Background Superficial cutaneous infection caused by the zoophilic dermatophyte Trichophyton benhamiae is often associated with a highly inflammatory immune response. As non‐professional immune cells, epidermal keratinocytes and dermal fibroblasts contribute to the first line of defence by producing pro‐inflammatory cytokines and antimicrobial peptides (AMP). Objective Purpose of this study was to gain a deeper understanding of the pathogenesis and the fungal–host interaction as not much is known about the innate immune response of these cutaneous cells against T. benhamiae. Methods Using a dermatophytosis model of fibroblasts and keratinocytes incubated with T. benhamiae DSM 6916, analyses included determination of cell viability and cytotoxicity, effects on the innate immune response including expression and secretion of pro‐inflammatory cytokines/chemokines and expression of AMP, as well as alterations of genes involved in cell adhesion. Results Trichophyton benhamiae DSM 6916 infection led to severe cell damage and direct induction of a broad spectrum of pro‐inflammatory cytokines and chemokines in both cutaneous cells. Only keratinocytes differentially up‐regulated AMP genes expression after T. benhamiae DSM 6916 infection. Expression of AMPs in fibroblasts was not inducible by fungal infection, whereas their absences potentially contributed to a continuous increase in the fungal biomass on fibroblasts, which in turn was reduced in keratinocytes possibly due to the antimicrobial actions of induced AMPs. On mRNA level, T. benhamiae DSM 6916 infection altered cell–cell contact proteins in keratinocytes, indicating that targeting specific cell–cell adhesion proteins might be part of dermatophytes’ virulence strategy. Conclusion This study showed that in addition to immune cells, keratinocytes and fibroblasts could participate in antimicrobial defence against an exemplary infection with T. benhamiae DSM 6916.

BACKGROUND-The role played by coronary perfusion in the maintenance of AF electrical sources that anchor to the posterior wall of the left atrium (PLA) has been incompletely investigated.

Aims: Emerging evidence is demonstrating that rapid regeneration of remnant liver elicited by associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) may be attenuated in fibrotic livers. However, the molecular mechanisms responsible for this process are largely unknown. It is widely acknowledged that the TGFβ1 signaling axis plays a major role in liver fibrosis. Therefore, the aims of this study were to elucidate the underlying mechanism of liver regeneration during ALPPS with or without fibrosis, specifically focusing on TGFβ1 signaling. Approach: ALPPS was performed in rat models with N -diethylnitrosamine-induced liver fibrosis and no fibrosis. Functional liver remnant regeneration and expression of TGFβ1 were analyzed during the ALPPS procedures. Adeno-associated virus-shTGFβ1 and the small molecule inhibitor LY2157299 (galunisertib) were used separately or in combination to inhibit TGFβ1 signaling in fibrotic rats. Results: Liver regeneration following ALPPS was lower in fibrotic rats than non-fibrotic rats. TGFβ1 was a key mediator of postoperative regeneration in fibrotic liver. Interestingly, AAV-shTGFβ1 accelerated the regeneration of fibrotic functional liver remnant and improved fibrosis, while LY2157299 only enhanced liver regeneration. Moreover, combination treatment elicited a stronger effect. Conclusions: Inhibition of TGFβ1 accelerated regeneration of fibrotic liver, ameliorated liver fibrosis, and improved liver function following ALPPS. Therefore, TGFβ1 is a promising therapeutic target in ALPPS to improve fibrotic liver reserve function and prognosis.

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Mutations in the human cardiac motor protein beta-myosin heavy chain (βMHC) have been long recognized as a cause of familial hypertrophic cardiomyopathy. Recently, mutations (P830L and A1004S) in the less abundant but faster isoform alpha-myosin heavy chain (αMHC) have been linked to dilated cardiomyopathy (DCM). In this study, we sought to determine the cellular contractile phenotype associated with these point mutations. Ventricular myocytes were isolated from 2 month male Sprague Dawley rats. Cells were cultured in M199 media and infected with recombinant adenovirus containing the P830L or the A1004S mutant human αMHC at a MOI of 500 for 18 h. Uninfected cells (UI), human βMHC (MOI 500, 18 h), and human αMHC (MOI 500, 18 h) were used as controls. Cells were loaded with fura-2 (1 μM, 15 min) after 48 h. Sarcomere shortening and calcium transients were recorded in CO buffered M199 media (36°±1 C) with and without 10 nM isoproterenol (Iso). The A1004S mutation resulted in decreased peak sarcomere shortening while P830L demonstrated near normal shortening kinetics at baseline. In the presence of Iso, the A1004S sarcomere shortening was identical to the βMHC shortening while the P830L was identical to the αMHC control. All experimental groups had identical calcium transients. Despite a shared association with DCM, the P830L and A1004S αMHC mutations alter myocyte contractility in completely different ways while at the same preserving peak intracellular calcium.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it. Video Link The video component of this article can be found at https://www.jove.com/video/60196/ 9. Consequently, using a single workstation this protocol allows for a thorough examination of singly myocyte excitation-contraction coupling. Protocol All methods and procedures described in this protocol have been approved by the Institutional Animal Care and Use Committee (IACUC) of Case Western Reserve University.

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Introduction: In 2016 the US government declared opioid misuse a public emergency. While most opioid related deaths can be attributed to overdose, recent data suggest a link between opioid use and cardiovascular disease. Further, epidemiological data suggest it may be sex dependent, with females (♀) being at a somewhat higher risk than males (♂). This is paradoxical because until menopause, ♀ have a lower risk of heart disease when compared to ♂. Healthy adult hearts express the δ and κ ORs, but not the μOR. Therefore, the overall goals of this study using a reductionist model were to: 1). To determine if there are sex specific differences in cardiac myocyte excitation-contraction coupling between ♂ and ♀ myocytes exposed to δ or κ OR agonists during normoxia/hypoxia. 2). Examine if 17β-Estradiol enhances or augments the effects of δ or κ OR stimulation. Methods: Myocytes were isolated from adult ♂ and ♀ Sprague-Dawley rats and cultured overnight in M199 HEPES media, or M199 HEPES media containing either 1 μM DADLE (δ agonist), 20 μM U50-488 (κ agonist), 100 nm 17β-Estradiol, 1μM DADLE + 100 nm 17β-Estradiol, or 20 μM U50-488 + 100 nm 17β-Estradiol. To simulate an infarct, a subset of the myocytes was incubated at 1%O 2 , 5% CO 2 for 4 hours + 10 nM isoproterenol followed by 60 min normoxia (21%O 2 , 5% CO 2 ) before recordings. Excitation-Contraction was measured using an IonOptix rig (IonOptix LLC, Milton MA) at 36±1° at 1 Hz pacing. Results: During normoxia, ♂ myocytes compared to ♀ myocytes had an increased peak contraction and peak Ca 2+ transient. The addition of 100 nM 17β-Estradiol did not affect peak contraction in either sex but decreased the Ca 2+ transient in ♂ myocytes while increasing it in ♀ myocytes. DADLE and U50-488 had no effect on ♀ myocyte contractility but decreased ♂ myocyte contractility by a minimum of 13% and a maximum of 26% with/without 100 nM 17β-Estradiol. ♀ myocytes were immune to the effects of hypoxia, even exhibiting a slight increase in their peak calcium compared to control conditions, arguably because of the addition of isoproterenol. ♂ myocytes on the other hand, had an overall decrease in excitation-contraction coupling during hypoxia which was made worse by OR stimulation. Conclusion: ♀ myocytes are immune to the detrimental effects of δ and κ OR stimulation.