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The endocardium interacts with the myocardium to promote proliferation and morphogenesis during the later stages of heart development. However, the role of the endocardium in early cardiac ontogeny remains under-explored. Given the shared origin, subsequent juxtaposition, and essential cell-cell interactions of endocardial and myocardial cells throughout heart development, we hypothesized that paracrine signaling from the endocardium to the myocardium is crucial for initiating early differentiation of myocardial cells. To test this, we generated an , endocardial-specific ablation model using the diphtheria toxin receptor under the regulatory elements of the genomic locus (). Early treatment of mouse embryoid bodies with diphtheria toxin efficiently ablated endocardial cells, which significantly attenuated the percentage of beating EBs in culture and expression of early and late myocardial differentiation markers. The addition of Bmp2 during endocardial ablation partially rescued myocyte differentiation, maturation and function. Therefore, we conclude that early stages of myocardial differentiation rely on endocardial paracrine signaling mediated in part by Bmp2. Our findings provide novel insight into early endocardial-myocardial interactions that can be explored to promote early myocardial development and growth.
© 2019. Published by The Company of Biologists Ltd.

The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen Candida albicans is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of C. albicans HSF1 induces morphogenesis in the absence of external cues. HSF1 depletion compromises Hsp90 function, thereby driving filamentation. HSF1 overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.

BACKGROUND - Influenza C virus (ICV) is associated with acute respiratory illness. Yet ICV remains under recognized, with most previous studies using only culture to identify cases.
OBJECTIVES - To develop a sensitive and specific real-time RT-PCR assay for ICV that allows for rapid and accurate detection in a clinical or research setting.
STUDY DESIGN - Multiple ICV sequences obtained from GenBank were analyzed, including 141 hemagglutinin-esterase (HE), 106 matrix (M), and 97 nucleoprotein (NP) sequences. Primers and probes were designed based on conserved regions. Multiple primer-probe sets were tested against multiple ICV strains.
RESULTS - The ICV M and NP genes offered the most conserved sequence regions. Primers and probes based on newer sequence data offered enhanced detection of ICV, especially for low titer specimens. An NP-targeted assay yielded the best performance and was capable of detecting 10-100 RNA copies per reaction. The NP assay detected multiple clinical isolates of ICV collected in a field epidemiology study conducted in Peru.
CONCLUSIONS - We report a new real-time RT-PCR assay for ICV with high sensitivity and specificity.
Copyright © 2017 Elsevier B.V. All rights reserved.

Clostridium difficile infection affects a significant number of hospitalized patients in the United States. Two homologous exotoxins, TcdA and TcdB, are the major virulence factors in C. difficile pathogenesis. The toxins are glucosyltransferases that inactivate Rho family-GTPases to disrupt host cellular function and cause fluid secretion, inflammation, and cell death. Toxicity depends on receptor binding and subsequent endocytosis. TcdB has been shown to enter cells by clathrin-dependent endocytosis, but the mechanism of TcdA uptake is still unclear. Here, we utilize a combination of RNAi-based knockdown, pharmacological inhibition, and cell imaging approaches to investigate the endocytic mechanism(s) that contribute to TcdA uptake and subsequent cytopathic and cytotoxic effects. We show that TcdA uptake and cellular intoxication is dynamin-dependent but does not involve clathrin- or caveolae-mediated endocytosis. Confocal microscopy using fluorescently labeled TcdA shows significant colocalization of the toxin with PACSIN2-positive structures in cells during entry. Disruption of PACSIN2 function by RNAi-based knockdown approaches inhibits TcdA uptake and toxin-induced downstream effects in cells indicating that TcdA entry is PACSIN2-dependent. We conclude that TcdA and TcdB utilize distinct endocytic mechanisms to intoxicate host cells.

BACKGROUND - Genome-wide association studies have implicated variants in SCN10A, which encodes Nav1.8, as modulators of cardiac conduction. Follow-up work has indicated the SCN10A sequence includes an intronic enhancer for SCN5A. Yet the role of the Nav1.8 protein in the myocardium itself is still unclear. To investigate this, we use homozygous knockout mice (Scn10a) generated by disruption of exons 4 and 5, leaving the Scn5a enhancer intact.
METHODS AND RESULTS - We previously reported that pharmacologic blockade of Nav1.8 in wild-type animals blunts action potential prolongation by ATX-II at slow drive rates (≤1 Hz). Here we present evidence of the same blunting in Scn10a compared to wild-type ventricular myocytes, supporting the conclusion that Nav1.8 contributes to late sodium current at slow rates. In contrast to earlier studies, we found no differences in electrocardiographic parameters between genotypes. Low-dose ATX-II exposure in lightly anesthetized animals and Langendorff-perfused hearts prolonged QTc and generated arrhythmias to the same extent in wild-type and Scn10a. RNA sequencing failed to identify full-length Scn10a transcripts in wild-type or knockout isolated ventricular myocytes. However, loss of late current in Scn10a myocytes was replicated independently in a blinded set of experiments.
CONCLUSIONS - While Scn10a transcripts are not detectible in ventricular cardiomyocytes, gene deletion results in reproducible loss of late sodium current under extreme experimental conditions. However, there are no identifiable consequences of this Scn10a deletion in the intact mouse heart at usual rates. These findings argue that common variants in SCN10A that affect ventricular conduction do so by modulating SCN5A.
© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Both molecular and serological assays have been used previously to determine the etiology of community-acquired pneumonia (CAP). However, the extent to which these methods are correlated and the added diagnostic value of serology for respiratory viruses other than influenza virus have not been fully evaluated. Using data from patients enrolled in the Centers for Disease Control and Prevention (CDC) Etiology of Pneumonia in the Community (EPIC) study, we compared real-time reverse transcription-PCR (RT-PCR) and serology for the diagnosis of respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza virus 1 to 3 (PIV1, PIV2, and PIV3), and adenovirus (AdV) infections. Of 5,126 patients enrolled, RT-PCR and serology test results were available for 2,023, including 1,087 children below the age of 18 years and 936 adults. For RSV, 287 (14.2%) patients were positive by RT-PCR and 234 (11.6%) were positive by serology; for HMPV, 172 (8.5%) tested positive by RT-PCR and 147 (7.3%) by serology; for the PIVs, 94 (4.6%) tested positive by RT-PCR and 92 (4.6%) by serology; and for AdV, 111 (5.5%) tested positive by RT-PCR and 62 (3.1%) by serology. RT-PCR provided the highest number of positive detections overall, but serology increased diagnostic yield for RSV (by 11.8%), HMPV (by 25.0%), AdV (by 32.4%), and PIV (by 48.9%). The method concordance estimated by Cohen's kappa coefficient (κ) ranged from good (for RSV; κ = 0.73) to fair (for AdV; κ = 0.27). Heterotypic seroresponses observed between PIVs and persistent low-level AdV shedding may account for the higher method discordance observed with each of these viruses. Serology can be a helpful adjunct to RT-PCR for research-based assessment of the etiologic contribution of respiratory viruses other than influenza virus to CAP.
Copyright © 2016 American Society for Microbiology.

Use of selective serotonin reuptake inhibitors (SSRIs) is common during pregnancy. Fetal exposure to SSRIs is associated with persistent pulmonary hypertension of the newborn (PPHN); however, a direct link between the two has yet to be established. Conversely, it is well known that PPHN can be caused by premature constriction of the ductus arteriosus (DA), a fetal vessel connecting the pulmonary and systemic circulations. We hypothesized that SSRIs could induce in utero DA constriction. Using isolated vessels and whole-animal models, we sought to determine the effects of two commonly prescribed SSRIs, fluoxetine and sertraline, on the fetal mouse DA. Cannulated vessel myography studies demonstrated that SSRIs caused concentration-dependent DA constriction and made vessels less sensitive to prostaglandin-induced dilation. Moreover, in vivo studies showed that SSRI-exposed mice had inappropriate DA constriction in utero. Taken together, these findings establish that SSRIs promote fetal DA constriction and provide a potential mechanism by which SSRIs could contribute to PPHN.
Copyright © 2016 the American Physiological Society.

Tityus serrulatus sting causes thousands of deaths annually worldwide. T. serrulatus-envenomed victims exhibit local or systemic reaction that culminates in pulmonary oedema, potentially leading to death. However, the molecular mechanisms underlying T. serrulatus venom (TsV) activity remain unknown. Here we show that TsV triggers NLRP3 inflammasome activation via K(+) efflux. Mechanistically, TsV triggers lung-resident cells to release PGE2, which induces IL-1β production via E prostanoid receptor 2/4-cAMP-PKA-NFκB-dependent mechanisms. IL-1β/IL-1R actions account for oedema and neutrophil recruitment to the lungs, leading to TsV-induced mortality. Inflammasome activation triggers LTB4 production and further PGE2 via IL-1β/IL-1R signalling. Activation of LTB4-BLT1/2 pathway decreases cAMP generation, controlling TsV-induced inflammation. Exogenous administration confirms LTB4 anti-inflammatory activity and abrogates TsV-induced mortality. These results suggest that the balance between LTB4 and PGE2 determines the amount of IL-1β inflammasome-dependent release and the outcome of envenomation. We suggest COX1/2 inhibition as an effective therapeutic intervention for scorpion envenomation.

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of triglyceride-rich apolipoprotein B-containing lipoproteins. Previous studies in our laboratory identified a novel splice variant of MTP in mice that we named MTP-B. MTP-B has a unique first exon (1B) located 2.7 kB upstream of the first exon (1A) for canonical MTP (MTP-A). The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study we report the identification of a second MTP splice variant (MTP-C), which contains both exons 1B and 1A. MTP-C is expressed in all the tissues we tested. In cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5'-UTR of MTP-C revealed seven ATGs upstream of the start site for MTP-A, which is the only viable start site in frame with the main coding sequence. One of those ATGs was located in the 5'-UTR for MTP-A. We generated reporter constructs in which the 5'-UTRs of MTP-A or MTP-C were inserted between an SV40 promoter and the coding sequence of the luciferase gene and transfected these constructs into HEK 293 cells. Luciferase activity was significantly reduced by the MTP-C 5'-UTR, but not by the MTP-A 5'-UTR. We conclude that alternative splicing plays a key role in regulating MTP expression by introducing unique 5'-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP levels and activity.

Nuclear Factor (NF)-κB is positioned to provide the interface between COPD and carcinogenesis through regulation of chronic inflammation in the lungs. Using a tetracycline-inducible transgenic mouse model that conditionally expresses activated IκB kinase β (IKKβ) in airway epithelium (IKTA), we found that sustained NF-κB signaling results in chronic inflammation and emphysema by 4 months. By 11 months of transgene activation, IKTA mice develop lung adenomas. Investigation of lung inflammation in IKTA mice revealed a substantial increase in M2-polarized macrophages and CD4+/CD25+/FoxP3+ regulatory T lymphocytes (Tregs). Depletion of alveolar macrophages in IKTA mice reduced Tregs, increased lung CD8+ lymphocytes, and reduced tumor numbers following treatment with the carcinogen urethane. Alveolar macrophages from IKTA mice supported increased generation of inducible Foxp3+ Tregs ex vivo through expression of TGFβ and IL-10. Targeting of TGFβ and IL-10 reduced the ability of alveolar macrophages from IKTA mice to induce Foxp3 expression on T cells. These studies indicate that sustained activation of NF-κB pathway links COPD and lung cancer through generation and maintenance of a pro-tumorigenic inflammatory environment consisting of alternatively activated macrophages and regulatory T cells.