Maturation of human induced pluripotent stem cell derived engineered cardiac tissues using chief transfection and chronic optical pacing.

The immaturity of engineered cardiac tissues (ECTs) limits their ability to regenerate damaged myocardium and to serve as in vitro disease models and surrogates for drug toxicity testing. Several chronic biomimetic conditioning protocols, including mechanical stretch, perfusion, and electrical stimulation promote ECT maturation but have significant technical limitations. Non-contacting chronic light stimulation using heterologously expressed light-sensitive ChIEF ion channels, termed optogenetics, may be an advantageous alternative to chronic electrical stimulation. As a proof of principle, we transfected ECTs using an AAV packaged ChIEF and then verified acute optical pacing (OP) by patch clamp. We then chronically OP ECTs for 7 days above the intrinsic beat rate. Chronic OP resulted in improved ECT electrophysiological properties; however, ECT force generation and histology remained unchanged. Some changes in cardiac relevant gene expression were noted. This work validates a novel chronic OP paradigm that can be used to identify strategies for optimal in vitro ECT maturation

Evidence from individual firm-bank relationships in Germany

What began as a financial crisis in the United States in 2007–2008 quickly evolved into a massive crisis of the global real economy. We investigate the importance of the bank lending and firm borrowing channel in the international transmission of bank distress to the real economy—in particular, to real investment and labor employment by nonfinancial firms. We analyze whether and to what extent firms are able to compensate for the shortage in loan supply by switching banks and by using other types of financing. The analysis is based on a unique matched data set for Germany that contains firm-level financial statements for the 2004–2010 period together with the financial statements of each firm’s relationship bank(s). We use instrumental variable estimations in first differences to eliminate firm- and bankspecific effects. The first stage results show that banks that suffered losses due to proprietary trading activities at the onset of the financial crisis reduced their lending more strongly than non-affected banks. In the second stage, we find that firms whose relationship banks reduce credit supply downsize their real investment and labor employment significantly. This effect is larger for firms that are unable to provide much collateral. We document that firms partially offset reduced credit supply by establishing new bank relationships, using internal funds, and issuing new equity

713-4 Inhibition of Vascular Superoxide Production in Hypercholesterolemic Rabbit Aorta by L-Arginine Contributes to Restored Endothelium-dependent Relaxation

Chronic oral administration of L-arginine (L-ARG) has been shown to enhance endothelial function in cholesterol (CHOL)-fed rabbits and to reduce atherogenesis. We investigated whether modulation of endogenous NO production (as assessed by urinary NO3-excretion) by L-ARG and the inhibitor of NO synthesis, L-NAME, affects vascular superoxide (O2-) production in hypercholesterolemic rabbits. Phorbol-myristate-acetate (PMA)-stimulated O2-production from isolated aortic rings was increased in rabbits given CHOL (+159±28%) or CHOL + L-NAME (+149±37%) as compared to controls (-22±7%), and endothelium-dependent relaxations by acetylcholine were diminished in both groups. In aortic rings from rabbits given CHOL + L-ARG, PMA-induced O2-production was restored to control levels (+14±17%; p<0.05), and endothelium-dependent cholinergic relaxations were also partly restored. Urinary NO3-excretion decreased in all animals fed a CHOL-enriched diet (p<0.01). As NO inactivated by O2-is also oxidized to NO3-, this indicates a decreased endothelial production of NO. NO3-excretion was further decreased by L-NAME (p<0.05 vs. CHOL), and partly restored by L-ARG (p<0.05). We conclude that both a decreased production of NO and an enhanced breakdown of NO by O2-contribute to the diminished biological activity of endothelial NO in hypercholesterolemia. L-ARG restores endothelial function by enhancing NO formation and by protecting NO from early breakdown by O2-

Serum antioxidants as predictors of the adult respiratory distress syndrome in septic patients

Adult respiratory distress syndrome (ARDS) can develop as a complication of various disorders, including sepsis, but it has not been possible to identify which of the patients at risk will develop this serious disorder. We have investigated the ability of six markers, measured sequentially in blood, to predict development of ARDS in 26 patients with sepsis. At the initial diagnosis of sepsis (6-24 h before the development of ARDS), serum manganese superoxide dismutase concentration and catalase activity were higher in the 6 patients who subsequently developed ARDS than in 20 patients who did not develop ARDS. These changes in antioxidant enzymes predicted the development of ARDS in septic patients with the same sensitivity, specificity, and efficiency as simultaneous assessments of serum lactate dehydrogenase activity and factor VIII concentration. By contrast, serum glutathione peroxidase activity and α1Pi-elastase complex concentration did not differ at the initial diagnosis of sepsis between patients who did and did not subsequently develop ARDS, and were not as effective in predicting the development of ARDS. Measurement of manganese superoxide dismutase and catalase, in addition to the other markers, should facilitate identification of patients at highest risk of ARDS and allow prospective treatment

Rare missense variants in Tropomyosin-4 (TPM4) are associated with platelet dysfunction, cytoskeletal defects, and excessive bleeding

Background: A significant challenge is faced for the genetic diagnosis of inherited platelet disorders in which candidate genetic variants can be found in more than 100 bleeding, thrombotic, and platelet disorder genes, especially within families in which there are both normal and low platelet counts. Genetic variants of unknown clinical significance (VUS) are found in a significant proportion of such patients in which functional studies are required to prove pathogenicity. Objective: To identify the genetic cause in patients with a suspected platelet disorder and subsequently perform a detailed functional analysis of the candidate genetic variants found. Methods: Genetic and functional studies were undertaken in three patients in two unrelated families with a suspected platelet disorder and excessive bleeding. A targeted gene panel of previously known bleeding and platelet genes was used to identify plausible genetic variants. Deep platelet phenotyping was performed using platelet spreading analysis, transmission electron microscopy, immunofluorescence, and platelet function testing using lumiaggregometry and flow cytometry. Results: We report rare conserved missense variants (p.R182C and p.A183V) in TPM4 encoding tromomyosin-4 in 3 patients. Deep platelet phenotyping studies revealed similar platelet function defects across the 3 patients including reduced platelet secretion, and aggregation and spreading defects suggesting that TPM4 missense variants impact platelet function and show a disordered pattern of tropomyosin staining. Conclusions: Genetic and functional TPM4 defects are reported making TPM4 a diagnostic grade tier 1 gene and highlights the importance of including TPM4 in diagnostic genetic screening for patients with significant bleeding and undiagnosed platelet disorders, particularly for those with a normal platelet count