Pharmacological Interference Research Articles

What evidence is there for implicating the brain orexin system in neuropsychiatric symptoms in dementia?

Neuropsychiatric symptoms (NPS) affect people with dementia (PwD) almost universally across all stages of the disease, and regardless of its exact etiology. NPS lead to disability and reduced quality of life of PwD and their caregivers. NPS include hyperactivity (agitation and irritability), affective problems (anxiety and depression), psychosis (delusions and hallucinations), apathy, and sleep disturbances. Preclinical studies have shown that the orexin neuropeptide system modulates arousal and a wide range of behaviors via a network of axons projecting from the hypothalamus throughout almost the entire brain to multiple, even distant, regions. Orexin neurons integrate different types of incoming information (e.g., metabolic, circadian, sensory, emotional) and convert them into the required behavioral output coupled to the necessary arousal status. Here we present an overview of the behavioral domains influenced by the orexin system that may be relevant for the expression of some critical NPS in PwD. We also hypothesize on the potential effects of pharmacological interference with the orexin system in the context of NPS in PwD.

Keloid Disorder: Genetic Basis, Gene Expression Profiles, and Immunological Modulation of the Fibrotic Processes in the Skin.

Keloid disorder, a group of fibroproliferative skin disorders, is clinically comprised of keloids, hypertrophic scars, keloidalis nuchae, and acne keloidalis. The prototype of these disorders is keloids, which manifest as cutaneous lesions with excessive deposition of collagen following an initiating trauma of varying degrees. The principal cell type responsible for collagen accumulation is the myofibroblast, and its gene expression is modulated by a network of regulatory factors, including cytokines, growth factors, and noncoding RNA species. In addition, keloids harbor a number of inflammatory cells, including macrophages and mast cells, that interact with fibroblastic cells by direct contact or by paracrine actions. Transforming growth factor-β1/Smad signaling regulates the expression of genes encoding extracellular matrix proteins and also controls cell proliferation and apoptosis. A key profibrotic molecule is the fibronectin splice variant cellular fibronectin extracellular domain A (cFN-EDA), which interacts with a number of cell-surface integrins and TLR4, contributing to the modulation of gene expression by lesional fibroblasts. Collectively, these complex cellular interactions result in accumulation of collagen with clinical development and growth of keloid lesions. Understanding of the precise pathomechanistic details of keloid formation will provide targets for pharmacological interference toward treatment of the keloid disorder, a group of currently difficult to treat skin diseases.

Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T‐cell lymphoma

Leukemic cutaneous T‐cell lymphomas (L‐CTCL) are lymphoproliferative disorders of skin‐homing mature T‐cells causing severe symptoms and high mortality through chronic inflammation, tissue destruction, and serious infections. Despite numerous genomic sequencing efforts, recurrent driver mutations have not been identified, but chromosomal losses and gains are frequent and dominant. We integrated genomic landscape analyses with innovative pharmacologic interference studies to identify key vulnerable nodes in L‐CTCL. We detected copy number gains of loci containing the STAT3/5 oncogenes in 74% (n = 17/23) of L‐CTCL, which correlated with the increased clonal T‐cell count in the blood. Dual inhibition of STAT3/5 using small‐molecule degraders and multi‐kinase blockers abolished L‐CTCL cell growth in vitro and ex vivo, whereby PAK kinase inhibition was specifically selective for L‐CTCL patient cells carrying STAT3/5 gains. Importantly, the PAK inhibitor FRAx597 demonstrated encouraging anti‐leukemic activity in vivo by inhibiting tumor growth and disease dissemination in intradermally xenografted mice. We conclude that STAT3/5 and PAK kinase interaction represents a new therapeutic node to be further explored in L‐CTCL.

DOCK2 and phosphoinositide-3 kinase δ mediate two complementary signaling pathways for CXCR5-dependent B cell migration.

Naive B cells use the chemokine receptor CXCR5 to enter B cell follicles, where they scan CXCL13-expressing ICAM-1+ VCAM-1+ follicular dendritic cells (FDCs) for the presence of antigen. CXCL13-CXCR5-mediated motility is mainly driven by the Rac guanine exchange factor DOCK2, which contains a binding domain for phosphoinositide-3,4,5-triphosphate (PIP3) and other phospholipids. While p110δ, the catalytic subunit of the class IA phosphoinositide-3-kinase (PI3K) δ, contributes to CXCR5-mediated B cell migration, the precise interdependency of DOCK2, p110δ, or other PI3K family members during this process remains incompletely understood. Here, we combined in vitro chemotaxis assays and in vivo imaging to examine the contribution of these two factors during murine naïve B cell migration to CXCL13. Our data confirm that p110δ is the main catalytic subunit mediating PI3K-dependent migration downstream CXCR5, whereas it does not contribute to chemotaxis triggered by CXCR4 or CCR7, two other chemokine receptors expressed on naïve B cells. The contribution of p110δ activity to CXCR5-driven migration was complementary to that of DOCK2, and pharmacological or genetic interference with both pathways completely abrogated B cell chemotaxis to CXCL13. Intravital microscopy of control and gene-deficient B cells migrating on FDCs confirmed that lack of DOCK2 caused a profound migration defect, whereas p110δ contributed to cell speed and directionality. B cells lacking active p110δ also displayed defective adhesion to ICAM-1; yet, their migration impairment was maintained on ICAM-1-deficient FDCs. In sum, our data uncover two complementary signaling pathways mediated by DOCK2 and p110δ, which enable CXCR5-driven naïve B cell examination of FDCs.

Behavioural reconsolidation interference not observed in a within-subjects design

Studies of reconsolidation interference posit that reactivation of a previously consolidated memory via a reminder brings it into an active, labile state, leaving it open for potential manipulation. If interfered with, this may disrupt the original memory trace. While evidence for pharmacological reconsolidation interference is widespread, it remains unclear whether behavioural interference using the presentation of competing information can engender it, especially in declarative memory. Almost all previous studies in this area have employed between-subjects designs, in which there are potential confounds, such as different retrieval strategies for the multiple conditions. In the current studies, within-subjects paradigms were applied to test the effects of reconsolidation interference on associative recognition and free recall. In Experiment 1, participants engaged in pair-associate learning of unrelated object pictures on Day 1, and after a reminder, interference, reminder + interference, or no manipulation (control) on Day 2, were tested on associative recognition of these pairs on Day 3. In Experiments 2 and 3, memoranda were short stories studied on Day 1. On Day 2, stories were assigned to either control, reminder, interference by alternative stories, or reminder + interference conditions. On Day 3 participants recalled the Day 1 stories, and answered yes/no recognition questions. Reminders improved subsequent memory, while interference was effective in reducing retrieval in differing degrees across the experiments. Importantly, the reminder + interference condition was no more effective in impairing retrieval than the interference-alone condition, contrary to the prediction of the behavioural reconsolidation-interference approach.

Therapeutic targeting of glutamate dehydrogenase 1 that links metabolic reprogramming and Snail-mediated epithelial–mesenchymal transition in drug-resistant lung cancer

Acquired drug resistance and epithelial–mesenchymal transition (EMT) mediated metastasis are two highly interacting determinants for non-small-cell lung cancer (NSCLC) prognosis. This study investigated the common mechanisms of drug resistance and EMT from the perspective of metabolic reprogramming, which may offer new ideas to improve anticancer therapy. Acquired resistant cells were found to grow faster and have a greater migratory and invasive capacity than their parent cells. Metabolomics analysis revealed that acquired resistant cells highly relied on glutamine utilization and mainly fluxed into oxidative phosphorylation energy production. Further mechanistic studies screened out glutamate dehydrogenase 1 (GLUD1) as the determinant of glutamine addiction in acquired resistant NSCLC cells, and provided evidence that GLUD1-mediated α-KG production and the accompanying reactive oxygen species (ROS) accumulation primarily triggered migration and invasion by inducing Snail. Pharmacological and genetic interference with GLUD1 in vitro significantly reversed drug resistance and decreased cell migration and invasion capability. Lastly, the successful application of R162, a selective GLUD1 inhibitor, to overcome both acquired resistance and EMT-induced metastasis in vivo, identified GLUD1 as a promising and druggable therapeutic target for malignant progression of NSCLC. Collectively, our study offers a potential strategy for NSCLC therapy, especially for drug-resistant patients with highly expressed GLUD1.

Hypoxia Triggers TAZ Phosphorylation in Basal A Triple Negative Breast Cancer Cells.

Hypoxia and HIF signaling drive cancer progression and therapy resistance and have been demonstrated in breast cancer. To what extent breast cancer subtypes differ in their response to hypoxia has not been resolved. Here, we show that hypoxia similarly triggers HIF1 stabilization in luminal and basal A triple negative breast cancer cells and we use high throughput targeted RNA sequencing to analyze its effects on gene expression in these subtypes. We focus on regulation of YAP/TAZ/TEAD targets and find overlapping as well as distinct target genes being modulated in luminal and basal A cells under hypoxia. We reveal a HIF1 mediated, basal A specific response to hypoxia by which TAZ, but not YAP, is phosphorylated at Ser89. While total YAP/TAZ localization is not affected by hypoxia, hypoxia drives a shift of [p-TAZ(Ser89)/p-YAP(Ser127)] from the nucleus to the cytoplasm in basal A but not luminal breast cancer cells. Cell fractionation and YAP knock-out experiments confirm cytoplasmic sequestration of TAZ(Ser89) in hypoxic basal A cells. Pharmacological and genetic interference experiments identify c-Src and CDK3 as kinases involved in such phosphorylation of TAZ at Ser89 in hypoxic basal A cells. Hypoxia attenuates growth of basal A cells and the effect of verteporfin, a disruptor of YAP/TAZ-TEAD–mediated transcription, is diminished under those conditions, while expression of a TAZ-S89A mutant does not confer basal A cells with a growth advantage under hypoxic conditions, indicating that other hypoxia regulated pathways suppressing cell growth are dominant.

Abstract PO019: Gas6/Axl signaling induces PRAME expression in hepatocellular carcinoma

Abstract The receptor tyrosine kinase Axl, binding to its ligand Gas6, fosters oncogenic effects during the progression of hepatocellular carcinoma (HCC) which is accompanied by increased mortality of patients. The specific role of Gas6/Axl signaling in neoplastic hepatocytes and its pathophysiological consequences are poorly understood at the current stage of research. We aimed at identifying and functionally analyzing targets of Gas6/Axl signaling in HCC by exploiting well-characterized human HCC tumor models. HCC cells transformed by epithelial to mesenchymal transition (EMT) and expressing high levels of Axl or harboring CRISPR/Cas9-mediated Axl deficiency, the latter in absence or presence of Axl reconstitution, were subjected to Gas6 stimulation and subsequent RNA-seq analysis. Bioinformatics and VENN relations were applied to identify novel targets of the Gas6/Axl axis. Expression of Gas6/Axl targets was assessed in publicly available HCC patient datasets and in HCC tissue microarrays. In vitro analysis was performed including gain and loss of functions studies to unravel target genes contribution to EMT and HCC progression. RNA-seq analyses revealed PRAME (preferentially expressed antigen in melanoma) and C15orf48 among others as novel targets of Gas6/Axl. Notably, these target genes showed elevated expression levels in Axl-stratified HCC patients and significant impact on patient survival. Pharmacological or genetic interference with Axl signaling resulted in strongly reduced expression of PRAME and C15orf48, suggesting bona fide targets of Gas6/Axl signaling. PRAME expression positively regulated 2D cell migration and invasion of 3D hepatospheres while proliferation and survival of HCC cells remained unaffected by altered PRAME levels. In accordance, PRAME expression strongly associated with a mesenchymal-like phenotype in human HCC. Intriguingly, HCC patients expressing PRAME correlated with Axl expression and vascular invasion in a large proportion of HCC patients. Our study demonstrates that PRAME is a novel target of Gas6/Axl signaling in HCC linked to EMT. Translation of in vitro findings into the HCC patient situation provides first insights that PRAME plays a crucial role in HCC progression. Citation Format: Viola Hedrich, Doris Chen, Heidemarie Huber, Wolfgang Mikulits. Gas6/Axl signaling induces PRAME expression in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO019.

Neuromodulatory and Protective Effects Induced by the Association of Herbal Extracts from Valeriana officinalis, Ziziphus jujuba, and Humulus lupulus with Melatonin: An Innovative Formulation for Counteracting Sleep Disorders

Background: The use of herbal extracts could represent an advantageous approach for treating sleeping disorders, especially in mild-to-moderate conditions, before the onset of a specific therapy with first-line drugs. Specifically, the focus was posed about the use of extracts from Valeriana officinalis, Ziziphus jujuba, and Humulus lupulus. Multiple studies demonstrated the efficacy of these medicinal plants to positively manage insomnia symptoms. Additionally, their efficacy in the treatment of sleeping disorders could also be improved by their pharmacological association. In the present study, extracts from Valeriana officinalis, Ziziphus jujuba, Humulus lupulus, melatonin, and their pharmacological association, Vagonotte® MEL, were studied for potential application in the treatment of insomnia. Methods: The extracts and melatonin were tested on hypothalamic neurons and tissue for evaluating biocompatibility and protective and neuromodulatory effects. The neuromodulatory effects were evaluated as orexin A gene expression and serotonin steady state level, in the hypothalamus. Results: The extracts and melatonin, although with evident differences, were effective as antioxidant and anti-inflammatory agents; additionally, they were also able to reduce the hypothalamic gene expression of orexin A and the steady state level of serotonin, playing master roles in wakefulness. It is noteworthy that the formulation displayed all the effects of the single ingredients, without any sign of toxicity and pharmacological interference in the hypothalamus. Conclusions: Concluding, the present study explored the biological effects of melatonin and herbal extracts with phytotherapy interest in V. officinalis, Z. jujuba, and H. lupulus. The study demonstrated their intrinsic scavenging/reducing activity, together with protective and neuromodulatory effects in the hypothalamus, with a significant reduction of both orexin A gene expression and serotonin steady state level. Additionally, the study also considered their pharmacological association, which displayed an overall pharmacological spectrum mirroring, including all the effects of the single ingredients, without showing any sign of toxicity in the brain and interference between the extracts and melatonin.

Effect of Extracellular Signal-Regulated Protein Kinase 5 Inhibition in Clear Cell Renal Cell Carcinoma

(1) Background: Extracellular signal-regulating kinase 5 (ERK5) has been implicated in many cellular functions, including survival, proliferation, and vascularization. Our objectives were to examine the expression and effect of ERK5 in clear cell renal cell carcinoma (ccRCC). (2) Methods: The expressions of ERK5 and its regulating micro-RNA miR-143 were investigated using immunohistochemistry and quantitative reverse transcriptase PCR in surgical specimens of ccRCC patients. With invitro and in vivo studies, we used pharmacologic ERK5 inhibitor XMD8-92, RNA interference, pre-miR-143 transduction, Western blotting, MTS assay, apoptosis assay, and subcutaneous xenograft model. (3) Results: A strong ERK5 expression in surgical specimen was associated with high-grade (p = 0.01), high-recurrence free rate (p = 0.02), and high cancer-specific survival (p = 0.03). Expression levels of ERK5 and miR-143 expression level were correlated (p = 0.049). Pre-miR-143 transduction into ccRCC cell A498 suppressed ERK5 expression. ERK5 inhibition enhanced cyclin-dependent kinase inhibitor p21 expression and decreased anti-apoptotic molecules BCL2, resulting in decreased cell proliferation and survival both in ccRCC and endothelial cells. In the xenograft model, ERK5 inhibitor XMD8-92 suppressed tumor growth. (4) Conclusions: ERK5 is regulated by miR-143, and ERK5 inhibition is a promising target for ccRCC treatment.

Onco-immunomodulatory properties of pharmacological interference with RAS-RAF-MEK-ERK pathway hyperactivation.

Hyperactivation of the RAS-RAF-MEK-ERK cascade - a mitogen-activated protein kinase pathway – has a well-known association with oncogenesis of leading tumor entities, including non-small cell lung cancer, colorectal carcinoma, pancreatic ductal adenocarcinoma, and malignant melanoma. Increasing evidence shows that genetic alterations leading to RAS-RAF-MEK-ERK pathway hyperactivation mediate contact- and soluble-dependent crosstalk between tumor, tumor microenvironment (TME) and the immune system resulting in immune escape mechanisms and establishment of a tumor-sustaining environment. Consequently, pharmacological interruption of this pathway not only leads to tumor-cell intrinsic disruptive effects but also modification of the TME and anti-tumor immunomodulation. At the same time, the importance of ERK signaling in immune cell physiology and potentiation of anti-tumor immune responses through ERK signaling inhibition within immune cell subsets has received growing appreciation. Specifically, a strong case was made for targeted MEK inhibition due to promising associated immune cell intrinsic modulatory effects. However, the successful transition of therapeutic agents interrupting RAS-RAF-MEK-ERK hyperactivation is still being hampered by significant limitations regarding durable efficacy, therapy resistance and toxicity. We here collate and summarize the multifaceted role of RAS-RAF-MEK-ERK signaling in physiology and oncoimmunology and outline the rationale and concepts for exploitation of immunomodulatory properties of RAS-RAF-MEK-ERK inhibition while accentuating the role of MEK inhibition in combinatorial and intermittent anticancer therapy. Furthermore, we point out the extensive scientific efforts dedicated to overcoming the challenges encountered during the clinical transition of various therapeutic agents in the search for the most effective and safe patient- and tumor-tailored treatment approach.

LRH‐1/NR5A2 interacts with the glucocorticoid receptor to regulate glucocorticoid resistance

Nuclear receptors are transcription factors with important functions in a variety of physiological and pathological processes. Targeting glucocorticoid receptor (GR) activity using glucocorticoids is a cornerstone in the treatment of patients with T cell acute lymphoblastic leukemia (T‐ALL), and resistance to GC‐induced cell death is associated with poor outcome and a high risk for relapse. Next to ligand‐binding, heterodimerization with other transcription factors presents an important mechanism for the regulation of GR activity. Here, we describe a GC‐induced direct association of the Liver Receptor Homolog‐1 (LRH‐1) with the GR in the nucleus, which results in reciprocal inhibition of transcriptional activity. Pharmacological and molecular interference with LRH‐1 impairs proliferation and survival in T‐ALL and causes a profound sensitization to GC‐induced cell death, even in GC‐resistant T‐ALL. Our data illustrate that direct interaction between GR and LRH‐1 critically regulates glucocorticoid sensitivity in T‐ALL opening up new perspectives for developing innovative therapeutic approaches to treat GC‐resistant T‐ALL.

Induction and modulation of the unfolded protein response during porcine deltacoronavirus infection

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that has the potential for cross-species infection. Many viruses have been reported to induce endoplasmic reticulum stress (ERS) and activate the unfolded protein response (UPR). To date, little is known about whether and, if so, how the UPR is activated by PDCoV infection. Here, we investigated the activation state of UPR pathways and their effects on viral replication during PDCoV infection. We found that PDCoV infection induced ERS and activated all three known UPR pathways (inositol-requiring enzyme 1 [IRE1], activating transcription factor 6 [ATF6], and PKR-like ER kinase [PERK]), as demonstrated by IRE1-mediated XBP1 mRNA cleavage and increased mRNA expression of XBP1s, ATF4, CHOP, GADD34, GRP78, and GRP94, as well as phosphorylated eIF2α expression. Through pharmacologic treatment, RNA interference, and overexpression experiments, we confirmed the negative role of the PERK–eIF2α pathway and the positive regulatory role of the ATF6 pathway, but found no obvious effect of IRE1 pathway, on PDCoV replication. Taken together, our results characterize, for the first time, the state of the ERS response during PDCoV infection and identify the PERK and ATF6 pathways as potential antiviral targets.

Localizing circuits of cocaine contextual memories

Addiction is characterized by compulsive drug seeking and taking behaviors. Cravings triggered by the presence of environmental cues that were once associated to drug use are a main contributor of relapse and once these drug‐cue memories are formed they are difficult to erase. Addictive drugs like cocaine engage molecular pathways of associate learning, in which recall of an established memory is followed by its reconsolidation, strengthening that memory. The reactivation period is a critical time wherein memories are labile and vulnerable to behavioral and pharmacological interference. Given the widespread negative impact of drug use in our society, it is of great importance to understand the mechanisms underlying drug seeking behaviors to generate effective preventions and treatments. The goal of this study is to elucidate the neural substrate in the nucleus accumbens crucial for the maintenance of cocaine contextual memories, and explore neural plasticity involved. Designer receptors exclusively activated by designer drugs (DREADDs) were used to inhibit neurons at the nucleus accumbens. To accomplish this, adult male mice underwent bilateral intracranial injections of an inhibitory DREADD followed by a cocaine conditioned place preference paradigm. Nucleus accumbens neurons were inhibited via systemic injection of clozapine N‐oxide immediately following reactivation of cocaine contextual memory, and mice were re‐tested for place preference 72 hours and 7 days later. A separate cohort of mice underwent the same cocaine conditioned place preference paradigm, 24 hours post preference testing animals were returned to the cocaine context in a drug‐free state to reactivate cocaine memories. Sixty minutes after, brains were collected and prepared for quantitative RT‐PCR to measure activity‐regulated cytoskeleton‐associate protein (Arc) mRNA. To evaluate the functional plasticity in medium spiny neurons at the nucleus accumbens, upon reconsolidation of cocaine contextual memories, we utilized the fosTRAP2‐Ai14 mouse model. A cohort of male and female fosTRAP2‐Ai14 mice underwent cocaine conditioned place preference exactly as in the previous experiment and brains collected 9 days after recall of cocaine contextual memory and 4‐hydroxytamoxifen induction. Dendritic spine density of fosTRAP cells activated by recall of cocaine context were analyzed. Results show that chemogenetic inhibition of the nucleus accumbens immediately following reactivation of cocaine contextual memory significantly reduced a once established preference for the cocaine context. Furthermore, Arc mRNA expression is upregulated at the nucleus accumbens following exposure to the prior cocaine‐paired context. Finally, we show a significant increase in dendritic spine density in medium spiny neurons at the nucleus accumbens following recall of cocaine context. These findings suggest the nucleus accumbens is important for the reconsolidation of cocaine contextual memories and exhibits dendritic neuroplastic changes upon reconsolidation of cocaine memories.

Loss of UCP1 function augments recruitment of futile lipid cycling for thermogenesis in murine brown fat

ObjectiveClassical ATP-independent non-shivering thermogenesis enabled by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) is activated, but not essential for survival, in the cold. It has long been suspected that futile ATP-consuming substrate cycles also contribute to thermogenesis and can partially compensate for the genetic ablation of UCP1 in mouse models. Futile ATP-dependent thermogenesis could thereby enable survival in the cold even when brown fat is less abundant or missing. MethodsIn this study, we explore different potential sources of UCP1-independent thermogenesis and identify a futile ATP-consuming triglyceride/fatty acid cycle as the main contributor to cellular heat production in brown adipocytes lacking UCP1. We uncover the mechanism on a molecular level and pinpoint the key enzymes involved using pharmacological and genetic interference. ResultsATGL is the most important lipase in terms of releasing fatty acids from lipid droplets, while DGAT1 accounts for the majority of fatty acid re-esterification in UCP1-ablated brown adipocytes. Furthermore, we demonstrate that chronic cold exposure causes a pronounced remodeling of adipose tissues and leads to the recruitment of lipid cycling capacity specifically in BAT of UCP1-knockout mice, possibly fueled by fatty acids from white fat. Quantification of triglyceride/fatty acid cycling clearly shows that UCP1-ablated animals significantly increase turnover rates at room temperature and below. ConclusionOur results suggest an important role for futile lipid cycling in adaptive thermogenesis and total energy expenditure.

Therapeutic Efficacy of Pharmacological Ascorbate on Braf Inhibitor Resistant Melanoma Cells In Vitro and In Vivo.

High-dose ascorbate paradoxically acts as a pro-oxidant causing the formation of hydrogen peroxide in an oxygen dependent manner. Tumor cells (in particular melanoma cells) show an increased vulnerability to ascorbate induced reactive oxygen species (ROS). Therefore, high-dose ascorbate is a promising pharmacological approach to treating refractory melanomas, e.g., with secondary resistance to targeted BRAF inhibitor therapy. BRAF mutated melanoma cells were treated with ascorbate alone or in combination with the BRAF inhibitor vemurafenib. Viability, cell cycle, ROS production, and the protein levels of phospho-ERK1/2, GLUT-1 and HIF-1α were analyzed. To investigate the treatment in vivo, C57BL/6NCrl mice were subcutaneously injected with D4M.3A (BrafV600E) melanoma cells and treated with intraperitoneal injections of ascorbate with or without vemurafenib. BRAF mutated melanoma cell lines either sensitive or resistant to vemurafenib were susceptible to the induction of cell death by pharmacological ascorbate. Treatment of BrafV600E melanoma bearing mice with ascorbate resulted in plasma levels in the pharmacologically active range and significantly improved the therapeutic effect of vemurafenib. We conclude that intravenous high-dose ascorbate will be beneficial for melanoma patients by interfering with the tumor’s energy metabolism and can be safely combined with standard melanoma therapies such as BRAF inhibitors without pharmacological interference.

Differential vulnerability of adult neurogenic niches to dosage of the neurodevelopmental-disorder linked gene Foxg1

The transcription factor FOXG1 serves pleiotropic functions in brain development ranging from the regulation of precursor proliferation to the control of cortical circuit formation. Loss-of-function mutations and duplications of FOXG1 are associated with neurodevelopmental disorders in humans illustrating the importance of FOXG1 dosage for brain development. Aberrant FOXG1 dosage has been found to disrupt the balanced activity of glutamatergic and GABAergic neurons, but the underlying mechanisms are not fully understood. We report that FOXG1 is expressed in the main adult neurogenic niches in mice, i.e. the hippocampal dentate gyrus and the subependymal zone/olfactory bulb system, where neurogenesis of glutamatergic and GABAergic neurons persists into adulthood. These niches displayed differential vulnerability to increased FOXG1 dosage: high FOXG1 levels severely compromised survival and glutamatergic dentate granule neuron fate acquisition in the hippocampal neurogenic niche, but left neurogenesis of GABAergic neurons in the subependymal zone/olfactory bulb system unaffected. Comparative transcriptomic analyses revealed a significantly higher expression of the apoptosis-linked nuclear receptor Nr4a1 in FOXG1-overexpressing hippocampal neural precursors. Strikingly, pharmacological interference with NR4A1 function rescued FOXG1-dependent death of hippocampal progenitors. Our results reveal differential vulnerability of neuronal subtypes to increased FOXG1 dosage and suggest that activity of a FOXG1/NR4A1 axis contributes to such subtype-specific response.

Neutrophil-Derived Myeloperoxidase and Hypochlorous Acid Critically Contribute to 20-Hydroxyeicosatetraenoic Acid Increases that Drive Postischemic Angiogenesis.

Compensatory angiogenesis is an important adaptation for recovery from critical ischemia. We recently identified 20-hydroxyeicosatetraenoic acid (20-HETE) as a novel contributor of ischemia-induced angiogenesis. However, the precise mechanisms by which ischemia promotes 20-HETE increases that drive angiogenesis are unknown. This study aims to address the hypothesis that inflammatory neutrophil-derived myeloperoxidase (MPO) and hypochlorous acid (HOCl) critically contribute to 20-HETE increases leading to ischemic angiogenesis. Using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry, Laser Doppler Perfusion Imaging, and Microvascular Density analysis, we found that neutrophil depletion and MPO knockout mitigate angiogenesis and 20-HETE production in the gracilis muscles of mice subjected to hindlimb ischemia. Furthermore, we found MPO and HOCl to be elevated in these tissues postischemia as assessed by immunofluorescence microscopy and in vivo live imaging of HOCl. Next, we demonstrated that the additions of either HOCl or an enzymatic system for generating HOCl to endothelial cells increase the expression of CYP4A11 and its product, 20-HETE. Finally, pharmacological interference of hypoxia inducible factor (HIF) signaling results in ablation of HOCl-induced CYP4A11 transcript and significant reductions in CYP4A11 protein. Collectively, we conclude that neutrophil-derived MPO and its product HOCl activate HIF-1α and CYP4A11 leading to increased 20-HETE production that drives postischemic compensatory angiogenesis. SIGNIFICANCE STATEMENT: Traditionally, neutrophil derived MPO and HOCl are exclusively associated in the innate immunity as potent bactericidal/virucidal factors. The present study establishes a novel paradigm by proposing a unique function for MPO/HOCl as signaling agents that drive critical physiological angiogenesis by activating the CYP4A11-20-HETE signaling axis via a HIF-1α-dependent mechanism. The findings from this study potentially identify novel therapeutic targets for the treatment of ischemia and other diseases associated with abnormal angiogenesis.

Design, synthesis and biological evaluation of biguanids and biguanid-sulfonamides as cholinesterase inhibitors

Recently, the inhibitory impact of enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) have emerged as a hopeful approach for pharmacological interference in various disorders such as epilepsy, Alzheimer's, Parkinson's disease and obesity. The biological and pharmacological properties of biguanidines and sulfonamides have made the compounds important. In this study, a series of biguanidine compounds (1–4) and their sulphonamide derivatives(1a-4a) were synthesized and their structures were elucidated by analytical and spectroscopic methods. The structure of compound 4 was determined by X-ray diffraction method. The synthesised compounds were showed for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory properties. The compounds were found to exhibit inhibitory properties for acetylcholinesterase and butyrylcholinesterase enzymes. amongst the tested compounds, it was determined that the sulfonamide compound, which contains benzamide in its structure, exhibited higher activity (IC50 138.47 µM) in AChE inhibition compared to other compounds.

Inducible epigenome editing probes for the role of histone H3K4 methylation in Arabidopsis heat stress memory.

Histone modifications play a crucial role in the integration of environmental signals to mediate gene expression outcomes. However, genetic and pharmacological interference often causes pleiotropic effects, creating the urgent need for methods that allow locus-specific manipulation of histone modifications, preferably in an inducible manner. Here, we report an inducible system for epigenome editing in Arabidopsis (Arabidopsis thaliana) using a heat-inducible dCas9 to target a JUMONJI (JMJ) histone H3 lysine 4 (H3K4) demethylase domain to a locus of interest. As a model locus, we target the ASCORBATE PEROXIDASE2 (APX2) gene that shows transcriptional memory after heat stress (HS), correlating with H3K4 hyper-methylation. We show that dCas9–JMJ is targeted in a HS-dependent manner to APX2 and that the HS-induced overaccumulation of H3K4 trimethylation (H3K4me3) decreases when dCas9–JMJ binds to the locus. This results in reduced HS-mediated transcriptional memory at the APX2 locus. Targeting an enzymatically inactive JMJ protein in an analogous manner affected transcriptional memory less than the active JMJ protein; however, we still observed a decrease in H3K4 methylation levels. Thus, the inducible targeting of dCas9–JMJ to APX2 was effective in reducing H3K4 methylation levels. As the effect was not fully dependent on enzyme activity of the eraser domain, the dCas9–JMJ fusion protein may act in part independently of its demethylase activity. This underlines the need for caution in the design and interpretation of epigenome editing studies. We expect our versatile inducible epigenome editing system to be especially useful for studying temporal dynamics of chromatin modifications.