSY‐5Y cells had a DC50 valueof 0.8 μM. In this research, suggeststhat anagyrine is an extremely potent desensitizer of neuronal nAChR which isin contrast to teratogenic piperidine alkaloids.

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USDA‐ARS
Discovery and Pharmacological Characterization of Selective Serotonin (5‐HT)2C Receptor Positive Allosteric Modulators (PAMs)
Authors: Wold Eric A. , Wild Christopher T. , McAllister Carrie , Ding Ye , Anastasio Noelle C. , Fox Robert G. , Stutz Sonja , White Mark A. , Chen Haiying , Allen John A. , Cunningham Kathryn A. , Zhou Jia
Published: November 2023

G protein‐coupled receptors (GPCRs) that are located in the central nervous system (CNS) maintain a fundamental role in modulating healthy and diseased brain states. The serotonin (5‐HT)2C receptor, a CNS predominant GPCR, is a regulatory component of neurobehavioral processes, and decreased signaling at this receptor may contribute to mental health disorders such as impulsivity disorders, drug addiction, schizophrenia and depression. The 5‐HT2C receptor is one of 14 functionally distinct 5‐HT receptors that share topologically similar orthosteric sites, lending importance to subtype selective modulation. Positive allosteric modulation of the 5‐HT2C receptor provides an attractive strategy to selectively fine‐tune binding and/or signaling in response to 5‐HT in a site‐ and event‐specific manner. To this end, we designed, synthesized and pharmacologically characterized a series of 4‐alkylpiperidine‐2‐carboxamides as positive allosteric modulators (PAMs) of the 5‐HT2C receptor. Our efforts have resulted in a series of small molecules that enhance 5‐HT‐evoked Cai2+ release through 5‐HT2C receptor signaling by up to 25% over 5‐HT alone, in vitro. Importantly, no significant effect on 5‐HT‐evoked Cai2+ release through 5‐HT2A receptor signaling was observed. Additionally, these PAMs maintained no intrinsic activity on either the 5‐HT2C receptor or the 5‐HT2A receptor in absence of 5‐HT, suggesting selective 5‐HT2C receptor positive allosteric modulation. Our most efficacious PAM in vitro, CYD‐1‐79, displayed favorable pharmacokinetic parameters and was screened to determine no interaction at all additional 5‐HT receptor orthosteric sites. Excitingly, behavioral studies in rat, using CYD‐1‐79, demonstrated suppression of impulsive action in a dose‐dependent manner by 60% at the highest dose (2 mg/kg), in a one‐choice serial reaction time (1‐CSRT) task, and significantly suppressed context‐induced and cue‐reinforced cocaine seeking (1 mg/kg). These efforts have yielded a selective and efficacious 5‐HT2C receptor PAM that exhibits promising pharmacokinetic properties and modulates 5‐HT2C receptor‐associated behaviors in vivo in a dose‐dependent manner. This novel series of 5‐HT2C receptor PAMs may provide valuable tool compounds to elucidate neuropathological serotonergic function and provides the framework for the development of novel neurotherapeutics.

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Supported by grants R01 DA038446 (JZ/KAC), K05 DA020087 (KAC), P30 DA028821 (KAC), T32 DA07287 (CTW, EAW), F31 DA038922 (CTW)
Impact of Paroxetine on Proximal β‐adrenergic Receptor Signaling
Authors: Guo Shuchi , Carter Rhonda , Tilley Douglas
Published: November 2023

β‐adrenergic receptor (βAR) signaling normally relays sympathetic signals to the heart, but during the development of heart failure, aberrant βAR signaling and desensitization can contribute to pathological remodeling. Thus, therapeutics targeting factors involved in the regulation of βAR signaling have been investigated extensively, including G protein‐coupled receptor kinase (GRK) inhibition. Via structural and biochemical analyses, the selective serotonin reuptake inhibitor (SSRI) paroxetine has recently been identified as a GRK2 inhibitor that enhances βAR‐dependent cardiomyocyte and cardiac contractility and reverses cardiac dysfunction and myocardial βAR expression in mouse models of heart failure, whereas another SSRI (fluoxetine) does not. Despite these functional outcomes, consistent with diminished βAR desensitization, the proximal βAR signaling mechanisms sensitive to paroxetine have not been reported. In this project, we aimed to determine whether paroxetine indeed prevents classic βAR desensitization mechanisms at a molecular level. Here we have applied fluorescence resonance energy transfer (FRET) assays to perform a real‐time comparative assessment of the effects of paroxetine versus fluoxetine on cyclic AMP (cAMP) production, β‐arrestin recruitment and interaction between βAR and epidermal growth factor receptor (EGFR). We also investigated whether paroxetine modulates βAR internalization and phosphorylation following catecholamine stimulation via fluorescent microscopy and western blotting. Our results indicate that paroxetine indeed inhibits βAR phosphorylation, β‐arrestin recruitment and internalization in response to catecholamine stimulation, while fluoxetine does not. Further, our data suggests that paroxetine impacts Gs protein/adenylyl cyclase‐dependent cAMP generation in response to catecholamine and acts to disrupt βAR association with EGFR. Thus, paroxetine inhibits classic βAR desensitization mechanisms consistent with GRK inhibition and may be a useful pharmacological tool for studying proximal GPCR signaling.
TUG‐891, an agonist of GPR120, inhibits lipid accumulation in hepatocytes
Authors: Park Soojin , Kang Saeromi , Im Dongsoon
Published: November 2023

Background

Omega‐3 polyunsaturated fatty acids have protective functions on non‐alcoholic fatty liver disease. Recently, a G protein‐coupled receptor named GPR120 was identified as a receptor for omega‐3 polyunsaturated fatty acids and its protective functions in insulin resistance and hepatic steatosis. Also, GPR120 expression in hepatocytes has also been shown from liver biopsies from child fatty liver disease patients.

METHODS

In order to assess functional roles of GPR120 in hepatocytes, an in vitro model of liver X receptor‐mediated hepatocellular steatosis was used.

RESULTS

GPR120 expression was confirmed in human hepatoma cells. A specific liver X receptor activator induced lipid accumulation and TUG‐891, an agonist of GPR120, inhibited the lipid accumulation. TUG‐891‐induced inhibition was ameliorated by treatment of siRNA transfection of GPR120 or AH7614 (GPR120 antagonist) treatment. SREBP‐1c, a key transcription factor for lipogenesis, was induced by liver X receptor activation. The SREBP‐1c induction was also inhibited by TUG‐891 at mRNA and protein levels. TUG‐891‐induced suppression of SREBP‐1c expression was again blunted by GPR120‐knockdown in the hepatocytes. Therefore, the present data suggest that GPR120 in hepatocytes may function as a receptor for omega‐3 polyunsaturated fatty acids to protect lipid accumulation in the liver.

CONCLUSION

Protective functions of omega‐3 polyunsaturated fatty acids on non‐alcoholic fatty liver disease may be mediated by interaction with GPR120 in hepatocytes.
Characterization of an In Vitro Assay for the Detection of Drug‐Induced Cholestasis Using the HepaRG Cell Line
Authors: Krause Caitlin , Blomme Eric , Liguori Michael
Published: November 2023

Drug‐induced cholestasis (DIC) is a relevant concern in the pharmaceutical industry, and reliable early detection of DIC is of great value in the discovery and development of safe drugs. Traditional animal models are poorly predictive of cholestasis risk in humans, partly because of biological differences. Moreover, limited robust in vitro methods to predict DIC are available. Recently, an in vitro model for the detection of DIC was developed using primary hepatocytes in sandwich configuration. Following incubation of cells with test compounds in the presence or absence of a concentrated bile acid mixture, a DIC Index (DICI) is calculated as a measure of the relative reduction in cellular function. This model was evaluated as part of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Innovative Medicine Initiative project MIP‐DILI. Here, we assessed several test compounds in a modified version of this assay using the HepaRG cell line. Several tool compounds with or without known cholestasis risk were tested at relevant concentrations: five compounds with clinical cholestasis reports (troglitazone, cyclosporin A, chlorpromazine, bosentan, and nefazodone), one toxicant inducing biliary injury (ANIT), two non‐cholestatic compounds known to induce hepatocyte injury (APAP, perhexiline), and one compound associated with rare instances of liver injury in humans (pioglitazone). Albumin and ATP production were assessed as parameters to evaluate the overall functionality and integrity of the cells and DICIs were calculated. Based on change in these parameters relative to the vehicle, the assay accurately predicted 6/6 cholestasis positive compounds and 2/3 cholestasis negative compounds. Results were repeatable. From these data, the HepaRG model for the detection of DIC may be beneficial for hazard identification (i.e., biliary injury) during lead optimization.

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Author Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.