Assessing Dose- and Sex-Dependent Antinociceptive Effects of Cannabidiol and Amitriptyline, Alone and in Combination, and Exploring Mechanism of Action Involving Serotonin 1A Receptors.

Inflammatory pain is caused by tissue hypersensitization and is a component of rheumatic diseases, frequently causing chronic pain. Current guidelines use a multimodal approach to pain and sociocultural changes have renewed interest in cannabinoid use, particularly cannabidiol (CBD), for pain. The tricyclic antidepressant amitriptyline (AT) is approved for use in pain-related syndromes, alone and within a multimodal approach. Therefore, we investigated sex- and dose-dependent effects of CBD and AT antinociception in the 2.5% formalin inflammatory pain model. Male and female C57BL/6J mice were pretreated with either vehicle, CBD (0.3-100 mg/kg), or AT (0.1-30 mg/kg) prior to formalin testing. In the acute phase, CBD induced antinociception after administration of 30-100 mg/kg in males and 100 mg/kg in females and in the inflammatory phase at doses of 2.5-100 mg/kg in males and 10-100 mg/kg in females. In the acute phase, AT induced antinociception at 10 mg/kg for all mice, and at 0.3 mg/kg in males and 3 mg/kg in female mice in the inflammatory phase. Combining the calculated median effective doses of CBD and AT produced additive effects for all mice in the acute phase and for males only in the inflammatory phase. Use of selective serotonin 1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1 piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY-100635) maleate (0.1 mg/kg) before co-administration of CBD and AT reversed antinociception in the acute and partially reversed antinociception in the inflammatory phase. Administration of AT was found to enhance cannabinoid receptor type 1mRNA expression only in female mice. These results suggest a role for serotonin and sex in mediating cannabidiol and amitriptyline-induced antinociception in inflammatory pain. SIGNIFICANCE STATEMENT: Inflammatory pain is an important component of both acute and chronic pain. We have found that cannabidiol (CBD) and amitriptyline (AT) show dose-dependent, and that AT additionally shows sex-dependent, antinociceptive effects in an inflammatory pain model. Additionally, the combination of CBD and AT was found to have enhanced antinociceptive effects that is partially reliant of serotonin 1A receptors and supports the use of CBD within a multimodal approach to pain.

Dishevelled 2 regulates cancer cell proliferation and T cell mediated immunity in HER2-positive breast cancer.

BACKGROUND: Dishevelled paralogs (DVL1, 2, 3) are key mediators of Wnt pathway playing a role in constitutive oncogenic signaling influencing the tumor microenvironment. While previous studies showed correlation of ß-catenin with T cell gene expression, little is known about the role of DVL2 in modulating tumor immunity. This study aimed to uncover the novel interaction between DVL2 and HER2-positive (HER2+) breast cancer (BC) in regulating tumor immunity and disease progression. METHODS: DVL2 loss of function studies were performed with or without a clinically approved HER2 inhibitor, Neratinib in two different HER2+ BC cell lines. We analyzed RNA (RT-qPCR) and protein (western blot) expression of classic Wnt markers and performed cell proliferation and cell cycle analyses by live cell imaging and flow cytometry, respectively. A pilot study in 24 HER2+ BC patients was performed to dissect the role of DVL2 in tumor immunity. Retrospective chart review on patient records and banked tissue histology were performed. Data were analyzed in SPSS (version 25) and GraphPad Prism (version 7) at a significance p < 0.05. RESULTS: DVL2 regulates the transcription of immune modulatory genes involved in antigen presentation and T cell maintenance. DVL2 loss of function down regulated mRNA expression of Wnt target genes involved in cell proliferation, migration, invasion in HER2+ BC cell lines (±Neratinib). Similarly, live cell proliferation and cell cycle analyses reveal that DVL2 knockdown (±Neratinib) resulted in reduced proliferation, higher growth arrest (G1), limited mitosis (G2/M) compared to non-targeted control in one of the two cell lines used. Analyses on patient tissues who received neoadjuvant chemotherapy (n = 14) further demonstrate that higher DVL2 expression at baseline biopsy pose a significant negative correlation with % CD8α levels (r = - 0.67, p < 0.05) while have a positive correlation with NLR (r = 0.58, p < 0.05), where high NLR denotes worse cancer prognosis. These results from our pilot study reveal interesting roles of DVL2 proteins in regulating tumor immune microenvironment and clinical predictors of survival in HER2+ BC. CONCLUSION: Our study demonstrates potential immune regulatory role of DVL2 proteins in HER2+ BC. More in-depth mechanistic studies of DVL paralogs and their influence on anti-tumor immunity may provide insight into DVLs as potential therapeutic targets benefiting BC patients.

Expression and Function of StAR in Cancerous and Non-Cancerous Human and Mouse Breast Tissues: New Insights into Diagnosis and Treatment of Hormone-Sensitive Breast Cancer.

Breast cancer (BC) is primarily triggered by estrogens, especially 17ß-estradiol (E2), which are synthesized by the aromatase enzyme. While all steroid hormones are derived from cholesterol, the rate-limiting step in steroid biosynthesis is mediated by the steroidogenic acute regulatory (StAR) protein. Herein, we demonstrate that StAR mRNA expression was aberrantly high in human hormone-dependent BC (MCF7, MDA-MB-361, and T-47D), modest in hormone-independent triple negative BC (TNBC; MDA-MB-468, BT-549, and MDA-MB-231), and had little to none in non-cancerous mammary epithelial (HMEC, MCF10A, and MCF12F) cells. In contrast, these cell lines showed abundant expression of aromatase (CYP19A1) mRNA. Immunofluorescence displayed qualitatively similar patterns of both StAR and aromatase expression in various breast cells. Additionally, three different transgenic (Tg) mouse models of spontaneous breast tumors, i.e., MMTV-Neu, MMTV-HRAS, and MMTV-PyMT, demonstrated markedly higher expression of StAR mRNA/protein in breast tumors than in normal mammary tissue. While breast tumors in these mouse models exhibited higher expression of ERα, ERß, and PR mRNAs, their levels were undetected in TNBC tumors. Accumulation of E2 in plasma and breast tissues, from MMTV-PyMT and non-cancerous Tg mice, correlated with StAR, but not with aromatase, signifying the importance of StAR in governing E2 biosynthesis in mammary tissue. Treatment with a variety of histone deacetylase inhibitors (HDACIs) in primary cultures of enriched breast tumor epithelial cells, from MMTV-PyMT mice, resulted in suppression of StAR and E2 levels. Importantly, inhibition of StAR, concomitant with E2 synthesis, by various HDACIs, at clinical and preclinical doses, in MCF7 cells, indicated therapeutic relevance of StAR in hormone-dependent BCs. These findings provide insights into the molecular events underlying the differential expression of StAR in human and mouse cancerous and non-cancerous breast cells/tissues, highlighting StAR could serve not only as a novel diagnostic maker but also as a therapeutic target for the most prevalent hormone-sensitive BCs.

SARS-CoV-2 and HIV: Impact on Pulmonary Epithelial Cells.

The SARS-CoV-2 pandemic provides a natural opportunity for the collision of coronavirus disease-2019 (COVID-19) with chronic infections, which place numerous individuals at high risk of severe COVID-19. Infection with Human Immunodeficiency Virus (HIV), a global epidemic, remains a major public health concern. Whether prior HIV+ status exacerbates COVID-19 warrants investigation. Herein, we characterized the impact of SARS-CoV-2 in human bronchial epithelial cells (HBECs) previously exposed to HIV. We optimized the air-liquid interface (ALI) cell culture technique to allow for challenges with HIV at the basolateral cell surface and SARS-CoV-2 spike protein on the apical surface, followed by genetic analyses for cellular stress/toxicity and innate/adaptive immune responses. Our results suggest that the IL-10 pathway was consistently activated in HBECs treated with spike, HIV, or a combination. Recombinant spike protein elicited COVID-19 cytokine storms while HIV activated different signaling pathways. HIV-treated HBECs could no longer activate NF-kB, pro-inflammatory TRAF-6 ubiquitination nor RIP1 signaling. Combinations of HIV and SARS-CoV-2 spike increased gene expression for activation of endoplasmic reticulum-phagosome pathway and downregulated non-canonical NF-kB pathways that are key in functional regulatory T cells and RNA Polymerase II transcription. Our in vitro studies suggest that prior HIV infection may not exacerbate COVID-19. Further in vivo studies are warranted to advance this field.

Chronic Administration of Cannabinoid Receptor 2 Agonist (JWH-133) Increases Ectopic Ovarian Tumor Growth and Endocannabinoids (Anandamide and 2-Arachidonoyl Glycerol) Levels in Immunocompromised SCID Female Mice.

Cannabinoid-based therapies are increasingly being used by cancer patients to treat chemotherapy-induced nausea and vomiting. Recently, cannabinoids have gained increased attention for their effects on cancer growth. Indeed, the effect of CB2 (JWH-015, JWH-133) agonists on breast cancer models have shown to reduce the size of breast cancer tumors. However, these studies assessing breast cancer progression were using CB2 agonist administered early into the cancer progression therefore assessing their effects on already established tumors is a critical need. In our study, we evaluate tumor growth using an ectopic xenograft ovarian (SKOV-3 and OVCAR-5) cancer model. The impact of chronic (30 days) administration of CB2 (JWH-133) agonist will be evaluated and started on 30 days of ectopic ovarian tumors. We will then evaluate and determine the mechanisms involved in ovarian cancer tumor growth by measuring levels of anandamide and 2-arachidonoyl glycerol as well as protein levels of CB1, CB2, ERα, ERß, GPER, TNFα, IL-1ß and IL-6 in ovarian and tumor tissues. Our results demonstrate a significant increase in ectopic ovarian tumor growth following chronic administration of JWH-133. Ovarian cancer tumor tissues chronically (30 days) treated with JWH-133 in comparison to vehicle treated groups showed an increase in endocannabinoid (AEA and 2-AG) and protein (CB2 and TNFα) levels with a decrease in GPER protein levels. Interestingly, our study emphasizes the importance of studying the impact of cannabinoid compounds on already established tumors to improve our understanding of cannabinoid-based therapies and, therefore better address clinical needs in cancer patients.

Dishevelled-1 DIX and PDZ domain lysine residues regulate oncogenic Wnt signaling.

DVL proteins are central mediators of the Wnt pathway and relay complex input signals into different branches of the Wnt signaling network. However, molecular mechanism(s) that regulate DVL-mediated relay of Wnt signals still remains unclear. Here, for the first time, we elucidate the functional significance of three DVL-1 lysines (K/Lys) which are subject to post-translational acetylation. We demonstrate that K34 Lys residue in the DIX domain regulates subcellular localization of ß-catenin, thereby influencing downstream Wnt target gene expression. Additionally, we show that K69 (DIX domain) and K285 (PDZ domain) regulate binding of DVL-1 to Wnt target gene promoters and modulate expression of Wnt target genes including CMYC, OCT4, NANOG, and CCND1, in cell line models and xenograft tumors. Finally, we report that conserved DVL-1 lysines modulate various oncogenic functions such as cell migration, proliferation, cell-cycle progression, 3D-spheroid formation and in-vivo tumor growth in breast cancer models. Collectively, these findings highlight the importance of DVL-1 domain-specific lysines which were recently shown to be acetylated and characterize their influence on Wnt signaling. These site-specific modifications may be subject to regulation by therapeutics already in clinical use (lysine deacetylase inhibitors such as Panobinostat and Vorinostat) or may possibly have prognostic utility in translational efforts that seek to modulate dysfunctional Wnt signaling.

Genomic profiling of DVL-1 and its nuclear role as a transcriptional regulator in triple negative breast cancer.

Dishevelled-1 (DVL-1) mediates Wnt signals critical for development and cellular homeostasis. DVL-1 is also linked with tumorigenesis, however its association with specific breast cancer (BC) subtypes and how it contributes to tumorigenicity remains poorly studied. Herein, using bioinformatics and genomics analyses, we investigate the role of DVL-1 in different molecular subtypes of BC. Our results demonstrate that DVL-1 is highly expressed in triple-negative BC compared to non-cancer tissues and associated with various clinical factors that may contribute to poor prognosis and survival rate. Another critical knowledge gap which remains poorly investigated involves the role of DVL-1 in the nucleus. While the cytoplasmic role of DVL-1 as a signaling hub has been extensively studied, the nuclear role of DVL-1 remains virtually unexplored. Herein for the first time, we have performed ChIP-Seq analyses to identify genomic regions targeted and regulated by DVL-1, thus highlighting its potential role as a regulator of transcription. Furthermore, we observed that DVL-1 peaks co-localize with H3K27me3 and EZH2, a repressive epigenetic mark and a histone methyltransferase respectively. Overall, our findings emphasize the importance of DVL-1 with TNBC-related pathology and identified unexpected gene targets of DVL-1, that may help explain the complexity of aberrant Wnt signaling in cancer.

Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth.

Dishevelled (DVL) critically regulates Wnt signaling and contributes to a wide spectrum of diseases and is important in normal and pathophysiological settings. However, how it mediates diverse cellular functions remains poorly understood. Recent discoveries have revealed that constitutive Wnt pathway activation contributes to breast cancer malignancy, but the mechanisms by which this occurs are unknown and very few studies have examined the nuclear role of DVL. Here, we have performed DVL3 ChIP-seq analyses and identify novel target genes bound by DVL3. We show that DVL3 depletion alters KMT2D binding to novel targets and changes their epigenetic marks and mRNA levels. We further demonstrate that DVL3 inhibition leads to decreased tumor growth in two different breast cancer models in vivo. Our data uncover new DVL3 functions through its regulation of multiple genes involved in developmental biology, antigen presentation, metabolism, chromatin remodeling, and tumorigenesis. Overall, our study provides unique insight into the function of nuclear DVL, which helps to define its role in mediating aberrant Wnt signaling.

The Versatility of Sirtuin-1 in Endocrinology and Immunology.

Sirtuins belong to the class III family of NAD-dependent histone deacetylases (HDAC) and are involved in diverse physiological processes that range from regulation of metabolism and endocrine function to coordination of immunity and cellular responses to stress. Sirtuin-1 (SIRT1) is the most well-studied family member and has been shown to be critically involved in epigenetics, immunology, and endocrinology. The versatile roles of SIRT1 include regulation of energy sensing metabolic homeostasis, deacetylation of histone and non-histone proteins in numerous tissues, neuro-endocrine regulation via stimulation of hypothalamus-pituitary axes, synthesis and maintenance of reproductive hormones via steroidogenesis, maintenance of innate and adaptive immune system via regulation of T- and B-cell maturation, chronic inflammation and autoimmune diseases. Moreover, SIRT1 is an appealing target in various disease contexts due to the promise of pharmacological and/or natural modulators of SIRT1 activity within the context of endocrine and immune-related disease models. In this review we aim to provide a broad overview on the role of SIRT1 particularly within the context of endocrinology and immunology.

Identification of Novel MeCP2 Cancer-Associated Target Genes and Post-Translational Modifications.

Abnormal regulation of DNA methylation and its readers has been associated with a wide range of cellular dysfunction. Disruption of the normal function of DNA methylation readers contributes to cancer progression, neurodevelopmental disorders, autoimmune disease and other pathologies. One reader of DNA methylation known to be especially important is MeCP2. It acts a bridge and connects DNA methylation with histone modifications and regulates many gene targets contributing to various diseases; however, much remains unknown about how it contributes to cancer malignancy. We and others previously described novel MeCP2 post-translational regulation. We set out to test the hypothesis that MeCP2 would regulate novel genes linked with tumorigenesis and that MeCP2 is subject to additional post-translational regulation not previously identified. Herein we report novel genes bound and regulated by MeCP2 through MeCP2 ChIP-seq and RNA-seq analyses in two breast cancer cell lines representing different breast cancer subtypes. Through genomics analyses, we localize MeCP2 to novel gene targets and further define the full range of gene targets within breast cancer cell lines. We also further examine the scope of clinical and pre-clinical lysine deacetylase inhibitors (KDACi) that regulate MeCP2 post-translationally. Through proteomics analyses, we identify many additional novel acetylation sites, nine of which are mutated in Rett Syndrome. Our study provides important new insight into downstream targets of MeCP2 and provide the first comprehensive map of novel sites of acetylation associated with both pre-clinical and FDA-approved KDACi used in the clinic. This report examines a critical reader of DNA methylation and has important implications for understanding MeCP2 regulation in cancer models and identifying novel molecular targets associated with epigenetic therapies.

PARP1 Deficiency Reduces Tumour Growth by Decreasing E2F1 Hyperactivation: A Novel Mechanism in the Treatment of Cancer.

In recent years, poly (ADP-ribose) polymerase (PARP) inhibitors have been evaluated for treating homologous recombination-deficient tumours, taking advantage of synthetic lethality. However, increasing evidence indicates that PARP1 exert several cellular functions unrelated with their role on DNA repair, including function as a co-activator of transcription through protein-protein interaction with E2F1. Since the RB/E2F1 pathway is among the most frequently mutated in many tumour types, we investigated whether the absence of PARP activity could counteract the consequences of E2F1 hyperactivation. Our results demonstrate that genetic ablation of Parp1 extends the survival of Rb-null embryos, while genetic inactivation of Parp1 results in reduced development of pRb-dependent tumours. Our results demonstrate that PARP1 plays a key role as a transcriptional co-activator of the transcription factor E2F1, an important component of the cell cycle regulation. Considering that most oncogenic processes are associated with cell cycle deregulation, the disruption of this PARP1-E2F1 interaction could provide a new therapeutic target of great interest and a wide spectrum of indications.

Análisis teórico de un posible futuro en la sociedad ciborg: robotización, estrés y epigenética / Theoretical analysis of a possible future in the cyborg society: robotization, stress and epigenetics / Análise teórica de um possível futuro na sociedade ciborgue: robotização, estresse e epigenética

La robótica ha generado, con el paso del tiempo, una enorme cantidad de imaginarios sociales presente en numerosas expresiones culturales. En el presente trabajo se hace un análisis preliminar de éstos para comprender mejor la nueva realidad social a la que nos podemos ver sometidos. A través de un análisis social teórico buscamos indagar elementos verosímiles que nos podemos encontrar en un futuro relativamente cercano. Concretamente nos centraremos en datos ofrecidos por la epigenética y analizaremos posibles realidades futuras (consecuencias) en base a esta información

Robotics has generated, over time, an enormous number of social imaginaries presents in plentiful cultural expressions. In the present work a preliminary analysis of these one is done to better understand the new social reality to which we can be subjected. Through a theoretical social analysis, we seek to investigate credible elements that we can find in a relatively close future. Specifically, we will focus on data offered by epigenetics and analyze possible future realities (consequences) based on this information

A robótica gerou ao longo do tempo uma enorme quantidade de imaginários sociais presentes em numerosas expressões culturais. No presente trabalho é feita uma análise preliminar destes para melhor entender a nova realidade social a qual podemos estar sujeitos. Através de uma análise social teórica, procuramos investigar elementos credíveis que podemos encontrar num futuro relativamente próximo. Especificamente, vamos nos concentrar nos dados oferecidos pela epigenética e analisar possíveis realidades futuras (consequências) com base nessas informações

Acetylation of conserved DVL-1 lysines regulates its nuclear translocation and binding to gene promoters in triple-negative breast cancer.

Dishevelled (DVL) proteins are central mediators of the Wnt signalling pathway and are versatile regulators of several cellular processes, yet little is known about their post-translational regulation. Acetylation is a reversible post-translational modification (PTM) which regulates the function of several non-histone proteins involved in tumorigenesis. Since we previously demonstrated that lysine deacetylase, SIRT-1, regulates DVL protein levels and its function, we reasoned that DVL could potentially be a substrate for SIRT-1 mediated deacetylation. To further examine the potential role of multiple families of lysine deacetylases in the post-translational regulation of DVL, we screened for novel acetylation sites using liquid chromatography mass-spectrometry (LC-MS/MS) analysis. Herein, we report 12 DVL-1 lysine residues that show differential acetylation in response to changes in oxygen tension and deacetylase inhibition in triple-negative breast cancer (TNBC). PTMs are well documented to influence protein activity, and cellular localization. We also identify that acetylation of two key lysine residues, K69 and K285, present on the DIX and PDZ domains respectively, promote nuclear over cytoplasmic localization of DVL-1, and influences its promoter binding and regulation of genes implicated in cancer. Collectively, these findings for the first time, uncover acetylation as a novel layer of regulation of DVL-1 proteins.

DVL1 and DVL3 differentially localize to CYP19A1 promoters and regulate aromatase mRNA in breast cancer cells.

The CYP19A1 gene encodes aromatase, an enzyme that converts androgens into estrogens and consequently directly contributes to both the depletion of androgens and the synthesis of estrogens in several organs. Aromatase is critical for diverse biological processes such as proliferation, regulation of fat metabolism and hormone signaling. Additionally, it is also overexpressed in diverse cancers and drives hormone-dependent tumor progression and increases 17-ß-estradiol (E2) within tumors and the tumor microenvironment. Although the inhibition of E2 production via aromatase inhibitors represents a major therapeutic paradigm in clinical oncology, fundamental questions regarding how cancer cells gain the capacity to overexpress aromatase remain unanswered. Multiple tissue-specific CYP19A1 promoters are known to be aberrantly active in tumors, yet how this occurs is unclear. Here, for the first time, we report that Dishevelled (DVL) proteins, which are key mediators of Wnt signaling, regulate aromatase expression in multiple breast cancer cell lines. We also report that DVL enters the nucleus and localizes to at least two different CYP19A1 promoters (pII and I.4) previously reported to drive overexpression in breast tumors and to a very distal CYP19A1 placental promoter (I.1) that remains poorly characterized. We go on to demonstrate that DVL-1 and DVL-3 loss of function leads to differential changes in various aromatase transcripts and in E2 production. The report, herein, uncovers a new regulator of CYP19A1 transcription and for the first time demonstrates that DVL, a critical mediator of WNT signaling, contributes to aberrant breast cancer-associated estrogen production.

Aromatase Acetylation Patterns and Altered Activity in Response to Sirtuin Inhibition.

Aromatase, a cytochrome P450 member, is a key enzyme involved in estrogen biosynthesis and is dysregulated in the majority of breast cancers. Studies have shown that lysine deacetylase inhibitors (KDI) decrease aromatase expression in cancer cells, yet many unknowns remain regarding the mechanism by which this occurs. However, advances have been made to clarify factors involved in the transcriptional regulation of the aromatase gene (CYP19A1). Yet, despite aromatase being a primary target for breast cancer therapy, its posttranslational regulation has been virtually unexplored. Acetylation is a posttranslational modification (PTM) known to alter the activity and stability of many oncoproteins, and given the role of KDIs in regulating aromatase expression, we postulate that aromatase acetylation acts as a novel posttranslational regulatory mechanism that impacts aromatase expression and/or activity in breast cancer. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that aromatase is basally acetylated on several lysine residues (108, 169, 242, 262, 334, 352, and 354) in MCF-7 cells, and treatment with a SIRT-1 inhibitor induced additional acetylation (376, 390, 440, and 448). These acetylated lysine residues are in regions critical for aromatase activity. Site-directed mutagenesis and overexpression studies demonstrated that K108R/Q or K440R/Q mutations significantly altered aromatase activity in breast cancer cells without altering its subcellular localization.Implications: These findings demonstrate a novel posttranslational regulation of aromatase and uncover novel anticancer effects of deacetylase inhibitors, thus providing new insight for ongoing development of deacetylase inhibitors as cancer therapeutics. Mol Cancer Res; 16(10); 1530-42. ©2018 AACR.

Dishevelled: A masterful conductor of complex Wnt signals.

The Dishevelled gene was first identified in Drosophila mutants with disoriented hair and bristle polarity [1-3]. The Dsh gene (Dsh/Dvl, in Drosophila and vertebrates respectively) gained popularity when it was discovered that it plays a key role in segment polarity during early embryonic development in Drosophila [4]. Subsequently, the vertebrate homolog of Dishevelled genes were identified in Xenopus (Xdsh), mice (Dvl1, Dvl2, Dvl3), and in humans (DVL1, DVL2, DVL3) [5-10]. Dishevelled functions as a principal component of Wnt signaling pathway and governs several cellular processes including cell proliferation, survival, migration, differentiation, polarity and stem cell renewal. This review will revisit seminal discoveries and also summarize recent advances in characterizing the role of Dishevelled in both normal and pathophysiological settings.

Lipopolysaccharide (LPS)-induced septic shock causes profound changes in myocardial energy metabolites in pigs.

INTRODUCTION: Energy deficiency is a cause for myocardial dysfunction during septic shock. In rodents, septic shock decreases the oxidation of long-chain fatty acids and glucose in the myocardium causing energy deficiency. However, the effect of septic shock on myocardial energy metabolites in large animals and human is unknown. OBJECTIVES: Investigate the effects of septic shock on myocardial energy metabolites in domestic pigs. METHODS: Seventeen female pigs divided into control and lipopolysaccharide (LPS)-induced septic shock groups. Myocardial metabolites were analyzed ex vivo by 1H nuclear magnetic resonance spectroscopy and liquid chromatography-tandem mass spectrometry. Gene and protein expression analysis were analyzed by real-time PCR and western blot. RESULTS: Septic shock was associated with an increase in myocardial levels of short- and medium-chain acylcarnitines, lactate, alanine, and pyruvate dehydrogenase kinase 4 gene expression. COX-2 and prostaglandin E4 receptor gene expression also increased in the septic myocardium, although the only elevated eicosanoid in the septic animals was thromboxane B2. Myocardial levels of niacin, taurine, glutamate, glutamine, and glutathione were higher, and hypoxanthine levels lower in septic pigs than controls. CONCLUSIONS: In pigs, septic shock induced by LPS caused myocardial changes directed to decrease the oxidation of medium- and short-chain fatty acid without an effect on long-chain fatty acid oxidation. The increase in myocardial levels of lactate, alanine, and pyruvate dehydrogenase kinase 4 gene expression suggest that septic shock decreases pyruvate dehydrogenase complex activity and glucose oxidation. Homeostasis of niacin, taurine, glutamate, glutamine, glutathione, hypoxanthine and thromboxane B2 is also affected in the septic myocardium.

Hydrogen sulphide inhibits Ca2+ release through InsP3 receptors and relaxes airway smooth muscle.

Hydrogen sulphide (H2S) is a signalling molecule that appears to regulate diverse cell physiological process in several organs and systems including vascular and airway smooth muscle cell (SMC) contraction. Decreases in endogenous H2S synthesis have been associated with the development of cardiovascular diseases and asthma. Here we investigated the mechanism of airway SMC relaxation induced by H2S in small intrapulmonary airways using mouse lung slices and confocal and phase-contrast video microscopy. Exogenous H2S donor Na2S (100 µm) reversibly inhibited Ca(2+) release and airway contraction evoked by inositol-1,4,5-trisphosphate (InsP3) uncaging in airway SMCs. Similarly, InsP3-evoked Ca(2+) release and contraction was inhibited by endogenous H2S precursor l-cysteine (10 mm) but not by l-serine (10 mm) or either amino acid in the presence of dl-propargylglycine (PPG). Consistent with the inhibition of Ca(2+) release through InsP3 receptors (InsP3Rs), Na2S reversibly inhibited acetylcholine (ACh)-induced Ca(2+) oscillations in airway SMCs. In addition, Na2S, the H2S donor GYY-4137, and l-cysteine caused relaxation of airways pre-contracted with either ACh or 5-hydroxytryptamine (5-HT). Na2S-induced airway relaxation was resistant to a guanylyl cyclase inhibitor (ODQ) and a protein kinase G inhibitor (Rp-8-pCPT-cGMPS). The effects of H2S on InsP3-evoked Ca(2+) release and contraction as well as on the relaxation of agonist-contracted airways were mimicked by the thiol-reducing agent dithiothreitol (DTT, 10 mm) and inhibited by the oxidizing agent diamide (30 µm). These studies indicate that H2S causes airway SMC relaxation by inhibiting Ca(2+) release through InsP3Rs and consequent reduction of agonist-induced Ca(2+) oscillations in SMCs. The results suggest a novel role for endogenously produced H2S that involves the modulation of InsP3-evoked Ca(2+) release - a cell-signalling system of critical importance for many physiological and pathophysiological processes.

Estudio clínico y molecular de cinco familias con resistencia a la acción de las hormonas tiroideas / Clinical and molecular study of five families with resistance to thyroid hormones

Fundamento y objetivo: La resistencia a la acción de las hormonas tiroideas (SRHT) es un síndrome causado mayoritariamente por mutaciones en el gen receptor beta de las hormonas tiroideas (THRB). Se estudian cinco familias con fenotipo de SRHT. Pacientes y método: Se realizó secuenciación de THRB. Se evaluó la respuesta a triyodotironina (T3) y efecto dominante negativo de los mutantes in vitro y se estudiaron los mecanismos de resistencia en sujetos sin mutación THRB cuantificando en cultivos de fibroblastos cambios de expresión en los genes regulator of calcineurin 2 (ZAKI4) y Kruppel-like factor 9 (BTEB). Resultados: Tres casos presentaron mutaciones en THRB: R243Q, R320C, R429Q, dando lugar a receptores TRβ con menor respuesta a T3. R243Q y R320C ejercen efecto dominante negativo. Un sujeto sin mutación THRB presentó cambios de expresión en ZAKI4 y BTEB similar a R230C, mientras que el otro mostró niveles de expresión superiores a los controles. Conclusiones: Mutaciones heterocigotas en THRB causaron tres de los casos de SRHT estudiados. Uno de los casos con SRHT sin mutación se comporta a nivel molecular como los portadores de mutación, mientras que en el otro la resistencia no está mediada por TRβ (AU)

Background and objective: Resistance to thyroid hormone (RTH) is a syndrome mostly caused by mutations in thyroid hormone receptor beta gen (THRB). We present five families with RTH phenotype. Patients and methods:THRB gene sequencing. In vitro studies to evaluate the mutants response to thyroid hormones and their dominant negative effect. Mechanism of resistance in patients with RTH without THRB mutations quantifying expression of regulator of calcineurin 2 (ZAKI4) and Kruppel-like factor 9 (BTEB) genes in patients fibroblast cultures.Results: THRB mutations were found in three cases: R243Q, R320C, R429Q. Mutants showed a decreased response to T3. R243Q and R320C had a strong dominant negative effect. One subject without THRB mutation showed changes in ZAKI4 and BTEB expression similar to R320C and the other showed expression levels higher than normal controls. Conclusions:Three cases of RTH were caused by THRB heterozygous mutations but in two cases mutations were not found. THRB mutation carriers and one of the patients without mutations share a similar mechanism of resistance and in the other subject RTH is TRβ independent (AU)

[Clinical and molecular study of five families with resistance to thyroid hormones]. / Estudio clínico y molecular de cinco familias con resistencia a la acción de las hormonas tiroideas.

BACKGROUND AND OBJECTIVE: Resistance to thyroid hormone (RTH) is a syndrome mostly caused by mutations in thyroid hormone receptor beta gen (THRB). We present five families with RTH phenotype. PATIENTS AND METHODS: THRB gene sequencing. In vitro studies to evaluate the mutants response to thyroid hormones and their dominant negative effect. Mechanism of resistance in patients with RTH without THRB mutations quantifying expression of regulator of calcineurin 2 (ZAKI4) and Kruppel-like factor 9 (BTEB) genes in patients fibroblast cultures. RESULTS: THRB mutations were found in three cases: R243Q, R320C, R429Q. Mutants showed a decreased response to T3. R243Q and R320C had a strong dominant negative effect. One subject without THRB mutation showed changes in ZAKI4 and BTEB expression similar to R320C and the other showed expression levels higher than normal controls. CONCLUSIONS: Three cases of RTH were caused by THRB heterozygous mutations but in two cases mutations were not found. THRB mutation carriers and one of the patients without mutations share a similar mechanism of resistance and in the other subject RTH is TRß independent.