bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoproteomic Identification of Bovine Pericardial Heart Valve Antigens
Authors: Gates Katherine Yvonne , Dalgliesh Ailsa , Griffiths Leigh
Published: November 2023

American Heart Association estimates the prevalence of valvular heart disease to be 2.5% in the United States. Biological heart valve replacements, such as glutaraldehyde‐fixed bovine pericardium (BP), are considered the current standard of care. The NHLBI xenotransplantation working group identified xenoantigenicity as the major barrier to expanding use of such xenogeneic tissues. Previous research has utilized an immunoproteomic approach based on two‐dimensional western blots to identify a small number of BP xenoantigens, which is time consuming and technically challenging. These limitations have resulted in an ever increasing use of shotgun proteomic approaches due to their superior throughput and reproducibility. However, traditional Liquid Chromatography‐Mass Spectrometry (LC‐MS) fails to separate proteins based on their antigenicity, and therefore it is impossible to determine which identified proteins are antigenic versus not. We hypothesized that an affinity chromatography method utilizing poly‐polyclonal serum would bridge this gap and represent an innovative shotgun proteomic antigen identification approach. Using affinity chromatography rabbit IgG antibodies were isolated from whole serum with Protein A HP SpinTrap (GE Healthcare). IgG capture was confirmed by serum protein electrophoresis and ELISA. Two types of antibody affinity columns were generated, with naive serum (D0) or serum from rabbits immunized with bovine pericardium (D84). Bovine pericardial protein extracts were run through both affinity columns, allowing for the specific capture of antigens, which were later eluted and collected. To confirm specificity of antigen capture, eluants were subjected to western blot, and probed with D84 serum. Affinity enriched antigenic proteins were identified by LC‐MS/MS, with statistical comparisons made between D0 and D84. This approach identified 133 putative antigenic proteins that were enriched on D84 compared to D0 columns. Ten of these antigens corresponded to previously published BP xenoantigens. Of the remaining 123 previously unidentified antigens, 30 have been indicated to be immunogenic in other fields such as cancer and autoimmunity. This method was then applied to three humans who had been implanted with bovine pericardial heart valves. Surprisingly, 300 to 500 proteins were identified from patient columns. Between 57 to 79% of the identified proteins were shared among the three individuals, as well as almost all of the proteins identified in the rabbit model were also present in the human. This work therefore represents the first high throughput method of antigen identification that can now be used to monitor currently implanted patients as well as inform the development of BP‐based biomaterials in the future.

Support or Funding Information

Research supported by grant number R01HL115205 from the National Heart Lung and Blood Institute (NHLBI) at the National Institutes of Health (NIH) and grant number 5T32OD010931‐10 from the NIH.
Sex, Age, and TNF Influence the Gut Microbiota in a Mouse Model of TNBS Colitis
Authors: Kozik Ariangela J
Published: November 2023

Studies of Crohn's Disease, a subtype of Inflammatory Bowel Disease (IBD), have suggested that the gut microbiota plays a pivotal role in disease development and perpetuation. However, data pertaining to mouse colitis and the gut microbiome are not always consistent or reproducible. It is thought that the variation in the ‘normal’ gut microbiota of humans and animals can be attributed to various host genetic and environmental factors. Several studies have shown that facilities, social stress, differences in handling, and diet have a direct impact on the mouse gut microbiome. However, the impact of sex and age on the microbiome of several commonly used strains of mice has not been well described. We hypothesize that mouse sex and age contribute significantly to differences in the gut microbiome and variation of the response to colitis. To address our hypothesis, we examined the gut microbiota of male and female wildtype (WT) and tumor necrosis factor (TNF) deficient (Tnf−/−) mice aged 4 weeks to 9 weeks, prior to and after the induction of acute trinitrobenzenesulfonic acid (TNBS) induced colitis. Fecal 16S rRNA gene microbial community sequence analysis pre and post colitis revealed statistically significant sex and age related differences in microbial diversity and composition. The Chao1 diversity metric estimating taxa richness (number different microbes present) within a community revealed that male mice had a lower Chao1 measure than female mice. Both WT and Tnf−/− mice also exhibited sex and age‐based differences in the gut microbiome composition. Male WT mice had higher relative proportions of Ruminococcus and Roseburia spp., while female WT mice had higher proportions of Peptostreptococcaceae. Male Tnf −/− mice had more Streptococcus, while female Tnf −/− mice had more Turicibacter and Clostridiaceae. We were also able to identify several taxa that are differentially abundant between 4, 6, 8, and 9 week old mice. Four week old WT mice had higher relative proportions of Lactobacillus and Proteobacteria while Tnf −/− mice had more Clostridiales and Firmicutes. Six and eight week WT mice had more Firmicutes, while Tnf −/− mice had more Clostridia. The gut microbiota of male and female mice also responded differently to acute TNBS colitis. Male WT mice developed more severe colitis than female WT mice and Tnf−/− mice of both sexes. Canonical Correspondence Analysis (CCA) also confirmed that sex and age, along with TNF, contribute significantly to variation in the mouse gut microbiome. The results of the microbial analyses and histopathological analyses support our hypothesis that sex and age influence the murine gut microbiota, and impacts the severity of acute colitis. It is crucial to consider these factors when using mouse models to study IBD, especially when these studies involve the microbiome.
Effect of Probiotic Pediococcus pentosaceus on intestinal permeability and occludin protein distribution in a murine model of colitis
Authors: Torres‐Aguilar Lorena , Rodriguez‐Fragoso Lourdes , Garcia‐Vazquez Francisco , Reyes‐Esparza Jorge
Published: November 2023

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon. In individuals with ulcerative colitis, redistribution and downregulation of several tight junction proteins has been observed in active disease as a molecular basis of increased epithelial permeability. A new alternatives for the treatment of UC is the use of probiotics. We have shown recently that Pediococcus pentosaceus treatment at the dose of 1×1010 CFU delayed the onset of some of the symptoms of colitis in a murine model of colitis. The aim of this study is to evaluate the effect of Pediococcus pentosaceus on intestinal permeability and distribution of tight junction occludin. Twenty‐one male BALB/c mice weighing 20–22 g were divided in three groups: Control, Colitis and Colitis+ P. pentosaceus (n = 7 each). Acute colitis was induced by administration of dextran sodium sulfate (DSS) (molecular weight 40 kDa) 3.5% wt/vol in drinking water ad libitum for 6 days. At day 2 during induction of colitis mice were intragastrically gavaged by P.pentosaceus (doses 1×1010 Colony Forming Units). At the seven day of study the intestinal permeability was measured by determination of the fluorescence intensity of FITC‐dextran (molecular weight 4.0 kDa) in serum after 4 hours of oral administration and the occludin distribution were studied by immunohistochemistry. The results showed a decrease of intestinal permeability in the Colitis+P. pentosaceus group with a 39% less of fluorescence intensity in serum compared with Colitis group. In addition, we could see the occluding distribution was exclusively localized at the apical junctional complex both at the surface and in the crypts. In conclusion Pediococcus pentosaceus treatment improve the intestinal barrier function decreasing the intestinal permeability and increasing the occludin distribution epithelial in a murine model of colitis.

Support or Funding Information

Lorena Torres Aguilar is fellowship holder from CONACYT
PAD4 Deficiency Limits Kidney Dysregulation in a Murine Model of Shock/Sepsis
Authors: Girard Bethany Biron , Chen Yaping , Chung Chun‐Shiang , Reichner Jonathan Biron , Ayala Alfred
Published: November 2023

Indirect Acute Respiratory Distress Syndrome (iARDS) is caused by a non pulmonary inflammatory process as a result from insults such as non‐pulmonary sepsis, hypotensive shock, etc. Patients with ARDS frequently develop Acute Kidney Injury, which in turn, increases overall mortality. Neutrophils are thought to have a significant role in mediating ARDS, with the development of iARDS being characterized by dysregulation and recruitment of activated neutrophils to the lung. PAD4 is an enzyme responsible for the citrullination of histones and subsequent neutrophil extracellular trap (NET) formation. Components of NETs including neutrophil derived circulating free DNA as well as circulating histones has been implicated in ARDS as well as AKI in ischemia/reperfusion injury models. However, it is unknown if there is any pathological significance of NET formation in AKI as a result from a “2 hit” model of traumatic shock (fixed‐pressure hemorrhage; Hem) followed by septic (CLP) insult (Hem/CLP)(iARDS). Previously, we have demonstrated that PAD4 gene deficiency improves overall survival as well as decreases pro‐inflammatory cytokines in the lung in this model of iARDS. Here we aim to determine if PAD4 deficient mice (PAD4−/− mice vs. WT mice) were also protected from AKI in an iARDS model. After Hem/CLP (the induction of iARDS) WT mice displayed increased vascular leakage in the kidney as well as increased circulating Blood Urea Nitrogen (BUN) levels. In contrast, PAD4 −/− mice were protected against increased vascular leakage within the kidney and displayed significant decreases in BUN levels.

Conclusion

These data taken together suggest that PAD4 deficiency protects against kidney dysfunction associated with ARDS, which in turn contributes to the overall improved survival in the Hem/CLP model of iARDS.
A study protocol for the comparative study of biological and metabolism bio‐marker between healthy population and patients with acne vulgaris
Authors: Kim Kyuseok , Ha Injin , Kim Eunok
Published: November 2023

Recent studies have reported that pro‐inflammatory mediators and cytokines related to innate immune responses and insulin resistance related factors, which induce sebaceous lipogenesis, sebocyte, and keratinocyte proliferation via milk consumption and hyperglycemic diets, are important roles in the pathogenesis of acne vulgaris. However, current level of evidence is poor to sufficiently explain the pathophysiology of acne vulgaris. Therefore, this study was designed to find out the molecular biological and metabolism bio‐marker between healthy population and patients with acne vulgaris. The study will consist of 40 healthy subjects and 60 patients with acne vulgaris. The molecular biological biomarker will be analyzed with the use of several cytokines (IL‐1α, IL‐1β, IL‐12 β, IL‐15, IL‐6 and TNF α) and chemokines (MCP‐1 and IL‐8) examinined by ELISA, real‐time PCR, or Western blot assay, whereas the metabolism biomarker will be assessed with steroidal hormones (DHEA, androstenedione, testosterone, DHT, estrone, and estradiol), saturated / trans‐fats (palmitic acid, elaidic acid, etc.) and untargeted markers related to TCA cycle by using the LC‐MS. We will examine skin sebum, skin surface temperature and skin viscosity elasticity in constant temperature and humidity room at Korean Medicine Clinical Trial Center, Kyung Hee University Korean Medicine Hospital (Seoul, Republic of Korea). In addition, we will collect the information including their diet pattern, menstruation (only female), medication history, smoking, and sleep pattern from subjects through a questionnaire.

Support or Funding Information

This study was supported by a grant from the Traditional Korean Medicine R&D Project, Ministry of Health & Welfare, Republic of Korea. (HI16C0801)
Involvement of RAGE signaling and inflammatory cytokine elaboration following in vitro exposure to electronic cigarette liquid
Authors: Hirschi Kelsey M , Lewis Joshua B , Ostergar Adam S. , Hall Parker D. , Broberg Dallin S. , Arroyo Juan A. , Reynolds Paul R.
Published: November 2023

The development of the electronic cigarette (e‐cig) has given rise to a new, largely unregulated market within the smoking industry. Proponents suggest they assist with smoking cessation as e‐cigs rely on vapor produced by heating an e‐liquid to deliver flavored nicotine. While generally assumed to be less harmful than traditional tobacco cigarette smoke, knowledge about the toxicity of the e‐liquid consumed via such electronic devices is still scarce. During use of an electronic cigarette, the oral cavity and respiratory system receives direct exposure to the chemical constituents of the e‐liquids. An additional area of concern is the effect of e‐cigs on the placenta due to a potential misconception that e‐cigs are safer to use during gestation than classical tobacco. Traditional cigarette smoke compromises health by increasing inflammation, mediated through receptor for advanced glycation end‐products (RAGE) signaling. Pro‐inflammatory profiles have been suggested following e‐cig exposure; however, the role of RAGE in e‐cig liquid‐induced inflammation has not been resolved. The purpose of this study was to determine the effects of e‐liquid on RAGE expression and levels of several pro‐inflammatory cytokines mediated at least in part by RAGE signaling such as interleukin (IL)‐1b, IL‐6, IL‐8 and tumor necrosis factor (TNF)‐a. Gingival epithelium (Ca9‐22), pulmonary epithelium (A549), and placental trophoblast (BeWo) cells were exposed to 0, 2, or 4% of Cuttwood™ e‐cigarette liquid, a popular national brand, for 24 hours. Cytotoxicity was measured by LDH assays. E‐cig induced RAGE upregulation was confirmed by quantitative RT‐PCR in A549 and Ca9‐22 cells while only a trend in RAGE upregulation was observed in BeWo cells. QPCR revealed increased transcription of IL‐1b, IL‐6, IL‐8 and TNF‐a in each cell type. Finally, a multiplex cytokine array was performed in order to precisely determine the secretion of these and other pro‐inflammatory modulators. In an effort to block RAGE signaling, we have synthesized a family of anionic, partially lipophilic sulfated polysaccharide derivatives known as semi‐synthetic glycosaminoglycan ethers (SAGEs). Co‐treatment of e‐cig liquid and SAGEs resulted in amelioration of inflammatory cytokine secretion. Together, these results provide a new perspective on a possible mechanism whereby e‐cigarette inflammation occurs.

Support or Funding Information

This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).
Investigating the Potential Role of North American Animals as Hosts for Zika Virus
Authors: Ragan Izabela , Blizzard Emily , Bowen Richard
Published: November 2023

The recent emergence of the mosquito‐borne Zika virus (ZIKV) in the Americas has become a global public health concern. While little is known about potential animal reservoirs for ZIKV, nonhuman primates are thought to play a role during enzootic transmission cycles. We describe a series of experimental infections designed to investigate whether species within certain taxonomic groups in North America have the potential to serve as ZIKV amplifying hosts or reservoirs. The objectives of this study were to (1) identify potential animal reservoirs in North America with an emphasis on wildlife and livestock, (2) examine ZIKV pathogenicity in neonatal and adult animals, and (3) evaluate the ability of animal models to recapitulate neonatal birth defects as seen in human patients. Species investigated include armadillos, cottontail rabbits, goats, mink, chickens, pigeons, ground hogs, deer mice, cattle, raccoons, ducks, Syrian Golden hamsters, garter snakes, leopard frogs, house sparrows, and pigs. Animals ranging in age from neonates to adults were inoculated with 105 PFU of one of two ZIKV strains (PRVA BC59 and FSS13025) by subcutaneous and intradermal injection. All animals were monitored daily for clinical signs of disease. Blood samples and body temperatures were collected on days 0–5, 7, 14, 21 and 28 post inoculation. Plaque assays, polymerase chain reaction (PCR), and plaque reduction neutralization tests were performed to detect viral presence and antibody production. Infectious virus was isolated from seven frogs and one armadillo. In addition, neutralizing antibodies were detected in goats, rabbits, ducks, frogs, and pigs. This study indicates that the animals tested to date are unlikely to act as animal reservoirs for ZIKV. However, goats, rabbits, frogs, and pigs could potentially serve as a sentinel species. Future work includes examining the role of amphibians in the transmission dynamics of ZIKV. Understanding the transmission cycle and maintenance of ZIKV in animals will help in developing effective surveillance programs and preventative measures for future outbreaks.

Support or Funding Information

This material is based upon work supported by Colorado State University Animal Models Core and the U.S. Department of Homeland Security under Grant Award Number DHS‐2010‐ST‐061‐AG0001. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Homeland Security.
Floating microelectrode arrays chronically implanted into the feline peripheral nerve elicit a characteristic tissue response
Authors: Kolarcik Christi L , Puglisi Kelly , Lesniak Andrew , Demetris Anthony J , Fisher Lee E , Gaunt Robert A
Published: November 2023

Neural interfacing technologies have the potential to significantly improve the quality of life of patients that are severely disabled or suffering from a variety of neurodegenerative conditions. To recapitulate proper physiological function, motor commands and sensory feedback must be considered. The spinal nerve is a unique location at which these complementary systems can be accessed. In this study, the dorsal root ganglion‐ventral root (DRG‐VR) complex was targeted with floating microelectrode arrays (FMAs) designed either to record from the VR or test stimulation paradigms in the DRG. Extensive characterization of the tissue response to these chronically implanted microelectrode arrays was performed to determine the reliability of these peripheral locations for interfacing applications. To accomplish these analyses, hematoxylin and eosin and Nissl/Luxol fast blue staining along with antibody‐based stains [NF200 (axons), S100 (Schwann cells), vimentin (fibroblasts, endothelial cells, astrocytes), MAC387(monocytes/macrophages), GLUT1 (glucose transport proteins)] were employed, regions of interest representing the entire area of the implanted array defined, and pixel‐based image analyses specific for each stain utilized. Implanted roots were compared to the non‐implanted (i.e., contralateral) roots from the same cohort of animals. The results indicate that the inflammatory reaction elicited with array insertion is not significantly greater than what is observed in a control (i.e., non‐implanted) spinal nerve. The absence of a significant immune response to these arrays suggests some level of immune privilege in the spinal nerve and bodes well for long‐term applications. In addition, no significant decreases in neuronal density or myelination were observed in the area associated with the array indicating that both neuronal cell bodies and axonal projections are maintained. Observations related to the status of the blood‐nerve barrier suggest it is preserved and/or reestablished at the chronic time points examined. Our data indicates that targeting the DRG‐VR complex is a viable option and that the associated tissue response in this location is an opportunity to achieve the overall goal of a long‐term, reliable interface with prosthetic limbs.

Support or Funding Information

This work was sponsored by the Defense Advanced Research Projects Agency (DARPA) Microsystems Technology Office under the auspices of Dr. Jack Judy through the Space and Naval Warfare Systems Center, Pacific Grant No. N66001‐11‐C‐4171.
Innate immunity and Alzheimer's disease
Authors: Affaneh Amira , Lahey Matthew , Langston Zachary , Cudaback Eiron
Published: November 2023

Alzheimer's disease (AD) is a devastating neurodegenerative disorder set to become one of the greatest public health challenges in modern history. Attempts at disease intervention have thus far proven unsuccessful, and so the need for identification of novel therapeutic targets has never been greater. The association between exaggerated brain inflammation and AD strongly suggests that a more complete understanding of the mechanisms underlying dysfunctional immune activity may allow for novel target identification. The APOE gene is uniquely polymorphic in humans and differentially modulates innate immune activity in brain, with the ɛ4 allele representing the greatest known genetic risk factor for development of AD. However, while genetic association helps to identify risk, it does little to inform us about the molecular mechanisms that underlie disease. Amyloid (A) β, a pleiotropic neurotoxic pepetide, aggregates to form insoluble senile plaques and represents the pathologic hallmark of AD. Indeed, significant evidence supports dysfunctional clearance of Aβ in the etiology and/or pathophysiology of AD. Furthermore, Aβ is a potent inflammagen, promoting neuroinflammation through direct activation of both microglia and astrocytes. Here we present data supporting APOE genotype‐dependent differential expression of key glial targets associated with AD pathophysiology.
APOE genotype‐dependent differential influences on MS
Authors: Lahey Matthew , Langston Zachary , Affaneh Amira , Cudaback Eiron
Published: November 2023

Multiple Sclerosis (MS) is an autoimmune disease that demyelinates the central nervous system (CNS) and leads to a loss of neuron conductivity. T helper (Th) cells promote inflammation in the CNS by responding to myelin antigens and activating microglia cells and astrocytes, the resident immune cells of the brain. Proinflammatory cytokines synthesized and secreted by Th cells have profound effects on the brain and represent a possible initiator and key mediator of MS pathogenesis. While the precise molecular and cellular mechanisms underlying CNS sensitivity to Th cells and their secreted products remain poorly understood, genetic risk factors offer important insight to the puzzle. APOE encodes apolipoprotein E and is uniquely polymorphic in humans. Inheritance of the ɛ4 allele is associated with increased disease severity and progression, as well as poorer outcomes in many neurodegenerative diseases, including MS. In addition, APOE genotype determines microglial and astrocyte responses to a wide range of inflammatory stimuli. Here we present data supporting APOE genotype‐dependent differential glial response to MS‐relevant stimuli.
Alzheimer's Disease: Prevention, Symptoms and Treatment
Authors: DiCarlo Marjorie L , Einstein George P , Tulp Orien L
Published: November 2023

The gradual slowing down of memory is assumed to be a normal consequence of aging. When memory dysfunction begins to interfere with the performance of daily activities, then there is a great chance that Alzheimer's disease [AD] or some other form of dementia may be present. Because AD is a very devastating disease, people who suffer from it, including families and friends of the individual so affected, experience frustration, not to mention the stress and pain as the individual is stripped of their abilities and cognitive functions. As AD progresses, in 5 to 8% of the people who are diagnosed with it may suffer from symptoms such as depression, insomnia and even anorexia. In order to stage Alzheimer's disease, the discovery of biomarkers and validation are considered the key aspects to support the clinical diagnosis and provide discriminatory power between different stages of the disorder. Usually after other neurological tests have been performed and other medical issues have been ruled out, then the individual is tested for AD. Upon the death of the person with the disease, an MRI of the brain can usually confirm the diagnosis by means of protein accumulation in the way of clumps and plaque in the brain's gray matter. Even though there is no cure for AD, it has several risk factors that are out of a person's control such as genetics and age. Controlling and identifying risk factors, can help to preserve cognitive abilities even though there is no cure or effective treatment. This challenging disease poses a challenge for policy‐makers which includes, the level of funding and direction of federally funded research, as well as the cost pressures on Medicare and Medicaid for long‐term care. These challenges will continue to increase in intensity as demographics change, particularly with the aging of the current baby boomer generation.

Support or Funding Information

Supported by Institutional Resources of USAT and Einstein Medical Institute
Ablation of Mitochondria Fusion Protein Mfn2 causes an Oxidative Stress Response and Eventual Neuronal Death in the Hippocampus and Cortex
Authors: Jiang Sirui , Wang Xinglong , Nandy Priya , Wang Chunyu , Torres Sandy , Siedlak Sandra L , Zhu Xiongwei
Published: November 2023

Mitochondria are the organelles responsible for energy metabolism and have a direct impact on neuronal function and survival. Mitochondrial abnormalities have been well characterized in Alzheimer disease, and significant advances have recently been made in the understanding of the changes in morphology and distribution of neuronal mitochondria in this devastating disease. It is believed that mitochondrial fragmentation, due to impaired fission and fusion balance, likely causes mitochondrial dysfunction that underlies many aspects of neurodegenerative changes in AD. It is clear that the mitochondrial fission and fusion proteins play a major role in maintaining the health and function of these important organelle. Mitofusion 2 (Mfn2) is one such protein that regulates mitochondrial fusion in which mutations lead to the development of neurodegeneration. To examine whether and how impaired mitochondrial fission/fusion balance causes neurodegeneration in AD, we developed a transgenic mouse model using the CAMKII promoter to knockout Mfn2 in the hippocampus and cortex. Electron micrographs of neurons from these mice show swollen mitochondria with cristae damage and mitochondria membrane abnormalities. Over time the mfn2 KO model demonstrates a progression of neurodegeneration via mitochondrial morphological changes, oxidative stress response, inflammatory changes, cell cycle induction, and loss of MAP2 in dendrites, leading to severe and selective neuronal death. In this model, hippocampal CA1 neurons were affected earlier and resulted in nearly total loss, while cortical neuronal death was associated with fewer neurons and decreased cortical size, but no changes in neuronal density. Overall, our findings indicate that impaired mitochondrial fission and fusion balance can cause many neurodegenerative changes and eventual neurodegeneration that characterize AD in the hippocampus and cortex which makes it a potential target for treatment strategies for AD.

Support or Funding Information

Thanks to the support of T32 NS077888, IIRG‐13‐284849, AARG‐16‐443584, R01 NS083385 and Dr. Robert M. Kohrman Memorial Fund.
Caffeine may affect apoptosis and autophagy activity to promote dopaminergic cells survival under the rotenone induced Parkinson disease model
Authors: Luo Chi‐Lun , Huang Yi‐Chieh , Huang Bin , Lu Tzongshi , Fu Yaw‐Syan
Published: November 2023

Introduction and hypothesis

Parkinson's disease (PD) is the most common neurodegenerative movement disorder caused by the loss of dopaminergic neurons in midbrain. Rotenone is a natural toxic compound extracted from Fabaceae plants and widely used as insecticide and pesticide. Rotenone is highly lipophilic and readily crosses the blood–brain barrier to impair oxidative phosphorylation in mitochondria and that may lead to dopaminergic neuron loss. In this study we investigated the protective effects of caffeine on the rotenone‐induced neurotoxicity of PD models.

Methods

Two dopaminergic cell lines, SH‐SY5Y cells and PC12 cells, were pretreated with caffeine (0~100μM) for 30mins and followed by treatment with rotenone (0~10μM) for 8hr~24hr respectively. The cell survival rate, proliferation, morphology were analysis by flow cytometry, ELISA, Cell Counting Kit‐8(CCK‐8), and histology studies. Cell apoptosis/necrosis were analyzed by Annexin V/PI staining using flow cytometry. The expressions of apoptosis and autophagy related proteins were analyzed by western blotting.

Results

Both dopaminergic cell lines treated with 10nM rotenone for 8hr may decrease the mitochondrial activity of around 60%. Caffeine treatments (0–100μM) had rarely toxic effect on both dopaminergic cell lines but could promote autophagy. When cells pretreated with caffeine (>10μM) for 30min and followed treated with 10nM rotenone for 8hr could significantly increase total cell numbers, but did not restored the mitochondrial activity. In addition, caffeine pretreatment (>10μM) could decrease the cell apoptotic ratio on rotenone‐induced neurotoxicity, but no effect on the necrosis ratio.

Conclusions

Our study data suggested that caffeine treatment can affect autophagy and have protect effects to promote cell survival on rotenone‐induced neurotoxicity and to decline the apoptosis, but it do not influence necrosis.

Support or Funding Information

KMU‐TP104E34, KMU‐NSYSU 105I001
The Use of Patient‐Oriented‐Problem‐Solving (POPS) in Pharmacology Exercises to Develop Simulated Scenes for Application during TBL
Authors: Hernandez Mark J , Foldy Samuel J , Abene Frank J , Lindsey Thomas J , Giannini John
Published: November 2023

Purpose

Teaching pharmacology with the use of pre‐recorded simulation scenes can be an effective, active‐learning pedagogy. To help teach basic pharmacology in a manner that can more easily integrate with clinical sciences, we have used simulated clinical scenarios. One unique approach involves a combination of simulation with exercises known as Patient‐Oriented Problem‐Solving (POPS) during Team Based Learning (TBL) sessions. POPS was developed in the 1980s as part of a movement to provide students with more problem‐solving sessions. Most recently, these exercises have been formatted so that they can be adapted to various course‐management software, with the objective that the POPS could be used by trainees in various health science professional programs (medicine, pharmacy, nursing, etc.), making them suitable for interprofessional education. Here, we describe a unique application of the cases in an educational setting using TBL where the episodes were re‐created with simulation and presented in the same order during the application exercises. This abstract describes the project and its outcomes.

Methods

According to an AAMC report, simulation is any educational activity that utilizes simulative tools and methods in order to create learning opportunities. Our goal at the Alabama College of Osteopathic Medicine is to maximize pharmacology learning and bridge that knowledge with more clinical concepts using simulated scenes. The preparation of the simulated scenes involved both basic and clinical faculty along with the active participation of third year medical student‐fellows who recorded the episodes. The episodes were then presented during the application exercises of TBL sessions delivered to first‐year and second‐year medical students. After each scene, there was opportunity for group discussion followed by questions. Audience response software was used to capture the overall group responses. Student participation was recorded using TurningPoint and learning was later assessed through examination. A Likert scale was used to determine student satisfaction with the use of this method.

Results

The incorporation of simulation helped bridge basic science and clinical sciences in a manner that made the group discussion more engaging. Our results indicate that more students were satisfied than dissatisfied with this method and more agreed that this was a valuable use of their time.

Conclusion

Faculty and student participation in these scenes was well‐received during the application exercises in TBL sessions. The educational approach described here encouraged discussion during the TBL session and promoted active learning, which led to successful outcomes and proved to be an efficient process.
A process‐oriented guided inquiry approach to teaching pharmacology
Authors: Pond Brooks Barnes
Published: November 2023

Process‐oriented guided inquiry learning (POGIL) is a team‐based learning method that uses guided inquiry through exploration of a model to actively engage students in the learning process. This unique teaching strategy was initially developed by college chemistry professors and is now employed by faculty in a variety of disciplines at the high school, college, and professional school levels. The POGIL approach encourages a deeper understanding of core concepts and application of those concepts, and much research within the scholarship of teaching and learning field has indicated that the POGIL approach also allows for better retention of the material by students. In this study, students enrolled in the second and third years of the pharmacy curriculum at the Bill Gatton College of Pharmacy at East Tennessee State University were given in‐class POGIL‐based activities to learn pharmacology. In some cases, the activities completely replaced traditional lecture; in others, short lectures either proceeded or followed a POGIL activity. Topics included general anesthetics, local anesthetics, antiseizure drugs, sedatives and hypnotics, alcohol, antipsychotics, drugs used to treat obesity, thyroid and antithyroid agents, drugs utilized for erectile dysfunction, and post‐menopausal hormone replacement therapy. In the Student Assessments of Instruction, when asked to rate the following statement on a 5 point Likert scale, “In‐class group activities were helpful in my learning of the course material”, all students either selected “strongly agree” or “agree”, with the mean being a 4.8 out of 5. In addition, the Exam Soft testing software was used to examine overall student performance on exam questions falling into higher levels of Bloom's Taxonomy, including those involving comprehension and application of the material. In conclusion, the POGIL technique enhances student perception of their learning experience and may be an effective active‐learning strategy for teaching pharmacology.
Are Exon 11‐Associated Variants of the OPRM Gene Involved in Alcohol Reward?
Authors: Brown Taylor , Martinez Shani , Xu Jin , Pan Ying‐Xian , Rossi Grace
Published: November 2023

Alcohol use remains a global problem with approximately 10–20% of chronic alcohol users becoming alcoholics. Despite severe consequences, alcohol continues to be a rewarding and highly addictive substance as other drugs of abuse. The opioid system has long been hypothesized to involve alcohol reward. Recent work in our laboratories has led to the generation of a knockout (KO) mouse model in which exon 11 (E11) of the mu opioid receptor gene (OPRM1) was disrupted. In E11‐KO mice, analgesia for several mu opioids, including M6G, fentanyl and heroin, was significantly reduced, while analgesic action for morphine and methadone was intact. Moreover, the analgesic action for a novel class of opioids, such as IBNtxA, that fails to produce the typical side effects associated with traditional opiates, was completely lost in E11‐KO mice. To investigate the role of E11‐associated variants in alcohol reward, we evaluated chronic alcohol self‐administration in wild‐type (WT) and E11 knockout (E11‐KO) mice using a two bottle choice paradigm. Total consumption was measured over a 30‐day period with escalating concentrations of alcohol (2–10%). Results showed E11‐KO mice drank significantly higher amounts of alcohol as compared to WT control mice, suggesting involvement of E11‐associated variants in the rewarding effects of alcohol. Although alcohol is presumed to have a very different mechanism of action than heroin, the physiological properties of reward might use a common E11 genetic map.

Support or Funding Information

This grant was funded in part by a Long Island University Monetary Grant.
Cortical circuit dynamics during punishment‐resistant alcohol drinking
Authors: Siciliano Cody A , Leow Yi Ning , Weele Caitlin Vander , Kimchi Eyal , Xu Derek , Chen Xinhong , Tye Kay M
Published: November 2023

A great deal of research has highlighted the role of the medial prefrontal cortex (mPFC) in encoding motivational value and decision making. Further, alcohol‐induced plasticity in mPFC has been implicated in aberrant alcohol drinking behaviors; however, we have little understanding of how neurons in mPFC are encoding alcohol related stimuli in real time, and what downstream circuits are involved. Here, we sought to dissect the role of projection‐defined subpopulations within mPFC in punishment resistant alcohol drinking. To allow for in vivo recordings of neural activity from defined populations, we delivered a Cre‐dependent viral vector carrying the genetically encoded calcium indicator GCaMP6m in the mPFC of male C57BL/6J mice. A second retrograde virus carrying Cre recombinase was injected into either the nucleus accumbens (NAc) or periaqueductal gray area (PAG) resulting in selective expression of the indicator in either mPFC:NAc or mPFC:PAG cells. By implanting a lens into the mPFC and attaching a miniature head‐mounted microscope we were then able to record calcium dynamics with single‐cell resolution during a Pavlovian conditioning task for alcohol, or alcohol adulterated with the bitter tastant quinine. We found that the activity of mPFC:PAG tracked stimulus value of both the conditioned and unconditioned stimulus throughout the course of alcohol and alcohol+quinine exposure. Indeed, mPFC:PAG cells were predominately inhibited by the presentation of an alcohol predictive cue, and during the consumption of alcohol. In animals that decreased their intake when quinine was present, mPFC:PAG showed excitation to the cue and during consumption, while animals that continued to drink despite quinine punishment continued to show an inhibitory response in this pathway. Additionally, we found that photostimulation of PFC‐PAG neurons produced robust avoidance, demonstrating a causal role for this pathway in encoding avoidance of aversive stimuli. Together, these results support a model where inhibition of the mPFC:PAG circuit encodes rewarding stimuli and promotes seeking, while activation encodes aversive stimuli and produces avoidance. Further, dysregulation of this pathway resulting from chronic alcohol use may blunt responsiveness to aversive stimuli and result in inflexible, punishment resistant alcohol drinking.
Novel Lipophilic Carbonic Anhydrase Activators Protect Mice from Recognition Memory Deficits Produced by Scopolamine
Authors: Sanku Rajesh Kishore Kumar , Draghici Bogdan , Pan Yang , Ilies Marc , Walker Ellen
Published: November 2023

Carbonic anhydrase (CA, EC 4.2.1.1) is a zinc enzyme responsible for the reversible hydration of carbon dioxide to bicarbonate which has a critical role in brain homeostasis and normal cerebral functions. In mammals 16 isozymes or CA‐related proteins have been described to date, with different catalytic activity, sub‐cellular localization, and tissue distribution. Carbonic anhydrase isozymes located in the CNS are believed to be involved in neuronal signaling and synaptic strengthening; suggesting that activation of these isozymes may enhance learning. The role played by carbonic anhydrase in learning and memory processes is not elucidated, predominantly due the fact that the large majority of carbonic anhydrase activators (CAA) available today are inefficient activators, unable to cross the blood‐brain barrier, lack appropriate pharmacokinetic profiles, and/or interact with other receptor systems. Therefore, we designed and synthesized several novel lipophilic carbonic anhydrase activators and we have profiled them in terms of carbonic anhydrase isozyme activation. The purpose of the current study was to determine cytotoxicity for a series of novel carbonic anhydrase activators in the neuron‐like differentiated SH‐SY5Y cell line. Solubility and lipophilicity and the activation profiles (KA) for key isozymes were also determined for the compounds. Representatives with different activation profiles and physicochemical properties were then assessed for recognition memory in mice in order to identify the main carbonic anhydrase isozymes involved in the process of learning and memory. Of the compounds screened, six activators were selected that possessed good lipophilicity, optimal solubility, low cytotoxicity, and were predominantly active against CA isozymes CA IV and CA XIV. When tested in the novel object recognition test, these CAAs protected mice from scopolamine‐induced recognition deficits when tested using a dose of 0.03 mg/kg, i.p. with a 30 min pretreatment. There were no signs of behavioral disruption or deficits in motor activity. Considering both the physicochemical and activation profiles, preliminary results gathered by testing CAAs in a mouse models of learning and memory revealed that CA isozymes CA IV and CA XIV are necessary for protection from drug‐induced learning deficits.
Role of Phosphodiesterase 4D in Memory and Cognition
Authors: Zhang Chong , Xu Ying , Gurney Mark , O'Donnell James
Published: November 2023

Inhibition of PDE4 has been proposed as a potential treatment for series of neuropsychological conditions including memory deficits. However, most centrally acting PDE4 inhibitors have accompanying tolerability issues such as emesis and diarrhea. Therefore, it is critical to elucidate the role of each subtype of PDE4 (namely PDE4A, B and D in the brain), so that unwanted adverse effects may be avoided. Using a selective PDE4D inhibitor, BPN14770, our data suggested that PDE4D might play a critical role in mediating behaviors related to memory and cognition. BPN14770 achieves its highly selective inhibition of PDE4D through allosteric binding to the phenylalanine, which is only present in the primates, in the UCR2 region of the enzyme. As a result, the compound inhibits human and mouse PDE4D with IC50s of 8 and 130 nM, respectively. In order to elucidate the involvement of PDE4D in the memory process in intact animals, we generated a “humanized” mouse line (PEDD) by mutating the mouse UCR2 tyrosine271 to phenylalanine. In the in vitro competition binding assay with 3H labeled rolipram, BPN14770 exhibited a preferred 2‐site binding mode with the PEDD but not the wild‐type mouse brain homogenate, suggesting that the compound may be able to bind with PDE4D with different affinities in the wild‐type and PEDD mouse brains. It was then demonstrated that in both the wild‐type and PEDD mice, BPN14770 were able to improve cognition, with the minimum effective doses of 1 and 0.01 mg/kg in the novel object recognition (NOR) test, respectively. However, such dose‐response shift was not present with rolipram, a non‐selective PDE4 inhibitor. These behavioral changes were supported by elevated brain cAMP levels in the mouse brain 1 hour after treatment, which showed a similar dose‐response shift between the wild‐type and PEDD mice. Moreover, BPN‐14770 enhanced the long‐term potential in hippocampal slices obtained from both the wild‐type and PEDD mice, with the minimum effective concentration of the latter being 10‐fold smaller. Neurochemical analyses of brain lysates from PEDD mice suggested that such behavioral changes may be due to altered synaptic function, especially in the post synapses, in the prefrontal cortex and hippocampus, as indicated by increased phosphorylation of synapsin and PSD95, as well as molecular targets downstream of cAMP, such as phosphorylation of CREB and BDNF. Taken together, our data suggested that PDE4D plays an indespensable role in regulation of memory and cognition.

Support or Funding Information

This work was supported by research grants from National Institute of Mental Health to Dr. M. E Gurney (No. R44MH091791).
Functional selectivity of GPCR‐directed drug action through location bias
Authors: Irannejad Roshanak
Published: November 2023

G protein‐coupled receptors (GPCRs) are increasingly recognized to function at intracellular membranes as well as the plasma membrane. We showed previously that the β2‐adrenergic GPCR promotes cyclic AMP (cAMP) signal generation by activating the heterotrimeric G protein, Gs, both from the plasma membrane and after ligand‐induced delivery of receptors to endosomes. We show here that the homologous β1‐adrenergic receptor mediates a second phase of intracellular Gs‐cAMP signaling from the Golgi apparatus rather than endosomes. The Golgi‐derived activation mechanism utilizes a pre‐existing receptor pool, contrasting with endosome‐localized activation that utilizes receptors internalized from the plasma membrane, thereby requiring ligands to access the Golgi‐localized pool separately from receptors. We show that epinephrine, a hydrophilic endogenous ligand, activates the Golgi‐localized receptor pool after facilitated transport requiring extraneuronal monoamine transporter (EMT/OCT3), conferring Golgi‐localized activation that is selectively inhibited by corticosterone. We also show that clinically relevant drugs differentially access the Golgi receptor pool according to hydrophobicity, presumably through passive diffusion across membranes, and that β‐blocker drugs presently used in the clinic differ significantly in their ability to inhibit Golgi relative to plasma membrane‐localized receptor activation. These results fundamentally expand the present understanding of intracellular GPCR activation, and reveal a remarkable distinction in the cellular signaling properties of closely homologous adrenoceptor subtypes based on location. We propose the principle of ‘location bias’ as a discrete cellular scheme for discriminating the signaling properties of related GPCRs when activated by endogenous ligands, and to impose functional selectivity on receptor‐mediated drug action.

Support or Funding Information

K99HL122508