Engraftment of connexin 43-expressing cells prevents post-infarct arrhythmia

Ventricular tachyarrhythmias are the main cause of sudden death in patients after myocardial infarction. Here we show that transplantation of embryonic cardiomyocytes (eCMs) in myocardial infarcts protects against the induction of ventricular tachycardia (VT) in mice. Engraftment of eCMs, but not skeletal myoblasts (SMs), bone marrow cells or cardiac myofibroblasts, markedly decreased the incidence of VT induced by in vivo pacing. eCM engraftment results in improved electrical coupling between the surrounding myocardium and the infarct region, and Ca2+ signals from engrafted eCMs expressing a genetically encoded Ca2+ indicator could be entrained during sinoatrial cardiac activation in vivo. eCM grafts also increased conduction velocity and decreased the incidence of conduction block within the infarct. VT protection is critically dependent on expression of the gap-junction protein connexin 43 (Cx43; also known as Gja1): SMs genetically engineered to express Cx43 conferred a similar protection to that of eCMs against induced VT. Thus, engraftment of Cx43-expressing myocytes has the potential to reduce life-threatening post-infarct arrhythmias through the augmentation of intercellular coupling, suggesting autologous strategies for cardiac cell-based therapy.     

Cytoskeleton-mediated templating of complex cellulose-scaffolded extracellular structure and its association with oikosins in the urochordate <Emphasis Type="Italic">Oikopleura</Emphasis>

Oriented cellulose deposition is critical to plant patterning and models suggest microtubules constrain cellulose synthase movements through the plasma membrane. Though widespread in plants, urochordates are the only animals that synthesize cellulose. We characterized the distinctive cellulose microfibril scaffold of the larvacean house and its interaction with house structural proteins (oikosins). Targeted disruption of cytoskeletal elements, secretory pathways, and plasma membrane organization, suggested a working model for templating extracellular cellulose microfibrils from animal cells that shows both convergence and differences to plant models. Specialized cortical F-actin arrays template microfibril orientation and glycosylphosphatidylinositol-anchored proteins in lipid rafts may act as scaffolding proteins in microfibril elongation. Microtubules deliver and maintain cellulose synthase complexes to specific cell membrane sites rather than orienting their movement through the membrane. Oikosins are incorporated into house compartments directly above their corresponding cellular field of expression and interact with the cellulose scaffold to a variable extent.     

Mutations in NNT encoding nicotinamide nucleotide transhydrogenase cause familial glucocorticoid deficiency

Using targeted exome sequencing, we identified mutations in NNT, an antioxidant defense gene, in individuals with familial glucocorticoid deficiency. In mice with Nnt loss, higher levels of adrenocortical cell apoptosis and impaired glucocorticoid production were observed. NNT knockdown in a human adrenocortical cell line resulted in impaired redox potential and increased reactive oxygen species (ROS) levels. Our results suggest that NNT may have a role in ROS detoxification in human adrenal glands.     

Amplification of siRNA in Caenorhabditis elegans generates a transgenerational sequence-targeted histone H3 lysine 9 methylation footprint

Exogenous double-stranded RNA (dsRNA) has been shown to exert homology-dependent effects at the level of both target mRNA stability and chromatin structure. Using C. elegans undergoing RNAi as an animal model, we have investigated the generality, scope and longevity of dsRNA-targeted chromatin effects and their dependence on components of the RNAi machinery. Using high-resolution genome-wide chromatin profiling, we found that a diverse set of genes can be induced to acquire locus-specific enrichment of histone H3 lysine 9 trimethylation (H3K9me3), with modification footprints extending several kilobases from the site of dsRNA homology and with locus specificity sufficient to distinguish the targeted locus from the other 20,000 genes in the C. elegans genome. Genetic analysis of the response indicated that factors responsible for secondary siRNA production during RNAi were required for effective targeting of chromatin. Temporal analysis revealed that H3K9me3, once triggered by dsRNA, can be maintained in the absence of dsRNA for at least two generations before being lost. These results implicate dsRNA-triggered chromatin modification in C. elegans as a programmable and locus-specific response defining a metastable state that can persist through generational boundaries.     

Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem of unknown etiology that varies in prevalence among ancestry groups. To identify genetic variants contributing to differences in hepatic fat content, we carried out a genome-wide association scan of nonsynonymous sequence variations (n = 9,229) in a population comprising Hispanic, African American and European American individuals. An allele in PNPLA3 (rs738409[G], encoding I148M) was strongly associated with increased hepatic fat levels (P = 5.9 x 10(-10)) and with hepatic inflammation (P = 3.7 x 10(-4)). The allele was most common in Hispanics, the group most susceptible to NAFLD; hepatic fat content was more than twofold higher in PNPLA3 rs738409[G] homozygotes than in noncarriers. Resequencing revealed another allele of PNPLA3 (rs6006460[T], encoding S453I) that was associated with lower hepatic fat content in African Americans, the group at lowest risk of NAFLD. Thus, variation in PNPLA3 contributes to ancestry-related and inter-individual differences in hepatic fat content and susceptibility to NAFLD.     

Autonomous translocation and intracellular trafficking of the cell-penetrating and immune-suppressive effector protein YopM

Extracellular Gram-negative pathogenic bacteria target essential cytoplasmic processes of eukaryotic cells by using effector protein delivery systems such as the type III secretion system (T3SS). These secretion systems directly inject effector proteins into the host cell cytoplasm. Among the T3SS-dependent Yop proteins of pathogenic Yersinia, the function of the effector protein YopM remains enigmatic. In a recent study, we demonstrated that recombinant YopM from Yersinia enterocolitica enters host cells autonomously without the presence of bacteria and thus identified YopM as a novel bacterial cell-penetrating protein. Following entry YopM down-regulates expression of pro-inflammatory cytokines such as tumor necrosis factor α. These properties earmark YopM for further development as a novel anti-inflammatory therapeutic. To elucidate the uptake and intracellular targeting mechanisms of this bacterial cell-penetrating protein, we analyzed possible routes of internalization employing ultra-cryo electron microscopy. Our results reveal that under physiological conditions, YopM enters cells predominantly by exploiting endocytic pathways. Interestingly, YopM was detected free in the cytosol and inside the nucleus. We could not observe any colocalization of YopM with secretory membranes, which excludes retrograde transport as the mechanism for cytosolic release. However, our findings indicate that direct membrane penetration and/or an endosomal escape of YopM contribute to the cytosolic and nuclear localization of the protein. Surprisingly, even when endocytosis is blocked, YopM was found to be associated with endosomes. This suggests an intracellular endosome-associated transport of YopM.     

Progesterone-induced blocking factor differentially regulates trophoblast and tumor invasion by altering matrix metalloproteinase activity

Invasiveness is a common feature of trophoblast and tumors; however, while tumor invasion is uncontrolled, trophoblast invasion is strictly regulated. Both trophoblast and tumor cells express high levels of the immunomodulatory progesterone-induced blocking factor (PIBF), therefore, we aimed to test the possibility that PIBF might be involved in invasion. To this aim, we used PIBF-silenced or PIBF-treated trophoblast (HTR8/Svneo, and primary trophoblast) and tumor (HT-1080, A549, HCT116, PC3) cell lines. Silencing of PIBF increased invasiveness as well as MMP-2,-9 secretion of HTR8/SVneo, and decreased those of HT-1080 cells. PIBF induced immediate STAT6 activation in both cell lines. Silencing of IL-4Rα abrogated all the above effects of PIBF, suggesting that invasion-related signaling by PIBF is initiated through the IL-4Rα/PIBF-receptor complex. In HTR-8/SVneo, PIBF induced fast, but transient Akt and ERK phosphorylation, whereas in tumor cells, PIBF triggered sustained Akt, ERK, and late STAT3 activation. The late signaling events might be due to indirect action of PIBF. PIBF induced the expression of EGF and HB-EGF in HT-1080 cells. The STAT3-activating effect of PIBF was reduced in HB-EGF-deficient HT-1080 cells, suggesting that PIBF-induced HB-EGF contributes to late STAT3 activation. PIBF binds to the promoters of IL-6, EGF, and HB-EGF; however, the protein profile of the protein/DNA complex is different in the two cell lines. We conclude that in tumor cells, PIBF induces proteins, which activate invasion signaling, while—based on our previous data—PIBF might control trophoblast invasion by suppressing proinvasive genes.