bluuebunny/crossref_metadata_2025 huggingface dataset:
Developing an In Vivo Model of Reinke's Edema
Authors: Durkes Abigail , Sivasankar Preeti
Published: November 2023

Approximately 7.5 million Americans suffer from difficulty using their voice. Phonatory difficulties are often multifactorial with Reinke's edema being a common underlying pathology. The most common cause of Reinke's edema is smoking. Reflux of stomach acids into the larynx and excessive voice use are also implicated in causing edema. There is currently no reproducible animal model of this common human laryngeal disease. We set out to determine if primary cigarette exposure could be 1) accomplished in an inhalation chamber designed for pigs; and 2) if cigarette smoke exposure could differentially affect the vocal folds of exposed pigs similarly to the human disorder of Reinke's edema. The pig was chosen as the animal model because the pig larynx offers the greatest structural, cellular, immunologic, and neuroanatomical similarity to the human larynx. Twelve Sinclair miniature pigs were randomly assigned to 2 treatment groups (baseline and high cigarette exposure). Six pigs assigned to the baseline cigarette smoke were exposed to 3 cigarettes per day for 20 days. Six pigs assigned to the high cigarette smoke were exposed to 15 cigarettes per day for 20 days. A week prior to treatment, each group of pigs was habituated to the chamber and human contact to reduce stress and ease treatment administration. Behavior modification was accomplished using positive reinforcement. Vocal fold, nasal mucosa, and trachea were examined histologically. Vocal fold ultrastructural alterations, epithelial intercellular space diameter, and microridge height were examined via transmission electron microscopy (TEM). Real‐time polymerase chain reaction was used to investigate genes associated with Reinke's edema in human beings. All animals tolerated the chamber without any signs of distress. Animals on average coughed more in the high exposure group compared with the low exposure (P<0.05). Histology and ultrastructure of the vocal fold were preserved even in the high exposure group of animals. Gene expression data is on‐going. The data obtained from this study offer a potential novel experimental methodology to simulate the development of Reinke's edema in healthy pigs where there is currently no reproducible animal model. The success of this methodology could impact future research on prevention, improving early diagnosis, and therapeutic options for Reinke's edema.

Support or Funding Information

NIH R01DC011759
The role of serine protease in diaphragm development
Authors: Hsu Yu‐Chen , Yang Shi‐Feng , Hung Chia‐Lu , Hung Chia‐Jui , Huang Hsiang‐Po , Yu I‐Shing , Lin Shu‐Wha
Published: November 2023

Congenital diaphragmatic hernias (CHD) are common birth defects that often have severe medical consequences. CHD occurs from the failure of diaphragm development, which leads to a herniation or elevation of abdominal components into the pleural cavity, and results in respiratory failure. Here we report that type II transmembrane serine protease‐1 (TMPRSS1)‐null mice exhibit defective diaphragms that mimic the phenotypes of the diaphragm elevation in humans. Histology and morphometry analyses showed that the tendinous muscle, but not the muscular layer in the diaphragm of the TMPRSS1‐null mice was significantly thinner than in the wild‐type mice. This phenomenon resulted in the position of the diaphragm and liver shifting upward, and the restricted expansion of the lung. We also observed that the pulmonary functions, such as lung resistance and compliance were also significantly impaired in the TMPRSS1‐null mice. To investigate the role of TMPRSS1 during the embryonic development of the diaphragm, we compared the area of diaphragm precursor structure, pleuroperitoneal folds (PPF) and the number of muscle progenitor cells and fibroblasts in the PPF between TMPRSS1‐null and wild‐type embryos (E13.5). The results showed that TMPRSS1‐null mice had no difference in the PPF area and the number of fibroblasts, while the number of muscle precursor cells was less than the wild type. These observations suggest that during early development, TMPRSS1 may play a role in maintaining the structure and function of the diaphragm and we would suggest that a genetic analysis of this modification be performed in patients with diaphragm elevation.
ATF3 protects LPS‐induced acute pancreatic inflammation via modulating NFκB‐mediated iNOS production in mice
Authors: Chen Yu‐Min , Tseng Ya‐Ping , Liu Chin‐Hung , Lin Heng , Cheng Ching‐Feng , Lai Pei‐Fang
Published: November 2023

A state of excessive endotoxin (lipopolysaccharide, LPS) in the circulation is called endotoxemia. An overproduction of proinflammatory mediators in acute pancreatic inflammation, which may lead to insulin resistance, pancreas dysfunction and even death. Activating transcription factor (ATF)‐3, a subunit belongs to ATF/cAMP responsive element‐binding protein (CREB) family of transcription factor protects against stress‐induced acute kidney injury. The previous results suggest that ATF3 contributes to the negative regulation of inflammatory cytokine gene expression by recruiting histone deacetylase (HDAC)‐1 to ATF/NFκB‐binding sites. Biological activities and pathological effects of ATF3 in sepsis is unclear. The aim of this study is to assess the possible protective effects of ATF3 in endotoxemia induced by LPS in vivo studies. ATF3‐knockout (KO) and wild‐type mice (WT) were injected with LPS (10 mg/kg, ip) to induce endotoxemia. Survival rate in mouse model of endotoxemia, circulating white blood cells (WBC) level, serum NO expression, glucose level, histology, and the mRNA expression of inflammatory factors were investigated. The decreased circulating WBC levels, increased serum NO and glucose levels were significantly obtained in KO mice after LPS induction. Furthermore, inflammatory factor expressions, macrophage infiltration and β cell damage in KO mice after LPS induction were significantly reversed by injected adeno‐associated virus (AAV) to restore ATF3 gene expression. These results demonstrated that ATF3 plays a protective role to prevent LPS‐induced pancreas inflammation in experimental sepsis.

Support or Funding Information

supported by MOST and Tzu Chi Medical Center
Model Systems to Study the Pathogenesis of Zika Virus‐Mediated Eye Disease
Authors: Martinez Laura Elizabeth , Contreras Deisy , Jones Melissa Kaye , Gangalapudi Vineela , Tang Jie , Wang Shaomei , Arumugaswami Vaithilingaraja
Published: November 2023

Zika virus (ZIKV) is a teratogenic vector‐borne viral pathogen that causes detrimental congenital eye disease, and other disorders such as microcephaly, intrauterine growth restriction, and sensorineural hearing loss. Eye malformations in newborns to mothers infected with ZIKV during pregnancy, such as macular pigment mottling, loss of foveal reflex, intra‐retinal hemorrhages, chorioretinal atrophy, and blindness have been reported. The disease manifestations of congenital ZIKV eye infection are not well understood. Our overall goal is to understand the mechanisms of ZIKV‐mediated ocular injury. We utilized both cell‐based in vitro and animal model systems to address this goal. We first evaluated the permissiveness of fetal retinal pigment epithelium (RPE) cells to ZIKV PRVABC59 (Asian genotype) infection. The RPE is an epithelial cell monolayer present in between photoreceptor cells and the highly vascularized choroid plexus. We observed that RPEs are highly susceptible to ZIKV infection that lead to reduced cell viability and apoptosis. Transcriptomic analysis of infected cells revealed the activation of inflammatory and VEGF signaling pathways, and dysregulation of factors involved in cell survival (PI3K/Akt signaling). Induced pluripotent stem cell‐derived retinal stem cells (iRSCs) supported ZIKV replication at a lower level compared to that of RPE cells. We then investigated the ZIKV disease pathogenesis using a mouse model. The type‐I interferon receptor knockout (ifnar1−/− or A129) mice were infected with ZIKV through subcutaneous route. Infected animals showed high viremia at 3 days post infection (dpi). Affected mice showed weight loss and signs of posterior paraplegia at 7 dpi, and reached 100% mortality. ZIKV did not establish efficient infection in wild‐type SJL mice and the infected mice did not show changes in body weight or mortality. Brain tissues from infected ifnar1−/− mice had the highest viral load, followed by spleen, thymus, eyes, kidney, and heart. Histopathological analysis showed neuroinflammation characterized by extensive perivascular cuffing with neutrophils and mononuclear cells. Infected eyes showed infiltration of cells in the vitreous space and ganglion cell layer. Immunohistochemistry revealed infiltration of macrophages (CD11) and T‐cells (CD4) in the infected retina. We are currently investigating the molecular mechanisms involved in ZIKV infection of the developing eye using a mouse pregnancy model. In conclusion, we provide evidence that fetal retinal epithelial cells and retinal stem cells are susceptible to ZIKV infection, and the described animal model is useful for studying the Zika virulence factors contributing to congenital eye injury and disease pathogenesis.

Support or Funding Information

Cedars‐Sinai Medical Center
Method Development for the Micromolar Analysis of Pyocyanin (PYO) in Blood and Urine via Mass Spectrometry (MS)
Authors: Mawhinney Thomas Patrick , Chance Deborah L. , Waters James K. , Mossine Valeri V.
Published: November 2023

The virulence factor, pyocyanin (PYO), is one of several toxins that are produced by the opportunistic Gram negative microbe Pseudomonas aeruginosa (PA) and is capable of affecting a broad range of cellular constituents and pathways. It is a soluble, redox‐active blue phenazine pigment that is zwitterionic and, at the pH of blood, freely moves through the cytoplasmic membrane and has been shown to be important in PA virulence. Intracellularly, PYO increases reactive oxygen species (ROS) which can directly and/or indirectly cause cell damage. Its cytotoxicity had been demonstrated on numerous cell lines which can result in morphological changes that include PYO‐induced cell vacuolation, along with a drop in the cellular viability, metabolic mitochondrial activity, and decreased transcriptional and translational activity. A major difficulty in research moving forward is the measurement of PA‐derived PYO in blood and urine from patients with bacteremia, urinary tract infections, cystic fibrosis, and others. In this study, we address the need for a more sensitive method of analysis by synthesizing internal standards (IS) of PYO with stable isotopes to be used for MS analysis.

Initial experiments demonstrated that by reacting phenazine methosulfate (PMS) with H2O18 (98% purity), PYO‐O18 (m/z, 213 M+H+) was produced in near quantitative yields. In a parallel fashion, by reacting phenazine with dimethyl sulfate‐D6, crystalline PMS(D3) was produced. When this product was individually reacted with H2O16 and H2O18, PYO‐D3 (m/z, 214 M+H+), and PYO‐D3O18 (m/z, 216 M+H+) were formed, respectively. With this library of isotopically labeled PYO analogues, differing in atomic mass units (amu) of 2, 3 and 5 (i.e., PYO‐O18, PYO‐D3 and PYO‐D3O18, respectively) from PYO (m/z, 211 M+H+), an analytical scheme was devised. Bovine serum, fortified with PYO, and spiked with PYO‐D3 as the IS, was first used to assist in the development of a PYO isolation scheme amenable to MS analysis (Orbitrap) via direct infusion, and by LC‐MS analysis. Preliminary results indicated that PYO, with a level of detection of ≤40 ng/mL, is achievable. Current research demonstrates that by utilizing both PYO‐D3 and PYO‐D3O18 the unambiguous identification and accurate measurement of bacterial exotoxin PYO in both human serum and urine is possible in the low ng/mL range.

Support or Funding Information

Supported by the Leda J. Sears Trust Fund, the Cystic Fibrosis Association of Missouri, and the University of Missouri Agricultural Experiment Station Laboratories.
Aberrant histone turnover in Alzheimer's disease
Authors: Dowell James A , Gitcho Michael A , Denu John M
Published: November 2023

Recent studies have shown a clear link between epigenetic alterations and the pathogenesis of Alzheimer's disease (AD). Specifically, AD‐dependent changes in histone acetylation alters transcription, resulting in learning and memory deficits. However, the majority of these studies have focused on the AD‐dependent effects on histone H4 acetylation and have not explored other changes in histone modifications during AD pathogenesis. Using a mass spectrometry‐based methodology, we have systematically assessed global histone PTM changes in APP/PS1 mouse hippocampus and cerebellum. These data reveal novel changes in histone H3.3 methylation and H2A.Z acetylation as well as an increase in the total histone H3.3. In the brain, both histone H3.3 and H2A.Z are deposited at transcriptionally active chromatin during neuronal stimulation and then subsequently evicted. This cycle of deposition/eviction is important for transcriptional control of genes involved in learning and memory. The hippocampal‐specific PTM changes we observe in H3.3 and H2A.Z indicate changes in histone dynamics that could be driving the aberrant transcription and downstream cognitive deficits associated with Alzheimer's pathogenesis.
Modeling the ischemic neurovascular unit in a dish using patient‐specific induced pluripotent stem cells
Authors: Page Shyanne
Published: November 2023

The blood‐brain barrier (BBB) is a component of the neurovascular unit formed by specialized brain microvascular endothelial cells (BMECs), astrocytes and neurons. It plays an important function in the maintenance of brain homeostasis by tightly regulating the entrance of water and solutes into the brain and by blocking the entrance of potentially neurotoxic molecules. Dysfunction of the BBB is a key element observed in various neurological disorders and, in particular, following stroke injury.

Stroke is the fifth cause of death in the US and the third leading cause of death in women. It is also a major cause of permanent disability. A key element of ischemic stroke (the most common type of stroke) pathophysiology is the opening of the BBB leading to cerebral edema onset and neural cell death by excitotoxicity. Notably, there is a growing literature highlighting the presence of a sexual dimorphism in stroke. Pre‐menopausal women have lesser stroke events than age‐matched men, however stroke outcome is worsened in post‐menopausal women. Interestingly, the nature such neuroprotection in pre‐menopausal women remains yet to be determined, as current in vitro models of the BBB cannot differentiate the effect of sex from the effects of sexual hormones.

In this project, we designed a sex‐specific in vitro model of the BBB using patient‐derived induced pluripotent stem cells (iPSCs), using one male (CTR66M) and one female cell line (IMR90). We were capable of generating three different cell types from these iPSCs: astrocytes, BMECs and neurons. We did not observe a sexual dimorphism in the differentiation of these cell types in normal conditions, suggesting that biological sex may have not an impact on the differentiation and maturation of the BBB.

Next, we challenged our cells to oxygen‐glucose deprivation (OGD) stress to model ischemic stroke injury in vitro. Cells were given DMEM –glucose and placed in a normobaric, hypoxic chamber for six hours. Following OGD stress, cells were given DMEM +glucose and re‐oxygenated for 24 hours to determine the effect of re‐oxygenation on barrier recovery. In order to assess barrier integrity, transendothelial electrical resistance (TEER) and permeability to sodium fluorescein was used. To determine any changes in cellular metabolism, an MTS or LDH assay was used.

Preliminary data suggests the ability of the different cell types to respond to OGD stress in similar fashion as in vivo studies, as we noted a reversible BBB opening, astrocyte activation and pruning of neurites. In conclusion, our study demonstrates the possibility to obtain a sex‐specific in vitro model of the neurovascular unit, as well as its ability to respond to OGD stress. We are currently investigating the role of the HIF1‐alpha pathway in the response to ischemic stroke in astrocytes, neurons and BMECs.
Regulator of G‐protein signaling 6 (RGS6) expression in human substantia nigra pars compacta (SNc) and loss in Parkinson's disease (PD)
Authors: Luo Zili , Ahlers Katelin E , Yang Jianqi , Chakravarti Bandana , Stevens Hanna E , Narayanan Nandakumar S , Fisher Rory A
Published: November 2023

Parkinson's disease (PD) is a devastating neurodegenerative disorder characterized by the progressive loss of dopamine (DA) neurons in the SNc. The reason for the greater sensitivity of these neurons to degeneration and their loss with aging in PD is unknown. Although there are treatments targeting symptoms of this disease there are no interventions that protect DA neurons from degeneration. We and our collaborators have recently discovered that RGS6 is restrictively expressed in and required for the adult survival of SNc DA neurons that undergo degeneration in PD. Mice lacking RGS6 exhibit a late onset age‐dependent loss of SNc DA neurons accompanied by reduced striatal and SNc DA content and motor deficits as well as increased sensitivity to 6‐OHDA‐induced neurodegeneration and behavioral deficits. These findings suggest a critical pathogenic link between RGS6 loss and DA neuron degeneration in PD. Here we tested this hypothesis by a quantitative analysis of RGS6 expressing neurons in the SNc of human control and PD patients (n = 5 each). Immunohistochemical analysis of RGS6 and tyrosine hydroxylase (TH) expression and unbiased sterology was used to quantify the numbers of RGS6+ and TH+ SNc neurons. We found that RGS6 is exclusively expressed in DA neurons in the SNc of humans and there is a dramatic (73%) and selective loss of these RGS6+ TH+ neurons in PD patients. Consistent with our hypothesis, all surviving SNc TH+ neurons in normal and PD patients express RGS6. Given our evidence that RGS6 is required for survival of SNc DA neurons in mice, these findings suggest that RGS6 loss in human SNc DA neurons renders them susceptible to neurodegeneration characteristic of PD. These findings provide new evidence suggesting that RGS6 may have a critical neuroprotective role in PD in both mice and humans. Together our findings identify RGS6 as a potential novel therapeutic target for PD treatment.

Support or Funding Information

NIH CA161882, MJ Fox 11551
Investigating the Underlying Mechanisms of Chemosensory Dysfunctions in Alzheimer's Disease Using Caenorhabditis elegans with Mutations in Presenilin 1
Authors: Parvand Mahraz , Bozorgmehr Tahereh , Rankin Catharine
Published: November 2023

Background

Many cases of familial Alzheimer's disease (FAD) are linked to mutations of the presenilin (PS) genes. These genes are orthologous with sel‐12 genes in Caenorhabditis elegans, a transparent roundworm. C.elegans have a small nervous system consisting of 302 neurons, making it an ideal candidate for system‐level genetic analysis of neural circuits and behavior. Olfactory dysfunction is an early symptom of AD. However, the underlying mechanisms of these dementia‐linked deficits are unknown.

Methods

Chemotaxis experiments were conducted on worms with a mutation in sel‐12 to gain a better understanding of the relationship between presenilin 1 (PS1) mutations in AD and olfactory deficits. To understand this phenomenon further, we examined various stages of the worm's life cycle to determine when this deficit appeared. This provided preliminary insight into the dynamics of PS1 mutations and its subsequent effect on cellular pathways involved in chemosensation. Since C. elegans are innately repulsed by the odorant octanol and attracted by the odorant diacetyl, we utilized these two odorants in chemotaxis assays to assess the olfactory function of sel‐12 mutants.

Results

Adult sel‐12 mutants had a significantly decreased sensitivity to octanol and diacetyl compared to wild‐type worms. Introducing human wild‐type PS1 into the nervous system of C. elegans rescued olfactory defects, while a PS1 mutant from Alzheimer's patients did not. Moreover, C. elegans sel‐12 mutants presented olfactory deficits from hatching, and this deficit increased as worms aged, similar to the neurodegenerative progression of AD.

Conclusions

This study demonstrates that mutations in C. elegans orthologue of PS1 are associated with altered chemosensation, and that these deficits were rescued by the wild‐type human PS gene in the nervous system.

Future Directions

In order to localize the region(s) where wild‐type sel‐12 function is required for normal olfaction, sel‐12 and PS1 rescues will be conducted in specific neurons that are responsible for the detection of octanol and diacetyl. We will also investigate the morphology of these neurons and compare them to those of wild‐type worms.
Anti‐Muscarinic Drug Effects on Murine Cognition: A Pilot Study
Authors: Theobald Robert , Blanchard Ashley , Morgan Cecil James
Published: November 2023

The importance of cholinergic activity in proper memory function has been well documented with reported memory improvement in mice associated with increased hippocampal cholinergic activity and neurogenesis (Dobryakova, et al, 2014; Jeong, et al, 2014). In the TgCRND8 mouse Alzheimer's Disease (AD) model, Proulx, et al, (2015) demonstrated a disruption in corticothalamic pathways of the prefrontal cortex, suggesting that AD was associated with a loss of intact cholinergic function. Many drugs used as mild sedatives, such as diphenhydramine (DPH), possess significant anti‐muscarinic action, an action that can produce cognitive impairment in mice (Budzynska, et al, 2015). This study tested memory changes by use of a Barnes Maze (BM), a nonstress measure of the ability of mice to remember the location of an escape box. Naïve mice were divided into 5 cohorts (C) and trained using the BM. They received either no drug (Control, C‐1), or oral administration of GDW (Sham, C‐2), atropine (ATR, 0.5 mg/kg, C‐3), DPH, (0.5 mg/kg, C‐4) or DAU 5884 (DAU, 0.25 mg/kg, C‐5). After 3 months treatment was stopped in 50% of mice in each cohort, but testing continued.

Results demonstrated a decrease in the ability of mice treated with ATR and DPH to find the escape box measured by increased time and/or distance travelled to escape. Only the ATR treated mice travelled a greater distance to the escape compartment, but both the ATR and the DPH treated mice took more time to find the escape compartment. There was a suggested recovery of memory when treatment was stopped, as seen in time and distance returning towards control. Results are compatible with previous studies with suggested clinical relevance to the choice of sedative agents with anti‐muscarinic effects likely to cause cognitive function decline, especially in the elderly. Additionally, the data supports previous studies indicating that use of M3 selective anti‐muscarinic agents for treatment in patients with overactive bladder to improve quality of life create minimal risk of cognitive decline.
A new testing paradigm for evaluating the motor and non‐motor symptoms of 6‐Hydroxydopamine lesions in rats
Authors: Spry Rebecca Marie
Published: November 2023

Parkinson's Disease (PD) is the second most common neurodegenerative disease throughout the United States. The symptoms of PD include impairments in both motor functions, such as rigidity, tremor, and postural instability, as well as non‐motor functions, such as mood and cognitive deficits. The motor symptoms of PD arise due to the selective destruction of nigrostriatal dopamine (DA) synthesis in nerve terminals of the striatum, including within the caudate putamen. Polyunsaturated fatty acids, including omega‐3 fatty acids, have been associated with stimulation of neurotransmitter synthesis and with neuroprotective roles towards limiting neuronal destruction in cell culture and animal models, including in PD. Moreover, polyunsaturated omega‐3 fatty acids have been shown to have beneficial effects on mood and cognition. Because of these findings, there is growing interest in examining the role of polyunsaturated omega‐3 fatty acids in both the motor and non‐motor symptoms of PD. To address this issue, we have generated a testing paradigm for use in Parkinsonian rats that allows for assessments of motor and non‐motor function within the same subject using a series of behavioral tasks. We have examined performance within this paradigm and shown that it closely approximates rat performance reported in the literature. We have begun to establish a rat model of PD using stereotaxic surgeries to directly infuse 6‐Hydroxydopamine (6‐OHDA) into the caudate putamen. In future studies, we will use the combination of our 6‐OHDA lesioning method and novel behavioral paradigm to determine the role of polyunsaturated omega‐3 fatty acids in the motor and non‐motor symptoms of PD.
Methamphetamine augments HIV‐1 Tat mediated memory deficits by altering the expression of synaptic proteins and neutrophic factors
Authors: Nookala Anantha Ram , Kumar Anil
Published: November 2023

Methamphetamine (METH) abuse is common among individuals infected with HIV‐1 and has been shown to affect disease progression towards AIDS. These HIV‐1 infected individuals also exhibit greater neuronal injury and higher cognitive decline. Several HIV‐1 proteins, specifically gp120 and HIV‐1 Tat have been shown to affect neurocognition. HIV‐1 Tat, a viral protein released early during HIV‐1 replication has been implicated in HIV‐associated neurotoxicity mediated through production of pro‐inflammatory cytokines, reactive oxygen species and dysregulation of neuroplasticity, among many others in the central nervous system. However, combined effect of METH and HIV‐1 Tat on neurocognition and its potential effect on neuroplasticity mechanisms is not well documented. The present study was undertaken to determine the combined effect of METH and HIV‐1 Tat on behavior and neuroplasticity markers. Doxycycline (DOX)‐inducible HIV‐1 Tat (1–86) transgenic mice were administered 6mg/kg MA twice a day. Expression of Tat in various brain regions was confirmed by RT‐PCR. Behavior studies were performed in both males and female mice after 3 weeks of MA administration. We employed Y maze and Morris water maze to determine cognition in these mice. Working memory, indicated by number of spontaneous alterations, was decreased significantly in Tat mice that were administered METH. In the water maze task, there was a significant increase in escape latency in all the groups compared to control mice, and the memory deficit was larger in Tat transgenic mice that were administered MA. There is significant decrease in the protein expression of synapsin1, synaptophysin, arg3.1, PSD95 and BDNF in different brain regions. We also looked at the expression levels of a second marker of synaptodendritic integrity, Calmodulin kinase II (CaMKII) by immunoblotting. The results indicate that CaMKII is significantly decreased in HIV‐1 Tat mice that were treated with METH in both sexes. This study therefore provides novel insights into the interaction of HIV‐1 Tat and METH on the dysregulated expression of various plastic proteins in different brain regions.

Support or Funding Information

The work was supported by the grants DA025528, DA025011 and AA020806 from National institute of health (NIH).
Cutting the brakes to prevent Type 2 Diabetes onset: augmenting insulin secretion and conserving pancreatic beta‐cell mass by deleting Gαz
Authors: Wisinski Jaclyn , Reuter Austin , Kimple Michelle
Published: November 2023

The hyperglycemia of Type 2 Diabetes (T2D) results from a combination of factors, including insulin resistance, impaired and inefficient insulin secretion, and destruction of pancreatic beta‐cells. The second messenger molecule, cyclic AMP (cAMP), amplifies glucose‐stimulated insulin secretion and has been suggested to preserve beta‐cell mass. Pathways that inhibit beta‐cell cAMP production are pathologically upregulated in T2D. One such pathway is mediated by Prostaglandin E2 (PGE2) receptor 3 (EP3), a G protein‐coupled receptor whose expression, as well as that of its natural ligand, PGE2, are both up‐regulated in islets collected from T2D mice and humans. While EP3 is ubiquitously expressed, in the beta‐cell it couples to a unique and non‐ubiquitously expressed G protein, Gz. We hypothesized the genetic elimination of the catalytic a‐subunit of Gz (Gαz) would augment beta‐cell function and mass, preventing or delaying the onset of hyperglycemia in the BTBR ob/ob mouse model of T2D. As expected, Gαz‐null ob/ob mice were as obese and as insulin resistant as wild‐type ob/ob mice, consistent with little to no liver, adipose, or muscle Gaz expression, but displayed near‐normal fasting blood glucose levels. Fasting euglycemia in Gaz‐null ob/ob mice correlated directly with elevated plasma insulin levels, suggesting that Gαz‐null ob/ob beta‐cells were able to better compensate for the increased insulin demand of obesity and insulin resistance. To test this directly, we isolated islets from Gaz‐null and wild‐type ob/ob mice, and found higher insulin content and insulin secretion from Gaz‐null ob/ob islets. To further elucidate the molecular mechanisms augmenting beta cell function in Gaz‐null ob/ob islets, we considered the influence of Gaz on glucagon‐like peptide‐1 (GLP‐1), which is a cAMP‐stimulatory gut/islet hormone. We postulated that, in the beta‐cell, Gaz signaling competes with GLP‐1 receptor signling to block the full effects of GLP‐1 on beta‐cell function and/or mass. To test this hypothesis, we subjected wild‐type and Gaz‐null ob/ob mice to intraperitoneal and oral glucose challenge, and found the Gaz‐null mutation partially restored glucose tolerance after an oral glucose challenge but not an intraperitoneal glucose challenge. Plasma levels of the GLP‐1 were not different between the genotypes, but intra‐islet GLP‐1 produced and secreted by alpha cells was altered in Gaz‐null ob/ob islets. Increased GLP‐1 responsiveness of Gaz‐null ob/ob islets was tested directly ex‐vivo by treatment with the stable GLP‐1 analog, exendin‐4. In sum, we identified a key role for Gαz in the islet pathophysiology of T2D and confirmed that deleting Gaz allowed for a more efficient response of the beta‐cell to a physiologic potentiator of insulin secretion and beta‐cell mass, GLP‐1.

Support or Funding Information

Molecular and Applied Nutrition Training Program T32
Targeting and Delivery of Heterotrimeric G‐proteins to Primary Cilia
Authors: Edwards Clare , Lambert Nevin
Published: November 2023

Primary cilia are fingerlike extensions of the plasma membrane important in cellular signaling and implicated in many disease processes. While much is known about localization and targeting of G‐protein coupled receptors (GPCRs) to cilia, little has been elucidated about the localization and targeting of heterotrimeric G‐proteins that reside downstream. This study aimed to determine if G‐proteins are targeted to the primary ciliary compartment and whether or not they possess the ability to move from the extaciliary membrane into this regulated space. Utilizing confocal microscopy, we imaged Gαi1‐Venus and SSTR3‐mTurq2 expression (a ciliary marker protein) in the primary cilia of NIH3t3 cells and quantified this expression as mean fluorescence in Imagej. Ciliary and membrane fluorescence were compared, and Fluorescence Recovery After Photo bleaching (FRAP) was completed to monitor movement of Gαi1‐V and SSTR3‐mTurq2. A student t‐test revealed that mean fluorescence of Gαi1‐V in the ciliary compartment (n=10, M=77.1, SD=45.8) is not significantly greater than extraciliary membrane fluorescence (n=10, M=75.4, SD=43.2); t(18)=0.08, p<0.05. Additionally, the difference between ciliary Gαi1‐V (n=10, M=77.1, SD=45.8) and membrane Gαi1‐V expression (n=10, M=75.4, SD=43.2) is significantly lower than the difference between expression of SSTR3‐mturq2 in the cilia (n=10, M=189.8, SD=71.5) and on the membrane (n=10, M=48.5, SD=30.1); t(18)=5.31, p<0.05. FRAP analysis of Gαi1‐V (n=6) returned a half recovery time of 12.8 ± 8.24 seconds and a mean mobile fraction of 0.60 ± 0.10, significantly higher than the mobile fraction of SSTR3‐mTurq2 (n=2, MF of 0.04 ± 0.014, t1/2 of 21.6 ± 15.3 seconds); t(6)=4.51, p<0.05. Our data indicate that Gαi1‐V expression is not significantly elevated in the primary cilia, and targeting of heterotrimeric G‐proteins to this space is unlikely. However, there is no evidence of exclusion of Gαi1‐V from the ciliary compartment as expression is visible and able to recover from photo bleaching in a time comparable to diffusion.

Support or Funding Information

Medical Scholars Program at Medical College of Georgia.
Quantitative Tissue Distribution and Pharmacokinetics of IW‐1973, a Novel Clinical‐Stage sGC Stimulator, After Oral Administration in Rats
Authors: Banijamali Ali , Wakefield James , Barden Tim , Carvalho Andrew , Zimmer Daniel R , Currie Mark , Hall Katherine , Solon Eric , Masferrer Jaime , Ribadeneira Maria
Published: November 2023

Soluble guanylate cyclase (sGC) catalyzes the conversion of guanosine‐5′‐triphosphate (GTP) to the secondary messenger cyclic guanosine‐3′,5′‐monophosphate (cGMP) upon binding of nitric oxide (NO). IW‐1973 is a sGC stimulator in clinical development that binds to sGC and enhances NO signaling resulting in an increase in cGMP production. Oral administration of IW‐1973 has been associated with elevations of cGMP‐sGC signaling in many different tissues including liver. Preclinical and clinical studies have shown that sGC stimulators relax smooth muscle, lower blood pressure, and have anti‐fibrotic effects. sGC stimulators with high distribution to tissues may have particular relevance for the treatment of vascular and fibrotic diseases of the lung, heart, liver, and kidney.

The tissue distribution and pharmacokinetics (PK) of total radioactivity in male Long‐Evans rats was evaluated by quantitative whole‐body autoradiography (qWBA) following administration of a single oral (PO) dose of 3 mg/kg and 200 μCi/kg [14C]IW‐1973. Tmax was 1 h post‐dose and the t1/2 of total plasma radioactivity was 23.7 h. Analysis of tissues at various timepoints post‐dose revealed rapid and widespread distribution of [14C]IW‐1973‐derived radioactivity. Tissue concentrations in 19 of 44 tissues were observed to be higher than in plasma at all timepoints. Of particular interest were the high tissue to plasma AUCall ratios observed for liver (172), white adipose (28), brown adipose (15), kidney (5–7), heart (5), and lung (3); and the distinctly lower ratios observed in the bone (0.4) and pigmented eye lens (0.1). The preferential tissue distribution of IW‐1973 to organs such as liver and kidney may be a favorable property to treat not only vascular diseases but also those associated with extensive tissue inflammation and fibrosis.

Support or Funding Information

Ironwood Pharmaceuticals
atRA upregulates SHP expression through RXR activation
Authors: Won Kyoung‐Jae , Jeong Hyunyoung
Published: November 2023

Retinoids (vitamin A) are used therapeutically as an anticancer agent or to treat dermatological diseases. Common side effects of retinoid include hyperlipidemia. Previous studies have shown that all‐trans retinoic acid (atRA; the bioactive retinoid) upregulates small heterodimer partner (SHP; a transcriptional repressor) and downregulates CYP7A1 (the rate‐limiting enzyme converting cholesterol to a bile acid intermediate), potentially responsible for the side effects. However, the detailed molecular mechanisms remain unclear. In this study, we investigated the role of retinoic acid receptor (RAR; the cognate nuclear receptor for atRA) and its binding partner, retinoid X receptor (RXR), in the transcriptional regulation of SHP and CYP7A1. In primary human hepatocytes and HepaRG cells, atRA increased SHP mRNA level by 12.8‐ and 6.6‐fold, respectively, while dramatically decreasing CYP7A1 mRNA level. In these cells, atRA also increased the levels of SHP pre‐mRNA, suggesting transcriptional activation of SHP by atRA. mRNA half‐life of SHP (determined after actinomycin D treatment in HepaRG cells and human hepatocytes) was not altered by atRA. In HepaRG cells, treatment with an RAR isoform‐specific agonist (i.e., BMS753, CD2314, and BMS961 for RARα, RARβ and RARγ, respectively) caused minimal changes in SHP expression. On the other hand, treatment with an RXR agonist (SR11237) led to increased SHP expression to an extent comparable to that by atRA (3.9‐ and 4.3‐fold, respectively), suggesting that SHP upregulation by atRA is mediated by RXR rather than RAR. mRNA expression of target genes of other permissive nuclear receptors that partner with RXR (i.e., CYP3A4, CYP2B6, and BSEP) was also increased in the cells treated by the RXR agonist to an extent similar to that by atRA. SHP knockdown led to significant de‐repression of CYP7A1 expression in atRA‐treated human hepatocytes (by 80% at 3 h), suggesting a key role of SHP in downregulation of CYP7A1 by atRA. Taken together, results from this study suggest an important role of RXR (rather than RAR) in mediating atRA action on the regulation of hepatic SHP and CYP7A1 expression, as well as expression of other target genes of permissive nuclear receptors that partner with RXR.

Support or Funding Information

This study was supported by NIH (R01 GM112746).
Impact on Drug Responses in Adult Life by Drug Treatment at the Neonatal Age
Authors: Piekos Stephanie , Chen Liming , Zhong Xiao‐bo
Published: November 2023

The cytochrome P450 enzymes (P450s) are responsible for the metabolism of 75% of drugs currently on the market. However, significant interindividual variations in the enzyme activities of several P450s, including CYP3A4, CYP2B6, CYP2C9, and CYP2C19 exist in the general population. The differences in drug metabolism rates between people alter plasma drug exposure, and may account for the high incidences of adverse drug reactions or low therapeutic efficacy, which are difficult to predict. Identification of factors contributing to these variations is critical to develop strategies to better predict drug response. Several studies in animal models over the past 30 years have proven that adult basal P450 expression is permanently induced following neonatal exposure to the antiepileptic drug phenobarbital. Recently, our own study demonstrated that neonatal treatment with phenobarbital resulted in decreased therapeutic efficacy of omeprazole in proton‐pump inhibition to raise gastric pH in stomachs of adult mice. This decreased efficacy of omeprazole correlated with an upregulation of the P450s that mediate omeprazole metabolism, CYP3A11 and CYP2C29, in the liver of adult mice that received treatment with phenobarbital at the neonatal age. There is now a critical need to investigate whether neonatal treatment with different drugs produces the same permanent variations in P450 expression and metabolism of other drugs administered later in life. We hypothesize that phenytoin, another antiepileptic drug that induces P450s in mice and humans, will also cause permanent overexpression of P450 genes following neonatal exposure, which will affect drug responses in adulthood. Our current ongoing study involves administering phenytoin to neonatal mice, then evaluating expression of CYP3A11, CYP2B10, and CYP2C29, and the responses to omeprazole treatment in adulthood. Gastric pH is measured to evaluate the therapeutic efficacy of omeprazole, livers are harvested for analysis of P450 expression and activity, and blood is collected to measure plasma concentration of omeprazole and its metabolites. If phenytoin produces the same permanent upregulation of several P450s following neonatal exposure with a correlating decrease in omeprazole efficacy, then this phenomenon is not limited to phenobarbital and may be a common consequence of neonatal anti‐epileptic drug treatment. Neonates and infants are commonly administered drugs to treat various indications, such as seizures, and many of these compounds can induce P450s. This work may help prove that induction of P450s due to drug administration in early life can leave lasting alterations in the efficacy of other drugs metabolized by the P450s in later life. To better predict how patients will respond to a drug, a patient's drug history may serve as a factor for predicting variations of P450‐mediated drug metabolism.

Support or Funding Information

NIH/NIGMS 1R01GM118367
In vitro metabolite profiling of sildenafil based on metabolomics approach
Authors: Kim Sunjoo , Jo Jun Hyun , Kwon Oh Kwang , Lee Sangkyu
Published: November 2023

Metabolomics combined with high‐resolution mass spectrometry (HR‐MS) and multivariate data analysis has broad applications in the study of xenobiotic metabolism. Although xenobiotic metabolism is essential to understand toxicity, pharmacokinetic parameters, and excretion pathways, it cannot predict all generated metabolites in biological fluids. In the present study, we used a metabolomics approach to further understand sildenafil phase 1 metabolism in human liver microsomes. Twelve phase 1 metabolites were identified in human liver microsomes, of which, M1–M5 were previously known. The chemical structures of novel metabolites were elucidated by MS2 fragmentation using HR‐MS as follows: M6, reduced sildenafil, M7, N,N‐deethylation and mono‐oxidation; M8, demethanamine, N,N‐deethylation and mono‐hydroxylation; M9, demethanamine and N,N‐deethylation; M10 and M11, mono‐oxidation in the piperazine ring after N‐demethylation; and M12, mono‐oxidation. All metabolites except M1 were formed by CYP3A4 and 3A5. In conclusion, we successfully updated the metabolic pathway of sildenafil in the human liver, including seven novel metabolites, using metabolomics combined with HR‐MS and multivariate data analysis.

Support or Funding Information

BK21 Plus KNU Multi‐Omics based Creative Drug Research Team
LXRa Inhibits Autophagy in Hepatocytes through ATG4B Inhibition
Authors: Kim Yun Seok , Nam Hyeon Joo , Kim Sang Geon
Published: November 2023

Liver steatosis is the most common feature of metabolic syndrome, and may progress to severe disease states. LXRα promotes SREBP‐1c‐dependent lipogenesis and fat accumulation in hepatocytes. However, the effect of LXRα on autophagy in hepatocytes and the consequent changes in energy homeostasis are elusive. This study investigated the role of LXRα modulations in autophagy in association with its impact on the progression of liver steatosis. Treatment of HepG2 cells or primary hepatocytes with T0901317 (an LXRα agonist) decreased the processing of LC3‐I to LC3‐II in a time‐ or concentration‐dependent manner. The effect of LXRα on lysosomal activity from its effect on autophagosome formation was verified using immunofluorescence confocal assays. In primary hepatocytes, LXRα agonist treatment visualized decrease of autophagic flux. Consistently, either ligand activation LXRα or LXRα overexpression decreased Atg4B levels both in vitro and in vivo. Our results provide evidence that LXRα activation inhibits autophagy in hepatocytes as mediated by the inhibition of Atg4B, implying that modulation of LXRα may affect the capacity of autophagy in hepatocytes and thereby contribute to the prevention and treatment of liver steatosis.

Support or Funding Information

Supported by the Korean government (NRF‐2015R1A2A1A10052663)
cAMP‐Phosphodiesterase PDE4D as a Target for Colon Cancer Therapy
Authors: Boyd Abigail , Baskar Ganga , Petty Tracy , Keeton Adam , Piazza Gary , Richter Wito
Published: November 2023

Type 4 cyclic nucleotide phosphodiesterases (PDE4s) are part of a superfamily of isoenzymes that hydrolyze and inactivate the second messenger cAMP. Recent studies have shown that treatment with non‐selective PDE4 inhibitors impairs cancer cell proliferation and tumor growth, cell migration, epithelial to mesenchymal transition and tumor angiogenesis in various types of cancers; suggesting that PDE4s represent a potential target for the development of novel cancer therapeutics. However, non‐selective PDE4 inhibitors are known to induce a number of side effects, including nausea and emesis, diarrhea and weight loss that limit their clinical utility. The PDE4 family comprises four subtypes/genes, PDE4A to D. As each plays unique and non‐overlapping physiological and pathophysiological roles in the body, targeting individual PDE4 subtypes may serve to dissect the therapeutic benefits from the side effects of non‐selective PDE4 inhibitors. To this end, we have explored the role of PDE4 and individual PDE4 subtypes in five human colon cancer cell lines. Treatment with the archetypal non‐selective PDE4 inhibitor Rolipram ablated the chief portion of total cAMP hydrolytic activity in cell extracts prepared from all five colon cancer cell lines, suggesting that PDE4s are the main cAMP‐PDE expressed in these cells. Treatment with non‐selective PDE4 inhibitors such as Rolipram significantly reduced the viability of colon cancer cells in the presence of low doses of the adenylyl cyclase activator forskolin; confirming the potential of PDE4 as a therapeutic target. In addition, treatment with Rolipram attenuated cancer cell migration assessed in wound scratch assays. Western blotting and immunoprecipitation with PDE4 subtype‐selective antibodies revealed that PDE4 activity and protein in colon cancer cells is largely due to PDE4D, with little to none of the other PDE4 subtypes (PDE4A‐C) being expressed. If colon cancer cells exclusively express PDE4D, then the effect of non‐selective PDE4 inhibitors on cell viability/motility must necessarily have been mediated via inhibition of PDE4D, thus identifying PDE4D as the preferred target for development of a therapeutic. We will be testing this hypothesis by assessing the effect of selective PDE4D inactivation on various cancer cell functions. The ultimate goal is to determine whether selective inhibition of PDE4D retains the cancer therapeutic benefits, but exhibits an improved safety profile compared to the non‐selective PDE4 inhibitors available to date.