Potential Drug Therapies Research Articles

Machine learning identifies key cells and therapeutic targets during ferroptosis after spinal cord injury.

Ferroptosis, a type of cell death that mainly involves iron metabolism imbalance and lipid peroxidation, is strongly correlated with the phagocytic response caused by bleeding after spinal cord injury. Thus, in this study, bulk RNA sequencing data (GSE47681 and GSE5296) and single-cell RNA sequencing data (GSE162610) were acquired from gene expression databases. We then conducted differential analysis and immune infiltration analysis. Atf3 and Piezo1 were identified as key ferroptosis genes through random forest and least absolute shrinkage and selection operator algorithms. Further analysis of single-cell RNA sequencing data revealed a close relationship between ferroptosis and cell types such as macrophages/microglia and their intrinsic state transition processes. Differences in transcription factor regulation and intercellular communication networks were found in ferroptosis-related cells, confirming the high expression of Atf3 and Piezo1 in these cells. Molecular docking analysis confirmed that the proteins encoded by these genes can bind cycloheximide. In a mouse model of T8 spinal cord injury, low-dose cycloheximide treatment was found to improve neurological function, decrease levels of the pro-inflammatory cytokine inducible nitric oxide synthase, and increase levels of the anti-inflammatory cytokine arginase 1. Correspondingly, the expression of the ferroptosis-related gene Gpx4 increased in macrophages/microglia, while the expression of Acsl4 decreased. Our findings reveal the important role of ferroptosis in the treatment of spinal cord injury, identify the key cell types and genes involved in ferroptosis after spinal cord injury, and validate the efficacy of potential drug therapies, pointing to new directions in the treatment of spinal cord injury.

The StuA Transcription Factor and Alternative Splicing Mechanisms Drive the Levels of MAPK Hog1 Transcripts in the Dermatophyte Trichophyton rubrum.

Trichophyton rubrum is a human fungal pathogen that causes dermatophytosis, an infection that affects keratinized tissues. Integrated molecular signals coordinate mechanisms that control pathogenicity. Transcriptional regulation is a core regulation of relevant fungal processes. Previous RNA sequencing data revealed that the absence of the transcription factor StuA resulted in the differential expression of the MAPK-related high glycerol osmolarity gene (hog1) in T. rubrum. Here we validated the role of StuA in regulating the transcript levels of hog1. We showed through RT-qPCR that transcriptional regulation controls hog1 levels in response to glucose, keratin, and co-culture with human keratinocytes. In addition, we also detected hog1 pre-mRNA transcripts that underwent alternative splicing, presenting intron retention in a StuA-dependent mechanism. Our findings suggest that StuA and alternative splicing simultaneously, but not dependently, coordinate hog1 transcript levels in T. rubrum. As a means of preventing and treating dermatophytosis, our results contribute to the search for new potential drug therapies based on the molecular aspects of signaling pathways in T. rubrum.

Incomplete reverse remodeling in aged mice exposed to pulmonary hypertension-induced right ventricular failure

Right ventricle (RV) function is the strongest predictor of survival in pulmonary hypertension (PH) and age-related HF; however no therapies exist to improve RV function. Understanding mechanisms by which the RV undoes pathological remodeling (reverse remodeling) might aid in identifying therapies for this unmet need. Our objective was to determine whether the aged RV can undergo reverse remodeling following pathological afterload (PH). We hypothesized that return to normoxia after hypoxia-induced PH would result in attenuation of RV mass and improved RV function. We exposed male and female aged (~18 months) C57Bl6 mice to hypobaric hypoxia (HH) for 4 weeks before returning them to normoxia for 3 (WK3RR) or 6 (WK6RR) weeks. HH induced RV hypertrophy by RV myocyte area which was attenuated at Wk3RR and WK6RR in both male (Con: 425 μm2 ± 104, HH: 740 μm2 ± 125, WK3RR: 447 μm2 ± 129, WK6RR: 530 μm2 ± 187) and female mice (Con: 340 μm2 ± 116, HH: 770 μm2 ± 35, WK3RR: 516 μm2 ± 186, WK6RR: 655 μm2 ± 224). HH stimulated fibrosis by collagen deposition that was also reversed during normoxia re-exposure in male mice only (Con: 4.1% ± 1.0, HH: 10.8% ± 0.8, WK3RR: 4.2% ± 0.8, WK6RR: 2.5% ± 0.7). However, HH decreased RV systolic function by fractional area change (FAC) that was also not rescued by normoxia re-exposure. The aged RV can undergo morphological reverse remodeling in response to HH; however RV function does not improve with resolution of afterload. Further investigation into the mechanisms of reverse remodeling may identify potential drug therapies for maladaptive RV remodeling. AHA-Predoctoral Fellowship 897622 (BDM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Not just for lymphoid cells: the role of the noncanonical NF-κB signaling pathway in early and late myelopoiesis with a focus on hypereosinophilic disorders.

The noncanonical NF-κB pathway is involved in lymphoid organ development, B-cell maturation, and cytokine production. However, new research has demonstrated that this pathway is also key for the orderly and sequential maturation of myeloid cells, including neutrophils and eosinophils. When this pathway is disrupted or constitutively activated, aberrations in hematopoietic stem and progenitor cell survival and proliferation, as well as subsequent granulopoiesis and eosinophilopoiesis, are affected. Disturbance of such a coordinated and delicate process can manifest in devastating clinical disease, including acute and chronic myeloid leukemias, preleukemic processes such as myelodysplastic syndrome, or hyperinflammatory conditions like hypereosinophilic syndrome. In this review, we discuss the molecular machinery within the noncanonical NF-κB pathway, crosstalk with the canonical NF-κB pathway, murine models of noncanonical signaling, and how aberrations in this pathway manifest in leukemic or hyperinflammatory disease with a focus on hypereosinophilic syndrome. Potential and promising drug therapies will also be discussed, emphasizing the noncanonical NF-κB pathway as a potential target for improved treatment for patients with leukemia or idiopathic hypereosinophilic syndrome. The hope is that review of such mechanisms and treatments may eventually result in findings that aid physicians in rapidly diagnosing and more accurately classifying patients with such complex and overlapping hematopoietic diseases.

A murine model of DC-SIGN humanization exhibits increased susceptibility against SARS-CoV-2

To generate a new murine model for virus, DC-SIGN gene in murine was humanized. In this study, we successfully generated a humanized C57BL/6N mouse model expressing human DC-SIGN (hDC-SIGN) using CRISPR/Cas9 technology, and evaluated its characters and susceptibility to virus. The humanized mice could survival as usual, and with normal physiological index just like the wild-type mice. Whereas, we found significant differences in the intestinal flora and metabolic profiles between wild-type mice and humanized mice. Following intranasal infection with SARS-CoV-2, hDC-SIGN mice exhibited significantly increased viral loads in the lungs and nasal turbinates, along with more severe lung damage. This phenomenon may be associated with differential lipid metabolism and Fcγ receptor-mediated phagocytosis in two mouse models. This study provides a useful tool for investigating the mechanisms of coronavirus infection and potential drug therapies against novel coronavirus.

Retraction Note: Functional modular networks identify the pivotal genes associated with morphine addiction and potential drug therapies

Retraction Note: Functional modular networks identify the pivotal genes associated with morphine addiction and potential drug therapies

Morphologic characteristics of painful varicose veins in women: possible role of disordered collagen and endothelial cells

ObjectiveThis study examines to what extent vein wall thickness, collagen arrangements, muscular layers distribution, immunohistochemical presentation of endothelial cell arrangement, and adventitia layer placement differed in patients with small painful veins vs large painful symptomatic varicose veins (VVs). MethodsThis study was conducted from June 2022 to September 2022 at Valley Vein Health Center, a rural phlebology outpatient clinic. Ten samples from each of the three subjects were collected (n = 3): five were small symptomatic veins, and five were large symptomatic veins. All tissue blocks were cut transversely, perpendicular to the vessel axis, into 5-μm-thick sections. Three stains were chosen: Masson trichrome, hematoxylin and eosin, and a cluster of differentiation 31 (CD31). Statistical analysis was performed with the GraphPad statistical program. Comparisons between vein wall thicknesses were made using Student’s t test. ResultsThe average thickness of small veins was less than that of large veins (426 μm ± 26.1 μm vs 480 μm ± 19.2 μm, respectively; P < .001). Histologic and immunohistochemical (CD31) analysis of small symptomatic VVs by hematoxylin and eosin and trichrome stain showed an endothelial layer overlying the media, mostly of elastic tissue fibers, and smooth muscle bundles. CD31 expression analysis demonstrated more endothelial channels in the tunica media of the enlarged veins compared with smaller vessels. The larger vein’s microscopic structure was variable with irregular collagen arrangement, clumped elastin layer appearance, feathered muscular layer distribution, and thickened adventitia layer placement. ConclusionsThe morphological distinctions in VVs highlighted in this study need to be considered to develop potential drug therapies tailored to women.

OR11-05 High-throughput Screening Identifies TR4 Modulators For Potential Therapeutic Application

Abstract Disclosure: S. Khowal: None. D. Zhang: None. R. Damoiseaux: None. K. Javaherian: None. A.P. Heaney: None. Cushing Disease (CD), due to an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor, leads to excess cortisol and is a life-threatening “orphan disease”. Although surgical removal can be effective, CD frequently recurs. Thereafter, repeat surgery, radiation therapy and/or bilateral adrenalectomy are not always successful and cause significant morbidity. Medical therapy has significantly improved but there is a clear unmet need for efficacious and safe therapies that target the pituitary corticotroph tumor itself to offer biochemical and tumor control. We previously demonstrated that the orphan testicular receptor 4 (TR4) regulates pro-opiomelanocortin gene transcription and ACTH secretion. Herein, we used the Flexi Vector system to identify TR4 modulators by fusing the TR4 LBD (380-615aa) to a 3’ GAL4 DBD to generate a chimeric fusion plasmid, pBIND-TR4-LBD. The pBIND-TR4-LBD construct and an exogenous luciferase reporter construct pGL4.35 (luc2P/9XGAL4UAS), containing 9 copies of a GAL4 responsive upstream activating sequence (UAS), were then transiently transfected into human embryonic kidney (HEK293) cells. Following treatment with TR4 ligands or modulators, our TR4-LBD undergoes a conformational change and recruits additional cofactors and RNA polymerase II to the complex to drive GAL4-directed firefly luciferase expression. In a high throughput screen (HTS) of 27,705 chemicals, we identified several potential “hit” TR4 modulators that induced or inhibited TR4 LBD-directed GAL4UAS-directed luciferase expression by &amp;gt;3 -fold. Compounds from the HTS included ATP-sensitive K+ and calcium channel blockers, steroid hormones and modulators of cell proliferation and inflammation and several agonist and antagonist “hit” compounds induced TR4 &amp;gt;10 fold or inhibited TR4 by 90% respectively with minimal cell toxicity (&amp;lt;10%). In silico analysis of interactions between the TR4 LBD and these potential ligands indicated that the GLU-534 &amp; ARG-565 may be crucial residues involved in TR4 ligand binding. Dose-response curves to calculate EC50 of these potential TR4 hit compounds and mode-of-action characterization are in progress. Our studies have identified several potent TR4 agonistic and antagonistic compounds and lay the framework for the discovery of safe and efficacious lead TR4 ligands that can be further advanced as potential drug therapies for CD and other diseases. Presentation: Friday, June 16, 2023

Addition of Neostigmine and Atropine to Conventional Management of Postdural Puncture Headache after Cesarean Section: A Randomized Controlled Study

Abstract Background Post dural puncture headache (PDPH) is a known side effect following spinal anesthesia occurring in 2.5-40% of patients, it typically starts after 2 days but may be delayed up to 2 weeks and almost resolves spontaneously within a few days. Classic symptoms of PDPH consist of photophobia, nausea, vomiting, neck stiffness, tinnitus, diplopia and dizziness. Objective The aim of the study is to know if the neostigmine and atropine can improve PDPH treatment when added to conventional management after cesarean sections. Patients and Methods After obtaining approval from research ethics committee of Faculty of Medicine, Ain Shams University and after obtaining informed written consents from the patients. The study was conducted in the operating theaters of Ain Shams University Hospitals. The study period was 6 months after approval of the protocol, computer generated simple randomization. Patients with PDPH and a VAS &amp;lt; 5 were randomly allocated to receive either: Group 1: Slow intravenous injection with 20 µg/Kg neostigmine and 10 µg/Kg atropine in 20 ml 0.9% Saline given over 5 minutes every 8 hours up to 72 hours, Group 2: 20 ml of 0.9% saline intravenous every 8 hours. Results In this study, we compared the efficacy of neostigmine versus placebo (Both groups received the conventional management) in treating the PDPH, All of the 2 groups (neostigmine and Placebo) were effective in lowering VAS scores at 24, 48 and 72 h after headache's onset but neostigmine alleviated the pain more effectively and with rapid onset. The mean VAS score was significantly lower in neostigmine group compared with other group 6, 12, 24, 36, 48 and 72 h after start of medications (p &amp;lt; 0.001 for all of them) Placebo group 6, 12, 24, 36, 48, and 72 h (p &amp;lt; 0.001 for all of them). This may cause the drug to be one of the potential new drug therapies for such distressing condition, especially with its different mechanism of action that differs from other drugs. Conclusion Neostigmine/atropine was effective in treating PDPH after only 2 doses. Neostigmine can pass the choroid plexus but not the blood–brain barrier. The central effects of both drugs influence both cerebrospinal fluid secretion and cerebral vascular tone, which are the primary pathophysiological changes in PDPH. The results are consistent with previous studies and clinical reports of neostigmine activity.

Abstract P1118: Engineering A Patient-specific Heart On A Chip Model Of Hypoplastic Left Heart Syndrome

Hypoplastic Left Heart Syndrome (HLHS) is the most expensive congenital cardiac disease and is characterized by underdevelopment of the left side of the heart. The surgical intervention leads to the developmentally disadvantaged right ventricle performing systemic circulation. As such, a subset of HLHS patients develops progressive heart failure that results in limited transplant-free survival. There are no animal models to study the molecular and functional properties of HLHS cardiomyocytes, limiting our understanding of disease mechanisms and human genotype-phenotype relationships. To address this, we are creating a physiologically and functionally relevant ”heart on a chip” model of HLHS hiPSC-derived cardiomyocytes (CMs). Induced Pluripotent Stem Cells (iPSCs) were derived from cord blood mononuclear cells isolated from the umbilical cord blood from 8 antenatally diagnosed HLHS children at birth. Control (iPSCs) from 3 healthy subjects were derived from peripheral blood mononuclear cells. We then differentiated ventricular CMs from these iPSCs using StemDiff ventricular CM kit. We observed HLHS iPSCs formed ~50% fewer CMs compared to healthy controls. We studied calcium handling by iPSC-CMs with Fluo-4 fluorescent calcium dye in HLHS and Healthy iPSC-CMs. We observed that calcium transients in HLHS-CMs have half the beat rate, 1.4 times longer rise time to rise, 1.5 times higher time constant (Tau), compared to Healthy CMs, suggesting that HLHS CMs have dysfunctional calcium handling compared to healthy CMs. Our results demonstrate that in an in vitro model of iPSC-derived CM, HLHS patient CMs demonstrate reduced formation and defective calcium handling, which may potentially explain the pathogenesis of heart failure in HLHS patients. In ongoing experiments, we are evaluating diastolic and systolic stress generation in these cells. We will also establish the underlying molecular mechanisms of functional defects in HLHS CMs and screen potential drug therapies for single-ventricle patients.

Comparative Roles of IL-1, IL-6, IL-10, IL-17, IL-18, 1L-22, IL-33, and IL-37 in Various Cardiovascular Diseases With Potential Insights for Targeted Immunotherapy.

In recent years, the study of interleukins (ILs), crucial cytokines involved in immune response and inflammation, has garnered significant attention within the sphere of cardiovascular diseases (CVDs). The research has provided insights into the involvement of ILs in diverse CVDs, including arrhythmias, myocardial infarction, atherosclerosis, and heart failure (HF). ILs have emerged as promising therapeutic targets for drug interventions through their involvement in disease development and progression. This comprehensive review provides a detailed overview of ILs, elucidating their functions within the immune system and offering insights into their specific contributions to various CVDs. Moreover, the article delves into the examination of current and potential drug therapies that selectively target ILs in the management of CVDs, presenting a comprehensive analysis of the advantages and disadvantages associated with these therapeutic approaches. A comprehensive literature review was conducted to investigate the involvement of ILs in CVDs. The relevant articles were searched on PubMed, PubMed Central, Medline, Cochrane, Google Scholar, and ScienceDirect databases. The search encompassed articles published from these databases' inception until July 12, 2023. We first examine generalized aspects of ILs, particularly CVDs. Then, we shift focus towards examining the direct impact of ILs on cardiac cells and tissue; on the immune system and inflammation; endothelial cells and vascular function; and finally, their interactions with other signaling pathways and molecules. Then, we discuss the molecular mechanisms of various ILs. Sequentially, we delve into a comprehensive analysis of the individualized role of each distinct IL in diverse CVDs, examining their specific contributions. Finally, we explore the potential for targeted drug therapy to modulate IL activity, aiming to enhance outcomes for patients burdened with CVD. The objective is the identification of gaps in current knowledge and highlight areas that require further investigation within the context of cardiovascular medicine. Through deepening our comprehension of the intricate involvement of ILs in CVDs and harnessing their potential for targeted drug therapy, novel treatment strategies can be devised, leading to improved patient outcomes in cardiovascular medicine.

The significance of PD-1/PD-L1 imbalance in ulcerative colitis.

To investigate the expression and significance of programmed cell death protein 1 (PD-1) and programmed cell death ligand-1 (PD-L1) in the mucosal tissues and peripheral blood of patients with ulcerative colitis (UC). Eighty patients with UC were recruited from January 2021 to August 2022 from the Shanxi Province People's Hospital. PD-1 and PD-L1 expression was assessed by immunohistochemistry in mucosal tissues. An enzyme-linked immunosorbent assay was used to measure soluble PD-1 and PD-L1 levels in peripheral blood serum, and the membrane-bound forms of PD-1 (mPD-1), (T-helper cell) Th1 and Th17, in peripheral blood were determined by flow cytometry. PD-1 expression was observed only in the monocytes of the mucosal lamina propria of UC patients, while PD-L1 was mainly located in both epithelial cells and monocytes on the cell membrane. The expression level of PD-1/PD-L1 in the monocytes and epithelial cells of mucosal lamina propria increased with disease activity (P < 0.05). The percentages of PD-1/T and PD-1/CD4+T in the peripheral blood of moderate UC patients (PD-1/T 12.83 ± 6.15% and PD-1/CD4+T 19.67 ± 9.95%) and severe UC patients (PD-1/T 14.29 ± 5.71% and PD-1/CD4+T 21.63 ± 11.44%) were higher than in mild UC patients (PD-1/T 8.17 ± 2.80% and PD-1/CD4+T 12.44 ± 4.73%; P < 0.05). There were no significant differences in PD-1/CD8+T cells between mild and severe UC patients (P > 0.05). There was a statistically significant difference in the expression level of sPD-L1 between the UC groups and healthy controls, and the expression level of sPD-L1 increased with disease severity (P < 0.05); however, there was no statistically significant difference in sPD-1 expression levels between the UC groups and healthy controls (P > 0.05). The correlation coefficients between Th1 and sPD-L1, PD-1/T, PD-1/CD4+T and PD-1/CD8+T were 0.427, 0.589, 0.486, and 0.329, respectively (P < 0.001). The correlation coefficients between Th17 and sPD-L1, PD-1/T, PD-1/CD4+T and PD-1/CD8+T were 0.323, 0.452, 0.320, and 0.250, respectively (P < 0.05). The expression level of PD-1/PD-L1 was correlated with UC disease activity, and two forms of PD-1 and PD-L1 may be used as a potential marker for predicting UC and assessing disease progression in UC patients. PD-1/PD-L1 imbalance was a significant phenomenon of UC immune dysfunction. Future research should focus on two forms of PD-1/PD-L1 signaling molecules to better understand the pathogenesis of UC and to identify potential drug therapies.

RETRACTED ARTICLE: Functional modular networks identify the pivotal genes associated with morphine addiction and potential drug therapies

Background Chronic morphine usage induces lasting molecular and microcellular adaptations in distinct brain areas, resulting in addiction-related behavioural abnormalities, drug-seeking, and relapse. Nonetheless, the mechanisms of action of the genes responsible for morphine addiction have not been exhaustively studied.MethodsWe obtained morphine addiction-related datasets from the Gene Expression Omnibus (GEO) database and screened for Differentially Expressed Genes (DEGs). Weighted Gene Co-expression Network Analysis (WGCNA) functional modularity constructs were analyzed for genes associated with clinical traits. Venn diagrams were filtered for intersecting common DEGs (CDEGs). Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for functional annotation. Protein–protein interaction network (PPI) and CytoHubba were used to screen for hub genes. Potential treatments for morphine addiction were figured out with the help of an online database.ResultsSixty-five common differential genes linked to morphine addiction were identified, and functional enrichment analysis showed that they were primarily involved in ion channel activity, protein transport, the oxytocin signalling pathway, neuroactive ligand-receptor interactions, and other signalling pathways. Based on the PPI network, ten hub genes (CHN2, OLIG2, UGT8A, CACNB2, TIMP3, FKBP5, ZBTB16, TSC22D3, ISL1, and SLC2A1) were checked. In the data set GSE7762, all of the Area Under Curve (AUC) values for the hub gene Receiver Operating Characteristic (ROC) curves were greater than 0.8. We also used the DGIdb database to look for eight small-molecule drugs that might be useful for treating morphine addiction.ConclusionsThe hub genes are crucial genes associated with morphine addiction in the mouse striatum. The oxytocin signalling pathway may play a vital role in developing morphine addiction.

Exploiting embryonic niche conditions to grow Wilms tumor blastema in culture.

Wilms Tumor (WT), or nephroblastoma, is the most common pediatric kidney cancer. Most WTs display a "favorable" triphasic histology, in which the tumor is comprised of blastemal, stromal, and epithelial cell types. Blastemal predominance after neoadjuvant chemotherapy or diffuse anaplasia ("unfavorable" histology; 5-8%) portend a worse prognosis. Blastema likely provide the putative cancer stem cells (CSCs), which retain molecular and histologic features characteristic of nephron progenitor cells (NPCs), within WTs. NPCs arise in the metanephric mesenchyme (MM) and populate the cap mesenchyme (CM) in the developing kidney. WT blastemal cells, like NPCs, similarly express markers, SIX2 and CITED1. Tumor xenotransplantation is currently the only dependable method to propagate tumor tissue for research or therapeutic screening, since efforts to culture tumors in vitro as monolayers have invariably failed. Therefore, a critical need exists to propagate WT stem cells rapidly and efficiently for high-throughput, real-time drug screening. Previously, our lab developed niche conditions that support the propagation of murine NPCs in culture. Applying similar conditions to WTs, we assessed our ability to maintain key NPC "stemness" markers, SIX2, NCAM, and YAP1, and CSC marker ALDHI in cells from five distinct untreated patient tumors. Accordingly, our culture conditions maintained the expression of these markers in cultured WT cells through multiple passages of rapidly dividing cells. These findings suggest that our culture conditions sustain the WT blastemal population, as previously shown for normal NPCs. As a result, we have developed new WT cell lines and a multi-passage in vitro model for studying the blastemal lineage/CSCs in WTs. Furthermore, this system supports growth of heterogeneous WT cells, upon which potential drug therapies could be tested for efficacy and resistance.

Towards Better Sinomenine-Type Drugs to Treat Rheumatoid Arthritis: Molecular Mechanisms and Structural Modification.

Sinomenine is the main component of the vine Sinomenium acutum. It was first isolated in the early 1920s and has since attracted special interest as a potential anti-rheumatoid arthritis (RA) agent, owing to its successful application in traditional Chinese medicine for the treatment of neuralgia and rheumatoid diseases. In the past few decades, significant advances have broadened our understanding of the molecular mechanisms through which sinomenine treats RA, as well as the structural modifications necessary for improved pharmacological activity. In this review, we summarize up-to-date reports on the pharmacological properties of sinomenine in RA treatment, document their underlying mechanisms, and provide an overview of promising sinomenine derivatives as potential RA drug therapies.

PMON149 Identification of TR4 Modulators for Treatment of Cushing Disease.

Abstract Cushing Disease (CD) is caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor that causes excess adrenal-derived cortisol. It is a life-threatening "orphan disease" with an annual health care cost &amp;gt;7-fold higher than average patients. Surgical removal is the current first-line therapy but the disease frequently recurs. Repeat surgery, radiation therapy and bilateral adrenalectomy are not always successful and associated with significant morbidity. Currently available drugs or those in clinical trials for CD do not target the pituitary corticotroph tumor itself and escape from control is common with long-term use. A clear unmet need for efficacious and safe therapies that offer biochemical and tumor control exists. We hypothesize that direct targeting of corticotroph tumors to modulate ACTH and in turn, glucocorticoid secretion is the optimal way to treat CD. We recently demonstrated that the orphan testicular receptor 4 (TR4, also known as NR2C2) is a potent regulator of hypothalamic-pituitary-adrenal (HPA) axis function and directly regulates pro-opiomelanocortin (POMC) gene transcription and ACTH secretion. To identify and characterize TR4 small molecule modulators, we used the Flexi Vector system to fuse the TR4 224-615aa LBD to the 3' GAL4 DBD to generate a chimeric fusion plasmid (pBIND-TR4-LBD) that constitutively expresses a Renilla luciferase serving as an internal reporter control. We then transiently transfected our chimeric pBIND-TR4-LBD construct into human embryonic kidney (HEK293) cells which express an exogenous reporter construct pGL4.35 (GloResponse, luc2P/9XGAL4UAS-HEK293 Cell Line, Promega) that is driven by multiple copies of a GAL4 responsive upstream activating sequence (UAS). Following treatment with TR4 ligands or modulators, our TR4-LBD undergoes a conformational change and recruits additional cofactors and RNA polymerase II to the complex to drive GAL4-directed firefly luciferase expression. This dual-luciferase format allows us to detect compound cytotoxicity or off-target change in expression during chemical screening, greatly improving data quality. In a pilot drug screen of a LOPAC library and using robust z-score statistics (z-score&amp;gt;3), representing a luciferase to renilla induction &amp;gt; 3 standard deviations of baseline variability, we have identified several G-protein coupled ion channel regulators as potential "hit" TR4 modulators. These identified GPCRs regulate phosphatidylinositol and cyclic AMP effector systems, and activate several signaling pathway kinases. Mode-of-action and dose-response evaluation to calculate EC50 of these potential TR4 hit compounds is under investigation and lead compounds will be further validated using a POMC transactivation assay in murine and human corticotroph tumor cells. Our studies have begun to identify and rigorously validate compounds that efficiently and specifically abrogate TR4 actions to inhibit ACTH secretion and may lead to the discovery of safe and efficacious lead TR4 inhibitory compounds that can be further advanced as potential drug therapies for CD. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Homozygous mutation of the LRRK2 ROC domain as a novel genetic model of parkinsonism

BackgroundParkinson’s disease (PD) is one of the most important neurodegenerative disorders in elderly people. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are found in a large proportion of the patients with sporadic and familial PD. Mutations can occur at different locations in the LRRK2. Patients with LRRK2 ROC-COR mutations face an increased risk of typical motor symptoms of PD, along with cognitive decline. An animal model with a monogenic LRRK2 gene mutation is a suitable model for exploring the pathophysiology of PD and identifying potential drug therapies. However, the effect of homozygous (HOM) LRRK2 in PD pathophysiology is unclear.MethodsWe established human LRRK2 (hLRRK2) R1441G HOM transgenic (Tg) mice to explore the phenotype and pathological features that are associated with hLRRK2 R1441G Tg mouse models and discuss the potential clinical relevance. The open field test (OFT) was performed to examine motor and nonmotor behaviors. A CatWalk analysis system was used to study gait function. [18F]FDOPA PET was used to investigate functional changes in the nigrostriatal pathway in vivo. Transmission electron microscopy was used to examine the morphological changes in mitochondria and lysosomes in the substantia nigra.ResultsThe R1441G HOM Tg mice demonstrated gait disturbance and exhibited less anxiety-related behavior and exploratory behavior than mice with hLRRK2 at 12 months old. Additionally, [18F]FDOPA PET showed a reduction in FDOPA uptake in the striatum of the HOM Tg mice. Notably, there was significant lysosome and autophagosome accumulation in the cytoplasm of dopaminergic neurons in R1441G hemizygous (HEM) and HOM mice. Moreover, it was observed using transmission electron microscopy (TEM) that the mitochondria of R1441G Tg mice were smaller than those of hLRRK2 mice.ConclusionThis animal provides a novel HOM hLRRK2 R1441G Tg mouse model that reproduces some phenotype of Parkinsonism in terms of both motor and behavioral dysfunction. There is an increased level of mitochondrial fission and no change in the fusion process in the group of HOM hLRRK2 R1441G Tg mouse. This mutant animal model of PD might be used to study the mechanisms of mitochondrial dysfunction and explore potential new drug targets.

Novel Drug Candidate Prediction for Intrahepatic Cholangiocarcinoma via Hub Gene Network Analysis and Connectivity Mapping

Simple SummaryOnly about 15% of intrahepatic cholangiocarcinoma (ICC) patients meet the criteria for resection at the time of diagnosis. For patients with advanced and/or metastatic disease, the development of novel therapeutic strategies is urgently needed. The aim of our study was to identify possible novel therapeutic targets and drugs for ICC by using transcriptomic profiles from the Gene Expression Omnibus databases and The Cancer Genome Atlas. The weighted co-expression gene network was constructed to screen hub genes. Potential drug candidates with promise in the treatment of ICC were identified by analyzing key protein–protein interaction (PPI) networks of the hub genes to identify potential interacting drugs based on the Connectivity Map database.Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy, and there is a need for effective systemic therapies. Gene expression profile-based analyses may allow for efficient screening of potential drug candidates to serve as novel therapeutics for patients with ICC. The RNA expression profile of ICC and normal biliary epithelial cells were downloaded from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Function annotation and enrichment pathway analyses of the differentially expressed genes (DEGs) were finished using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A weighted gene co-expression network (WGCN) was constructed by WGCN analysis (WGCNA). Key genes from the DEGs and co-expression gene modules were analyzed to generate a protein–protein interaction (PPI) network. The association between the top 10 screened hub genes and the overall and disease-free survival of ICC patients was examined. The Connectivity Map (cMap) analysis was performed to identify possible drugs for ICC using hub genes. A total of 151 key genes were selected from the overlapping genes of 1287 GSE-DEGs, 8183 TCGA-DEGs and 1226 genes in the mixed modules. A total of 10 hub genes of interest (CTNNB1, SPP1, COL1A2, COL3A1, SMAD3, SRC, VCAN, PKLR, GART, MRPS5) were found analyzing protein–protein interaction. Using the cMap, candidate drugs screened with potential efficacy for ICC included three tyrosine kinase inhibitors (dasatinib, NVP-BHG712, tivantinib), two cannabinoid receptor agonists (palmitoylethanolamide, arachidonamide), two antibiotics (moxifloxacin, amoxicillin), one estrogen receptor agonist (levonorgestrel), one serine/threonine protein kinase inhibitor (MK-2206) and other small molecules. Key genes from network and PPI analysis allowed us to identify potential drugs for ICC. The identification of novel gene expression profiles and related drug screening may accelerate the identification of potential novel drug therapies for ICC.

Vaccine-Linked Chemotherapy Approach: Additive Effects of Combining the Listeria monocytogenes-Based Vaccine Lm3Dx_NcSAG1 With the Bumped Kinase Inhibitor BKI-1748 Against Neospora caninum Infection in Mice.

The apicomplexan parasite Neospora (N.) caninum causes neosporosis in numerous host species. There is no marketed vaccine and no licensed drug for the prevention and/or treatment of neosporosis. Vaccine development against this parasite has encountered significant obstacles, probably due to pregnancy-induced immunomodulation hampering efficacy, which has stimulated the search for potential drug therapies that could be applied to limit the effects of neosporosis in dams as well as in offspring. We here investigated, in a pregnant neosporosis mouse model, the safety and efficacy of a combined vaccination-drug treatment approach. Mice were vaccinated intramuscularly with 1 × 107 CFU of our recently generated Listeria (L.) monocytogenes vaccine vector expressing the major N. caninum tachyzoite surface antigen NcSAG1 (Lm3Dx_SAG1). Following mating and experimental subcutaneous infection with 1 × 105 N. caninum (NcSpain-7) tachyzoites on day 7 of pregnancy, drug treatments were initiated using the bumped kinase inhibitor BKI-1748 at 20 mg/kg/day for 5 days. In parallel, other experimental groups were either just vaccinated or only treated. Dams and offspring were followed-up until day 25 post-partum, after which all mice were euthanized. None of the treatments induced adverse effects and neither of the treatments affected fertility or litter sizes. Cerebral infection in dams as assessed by real-time PCR was significantly reduced in the vaccinated and BKI-1748 treated groups, but was not reduced significantly in the group receiving the combination. However, in non-pregnant mice, all three treatment groups exhibited significantly reduced parasite burdens. Both, vaccination as well BKI-1748 as single treatment increased pup survival to 44 and 48%, respectively, while the combination treatment led to survival of 86% of all pups. Vertical transmission in the combination group was 23% compared to 46 and 50% in the groups receiving only BKI-treatment or the vaccine, respectively. In the dams, IgG titers were significantly reduced in all treatment groups compared to the untreated control, while in non-pregnant mice, IgG titers were reduced only in the group receiving the vaccine. Overall, vaccine-linked chemotherapy was more efficacious than vaccination or drug treatment alone and should be considered for further evaluation in a more relevant experimental model.

PCOS and Depression: Common Links and Potential Targets.

PCOS or polycystic ovary syndrome is a common endocrine disorder that occurs during the reproductive age in females. It manifests in the form of a wide range of symptoms including (but not limited to) hirsutism, amenorrhea, oligomenorrhea, obesity, acne vulgaris, infertility, alopecia, and insulin resistance. The incidence of depression in PCOS population is increasing as compared to the general population. Increased depression in PCOS significantly alters the quality of life (QOL) of affected females. Also, self-esteem is found to be low in both depression and PCOS. The loss in self-esteem in such patients can be largely attributed to the associated factors including (but not limited to) obesity, acne, androgenic alopecia, and hirsutism. The reason behind the occurrence of depression in PCOS remains elusive to date. Literature suggests that there is an overlap of clinical symptoms between depression and PCOS. As the symptoms overlap, there is a possibility of common associations between depression, PCOS, and PCOS-associated abnormalities including insulin resistance (IR), obesity, CVD, and androgen excess. Studies demonstrate that depression is an inflammatory disorder marked with increased levels of inflammatory markers. On the other hand, PCOS is also regarded as a pro-inflammatory state that is characterized by increased levels of pro-inflammatory markers. Thus, there is a possibility of an inflammatory relationship existing between depression and PCOS. It is also possible that the inflammatory markers in PCOS can cross the blood-brain barrier (BBB) leading to the development of depression. Through the present review, we have attempted to shed light on common associations/shared links between depression and PCOS with respect to the levels of cortisol, androgen, vitamin D, neurotransmitters, monoaminoxidase (MAO), and insulin-like growth factor-1 (IGF-1). Tracking down common associations between depression and PCOS will help find potential drug therapies and improve the QOL of females with depression in PCOS.