Cells of Bacillus subtilis can either be motile or sessile, depending on the expression of mutually exclusive sets of genes that are required for flagellum or biofilm formation, respectively. Both activities are coordinated by the master regulator SinR. We have analyzed the role of the previously uncharacterized ymdB gene for bistable gene expression in B. subtilis. We observed a strong overexpression of the hag gene encoding flagellin and of other genes of the D -dependent motility regulon in the ymdB mutant, whereas the two major operons for biofilm formation, tapA-sipW-tasA and epsA-O, were not expressed. As a result, the ymdB mutant is unable to form biofilms. An analysis of the individual cells of a population revealed that the ymdB mutant no longer exhibited bistable behavior; instead, all cells are short and motile. The inability of the ymdB mutant to form biofilms is suppressed by the deletion of the sinR gene encoding the master regulator of biofilm formation, indicating that SinR-dependent repression of biofilm genes cannot be relieved in a ymdB mutant. Our studies demonstrate that lack of expression of SlrR, an antagonist of SinR, is responsible for the observed phenotypes. Overexpression of SlrR suppresses the effects of a ymdB mutation.

V ascular smooth muscle cell (VSMC) activation and phenotypic switching are critical for remodeling processes in vascular proliferative disorders, including intimal hyperplasia. Both the migratory and proliferative activities of VSMCs, as well as the interplay between the extracellular matrix (ECM) and integrin receptors essentially, contribute to neointimal hyperplasia and restrictive remodeling processes in the vessels.1 Among integrins, the particular role of integrin α V β 3 in the induction of VSMC responses has been shown both in vivo and in vitro.

Human pluripotent stem cell (hPSC)-derived cardiomyocytes hold great potential for in vitro modeling of diseases like cardiomyopathies. Yet, knowledge about expression and functional impact of sarcomeric protein isoforms like the myosin heavy chain (MyHC) in hPSC-cardiomyocytes is scarce. We hypothesized that ventricular β-MyHC expression alters contraction and calcium kinetics and drives morphological and electrophysiological differentiation towards ventricular-like cardiomyocytes. To address this, we (1) generated human embryonic stem cell-derived cardiomyocytes (hESC-CMs) that switched towards exclusive β-MyHC, and (2) functionally and morphologically characterized these hESC-CMs at the single-cell level. MyHC-isoforms and functional properties were investigated during prolonged in vitro culture of cardiomyocytes in floating cardiac bodies (soft conditions) vs. culture on a stiff matrix. Using a specific anti-β-MyHC and a newly generated anti-α-MyHC-antibody, we found individual cardiomyocytes grown in cardiac bodies to mostly express both α- and β-MyHC-protein isoforms. Yet, 35 and 75 days of cultivation on laminin-coated glass switched 66 and 87 % of all cardiomyocytes to exclusively express β-MyHC, respectively. Twitch contraction and calcium transients were faster for CMs on laminin-glass. Surprisingly, both parameters were only little affected by the MyHC-isoform, although hESC-CMs with only β-MyHC had much lower ATP-turnover and tension cost, just as in human ventricular cardiomyocytes. Spontaneous contractions and no strict coupling of β-MyHC to ventricular-like action potentials suggest that MyHC-isoform expression does not fully determine the hESC-CM differentiation status. Stiff substrate-induced pure β-MyHC-protein expression in hESC-CMs, with several contractile parameters close to ventricular cardiomyocytes, provides a well-defined in vitro system for modeling of cardiomyopathies and drug screening approaches.

Evidence suggests that autophagy promotes the development of cellular senescence. Because cellular senescence contributes to renal aging and promotes the progression from AKI to CKD, we investigated the potential effect of tubular autophagy on senescence induction. Compared with kidneys from control mice, kidneys from mice with conditional deletion of autophagy-related 5 (Atg5) for selective ablation of autophagy in proximal tubular S3 segments (Atg5 Dflox/Dflox ) presented with significantly less tubular senescence, reduced interstitial fibrosis, and superior renal function 30 days after ischemia/reperfusion injury. To correlate this long-term outcome with differences in the early injury process, kidneys were analyzed 2 hours and 3 days after reperfusion. Notably, compared with kidneys of control mice, Atg5 Dflox/Dflox kidneys showed more cell death in outer medullary S3 segments at 2 hours but less tubular damage and inflammation at day 3. These data suggest that the lack of autophagy prevents early survival mechanisms in severely damaged tubular cells. However, if such compromised cells persist, then they may lead to maladaptive repair and proinflammatory changes, thereby facilitating the development of a senescent phenotype and CKD.

BackgroundInterstitial lung disease occurring in children is a condition characterized by high frequency of cases due to genetic aberrations of pulmonary surfactant homeostasis, that are also believed to be responsible of a fraction of familial pulmonary fibrosis. To our knowledge, ABCA3 gene was not previously reported as causative agent of fibrosis affecting both children and adults in the same kindred.MethodsWe investigated a large kindred in which two members, a girl whose interstitial lung disease was first recognized at age of 13, and an adult, showed a diffuse pulmonary fibrosis with marked differences in terms of morphology and imaging. An additional, asymptomatic family member was detected by genetic analysis. Surfactant abnormalities were investigated at biochemical, and genetic level, as well as by cell transfection experiments.ResultsBronchoalveolar lavage fluid analysis of the patients revealed absence of surfactant protein C, whereas the gene sequence was normal. By contrast, sequence of the ABCA3 gene showed a novel homozygous G > A transition at nucleotide 2891, localized within exon 21, resulting in a glycine to aspartic acid change at codon 964. Interestingly, the lung specimens from the girl displayed a morphologic usual interstitial pneumonitis-like pattern, whereas the specimens from one of the two adult patients showed rather a non specific interstitial pneumonitis-like pattern.ConclusionsWe have detected a large kindred with a novel ABCA3 mutation likely causing interstitial lung fibrosis affecting either young and adult family members. We suggest that ABCA3 gene should be considered in genetic testing in the occurrence of familial pulmonary fibrosis.