The Drosophila IKK-related kinase (Ik2) and Spindle-F proteins are part of a complex that regulates cytoskeleton organization during oogenesis

<p>Abstract</p> <p>Background</p> <p>IkappaB kinases (IKKs) regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. The <it>Drosophila </it>genome encodes two members of the IKK family. Whereas the first is a kinase essential for activation of the NF-kappaB pathway, the latter does not act as IkappaB kinase. Instead, recent findings indicate that Ik2 regulates F-actin assembly by mediating the function of nonapoptotic caspases via degradation of DIAP1. Also, it has been suggested that <it>ik2 </it>regulates interactions between the minus ends of the microtubules and the actin-rich cortex in the oocyte. Since <it>spn-F </it>mutants display oocyte defects similar to those of <it>ik2 </it>mutant, we decided to investigate whether Spn-F could be a direct regulatory target of Ik2.</p> <p>Results</p> <p>We found that Ik2 binds physically to Spn-F, biomolecular interaction analysis of Spn-F and Ik2 demonstrating that both proteins bind directly and form a complex. We showed that Ik2 phosphorylates Spn-F and demonstrated that this phosphorylation does not lead to Spn-F degradation. Ik2 is localized to the anterior ring of the oocyte and to punctate structures in the nurse cells together with Spn-F protein, and both proteins are mutually required for their localization.</p> <p>Conclusion</p> <p>We conclude that Ik2 and Spn-F form a complex, which regulates cytoskeleton organization during <it>Drosophila </it>oogenesis and in which Spn-F is the direct regulatory target for Ik2. Interestingly, Ik2 in this complex does not function as a typical IKK in that it does not direct SpnF for degradation following phosphorylation.</p

Obstructive Sleep Apnea Syndrome In Vitro Model: Controlled Intermittent Hypoxia Stimulation of Human Stem Cells-Derived Cardiomyocytes

Cardiovascular morbidity is the leading cause of death of obstructive sleep apnea (OSA) syndrome patients. Nocturnal airway obstruction is associated with intermittent hypoxia (IH). In our previous work with cell lines, incubation with sera from OSA patients induced changes in cell morphology, NF-&kappa;B activation and decreased viability. A decrease in beating rate, contraction amplitude and a reduction in intracellular calcium signaling was also observed in human cardiomyocytes differentiated from human embryonic stem cells (hESC-CMs). We expanded these observations using a new controlled IH in vitro system on beating hESC-CMs. The Oxy-Cycler system was programed to generate IH cycles. Following IH, we detected the activation of Hif-1&alpha; as an indicator of hypoxia and nuclear NF-&kappa;B p65 and p50 subunits, representing pro-inflammatory activity. We also detected the secretion of inflammatory cytokines, such as MIF, PAI-1, MCP-1 and CXCL1, and demonstrated a decrease in beating rate of hESC-CMs following IH. IH induces the co-activation of inflammatory features together with cardiomyocyte alterations which are consistent with myocardial damage in OSA. This study provides an innovative approach for in vitro studies of OSA cardiovascular morbidity and supports the search for new pharmacological agents and molecular targets to improve diagnosis and treatment of patients

Functional defects in hiPSCs-derived cardiomyocytes from patients with a PLEKHM2-mutation associated with dilated cardiomyopathy and left ventricular non-compaction

Abstract Dilated cardiomyopathy (DCM) is a primary myocardial disease, leading to heart failure and excessive risk of sudden cardiac death with rather poorly understood pathophysiology. In 2015, Parvari's group identified a recessive mutation in the autophagy regulator, PLEKHM2 gene, in a family with severe recessive DCM and left ventricular non-compaction (LVNC). Fibroblasts isolated from these patients exhibited abnormal subcellular distribution of endosomes, Golgi apparatus, lysosomes and had impaired autophagy flux. To better understand the effect of mutated PLEKHM2 on cardiac tissue, we generated and characterized induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) from two patients and a healthy control from the same family. The patient iPSC-CMs showed low expression levels of genes encoding for contractile functional proteins (α and β-myosin heavy chains and 2v and 2a-myosin light chains), structural proteins integral to heart contraction (Troponin C, T and I) and proteins participating in Ca2+ pumping action (SERCA2 and Calsequestrin 2) compared to their levels in control iPSC-derived CMs. Furthermore, the sarcomeres of the patient iPSC-CMs were less oriented and aligned compared to control cells and generated slowly beating foci with lower intracellular calcium amplitude and abnormal calcium transient kinetics, measured by IonOptix system and MuscleMotion software. Autophagy in patient’s iPSC-CMs was impaired as determined from a decrease in the accumulation of autophagosomes in response to chloroquine and rapamycin treatment, compared to control iPSC-CMs. Impairment in autophagy together with the deficiency in the expression of NKX2.5, MHC, MLC, Troponins and CASQ2 genes, which are related to contraction-relaxation coupling and intracellular Ca2+ signaling, may contribute to the defective function of the patient CMs and possibly affect cell maturation and cardiac failure with time

Reactive Rules Inference from Dynamic Dependency Models

Defining dependency models is sometimes an easier, more intuitive way for ontology representation than defining reactive rules directly, as it provides a higher level of abstraction. We will shortly introduce the ADI (Active Dependency Integration) model capabilities, emphasizing new developments: 1. Support of automatic dependencies instantiation from an abstract definition that expresses a general dependency in the ontology, namely a &quot;template&quot;

Dock10 Regulates Cardiac Function under Neurohormonal Stress

Dedicator of cytokinesis 10 (Dock10) is a guanine nucleotide exchange factor for Cdc42 and Rac1 that regulates the JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) signaling cascades. In this study, we characterized the roles of Dock10 in the myocardium. In vitro: we ablated Dock10 in neonatal mouse floxed Dock10 cardiomyocytes (NMCMs) and cardiofibroblasts (NMCFs) by transduction with an adenovirus expressing Cre-recombinase. In vivo, we studied mice in which the Dock10 gene was constitutively and globally deleted (Dock10 KO) and mice with cardiac myocyte-specific Dock10 KO (Dock10 CKO) at baseline and in response to two weeks of Angiotensin II (Ang II) infusion. In vitro, Dock10 ablation differentially inhibited the &alpha;-adrenergic stimulation of p38 and JNK in NMCM and NMCF, respectively. In vivo, the stimulation of both signaling pathways was markedly attenuated in the heart. The Dock10 KO mice had normal body weight and cardiac size. However, echocardiography revealed mildly reduced systolic function, and IonOptix recordings demonstrated reduced contractility and elevated diastolic calcium levels in isolated cardiomyocytes. Remarkably, Dock10 KO, but not Dock10 CKO, exaggerated the pathological response to Ang II infusion. These data suggest that Dock10 regulates cardiac stress-related signaling. Although Dock10 can regulate MAPK signaling in both cardiomyocytes and cardiofibroblasts, the inhibition of pathological cardiac remodeling is not apparently due to the Dock10 signaling in the cardiomyocyte