Subchronic inhalation exposure to ultrafine particulate matter alters the intestinal microbiome in various mouse models.

Exposure to ultrafine particles (UFPs) has been associated with multiple adverse health effects. Inhaled UFPs could reach the gastrointestinal tract and influence the composition of the gut microbiome. We have previously shown that oral ingestion of UFPs alters the gut microbiome and promotes intestinal inflammation in hyperlipidemic Ldlr-/- mice. Particulate matter (PM)2.5 inhalation studies have also demonstrated microbiome shifts in normolipidemic C57BL/6 mice. However, it is not known whether changes in microbiome precede or follow inflammatory effects in the intestinal mucosa. We hypothesized that inhaled UFPs modulate the gut microbiome prior to the development of intestinal inflammation. We studied the effects of UFP inhalation on the gut microbiome and intestinal mucosa in two hyperlipidemic mouse models (ApoE-/- mice and Ldlr-/- mice) and normolipidemic C57BL/6 mice. Mice were exposed to PM in the ultrafine-size range by inhalation for 6 h a day, 3 times a week for 10 weeks at a concentration of 300-350 µg/m3.16S rRNA gene sequencing was performed to characterize sequential changes in the fecal microbiome during exposures, and changes in the intestinal microbiome at the end. PM exposure led to progressive differentiation of the microbiota over time, associated with increased fecal microbial richness and evenness, altered microbial composition, and differentially abundant microbes by week 10 depending on the mouse model. Cross-sectional analysis of the small intestinal microbiome at week 10 showed significant changes in α-diversity, ß-diversity, and abundances of individual microbial taxa in the two hyperlipidemic models. These alterations of the intestinal microbiome were not accompanied, and therefore could not be caused, by increased intestinal inflammation as determined by histological analysis of small and large intestine, cytokine gene expression, and levels of fecal lipocalin. In conclusion, 10-week inhalation exposures to UFPs induced taxonomic changes in the microbiome of various animal models in the absence of intestinal inflammation.

Dissecting mechanisms of chamber-specific cardiac differentiation and its perturbation following retinoic acid exposure.

The specification of distinct cardiac lineages occurs before chamber formation and acquisition of bona fide atrial or ventricular identity. However, the mechanisms underlying these early specification events remain poorly understood. Here, we performed single cell analysis at the murine cardiac crescent, primitive heart tube and heart tube stages to uncover the transcriptional mechanisms underlying formation of atrial and ventricular cells. We find that progression towards differentiated cardiomyocytes occurs primarily based on heart field progenitor identity, and that progenitors contribute to ventricular or atrial identity through distinct differentiation mechanisms. We identify new candidate markers that define such differentiation processes and examine their expression dynamics using computational lineage trajectory methods. We further show that exposure to exogenous retinoic acid causes defects in ventricular chamber size, dysregulation in FGF signaling and a shunt in differentiation towards orthogonal lineages. Retinoic acid also causes defects in cell-cycle exit resulting in formation of hypomorphic ventricles. Collectively, our data identify, at a single cell level, distinct lineage trajectories during cardiac specification and differentiation, and the precise effects of manipulating cardiac progenitor patterning via retinoic acid signaling.

PPARdelta activation induces metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

Pluripotent stem-cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease, but they are functionally and structurally immature. Here, we induce efficient human PSC-CM (hPSC-CM) maturation through metabolic-pathway modulations. Specifically, we find that peroxisome-proliferator-associated receptor (PPAR) signaling regulates glycolysis and fatty acid oxidation (FAO) in an isoform-specific manner. While PPARalpha (PPARa) is the most active isoform in hPSC-CMs, PPARdelta (PPARd) activation efficiently upregulates the gene regulatory networks underlying FAO, increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation, and augments FAO flux. PPARd activation further increases binucleation, enhances myofibril organization, and improves contractility. Transient lactate exposure, which is frequently used for hPSC-CM purification, induces an independent cardiac maturation program but, when combined with PPARd activation, still enhances oxidative metabolism. In summary, we investigate multiple metabolic modifications in hPSC-CMs and identify a role for PPARd signaling in inducing the metabolic switch from glycolysis to FAO in hPSC-CMs.

A library of induced pluripotent stem cells from clinically well-characterized, diverse healthy human individuals.

A library of well-characterized human induced pluripotent stem cell (hiPSC) lines from clinically healthy human subjects could serve as a useful resource of normal controls for in vitro human development, disease modeling, genotype-phenotype association studies, and drug response evaluation. We report generation and extensive characterization of a gender-balanced, racially/ethnically diverse library of hiPSC lines from 40 clinically healthy human individuals who range in age from 22 to 61 years. The hiPSCs match the karyotype and short tandem repeat identities of their parental fibroblasts, and have a transcription profile characteristic of pluripotent stem cells. We provide whole-genome sequencing data for one hiPSC clone from each individual, genomic ancestry determination, and analysis of mendelian disease genes and risks. We document similar transcriptomic profiles, single-cell RNA-sequencing-derived cell clusters, and physiology of cardiomyocytes differentiated from multiple independent hiPSC lines. This extensive characterization makes this hiPSC library a valuable resource for many studies on human biology.

Physiologic, Pathologic, and Therapeutic Paracrine Modulation of Cardiac Excitation-Contraction Coupling.

Cardiac excitation-contraction coupling (ECC) is the orchestrated process of initial myocyte electrical excitation, which leads to calcium entry, intracellular trafficking, and subsequent sarcomere shortening and myofibrillar contraction. Neurohumoral ß-adrenergic signaling is a well-established mediator of ECC; other signaling mechanisms, such as paracrine signaling, have also demonstrated significant impact on ECC but are less well understood. For example, resident heart endothelial cells are well-known physiological paracrine modulators of cardiac myocyte ECC mainly via NO and endothelin-1. Moreover, recent studies have demonstrated other resident noncardiomyocyte heart cells (eg, physiological fibroblasts and pathological myofibroblasts), and even experimental cardiotherapeutic cells (eg, mesenchymal stem cells) are also capable of altering cardiomyocyte ECC through paracrine mechanisms. In this review, we first focus on the paracrine-mediated effects of resident and therapeutic noncardiomyocytes on cardiomyocyte hypertrophy, electrophysiology, and calcium handling, each of which can modulate ECC, and then discuss the current knowledge about key paracrine factors and their underlying mechanisms of action. Next, we provide a case example demonstrating the promise of tissue-engineering approaches to study paracrine effects on tissue-level contractility. More specifically, we present new functional and molecular data on the effects of human adult cardiac fibroblast conditioned media on human engineered cardiac tissue contractility and ion channel gene expression that generally agrees with previous murine studies but also suggests possible species-specific differences. By contrast, paracrine secretions by human dermal fibroblasts had no discernible effect on human engineered cardiac tissue contractile function and gene expression. Finally, we discuss systems biology approaches to help identify key stem cell paracrine mediators of ECC and their associated mechanistic pathways. Such integration of tissue-engineering and systems biology methods shows promise to reveal novel insights into paracrine mediators of ECC and their underlying mechanisms of action, ultimately leading to improved cell-based therapies for patients with heart disease.

The Importance of Non-neuronal Cell Types in hiPSC-Based Disease Modeling and Drug Screening.

Current applications of human induced pluripotent stem cell (hiPSC) technologies in patient-specific models of neurodegenerative and neuropsychiatric disorders tend to focus on neuronal phenotypes. Here, we review recent efforts toward advancing hiPSCs toward non-neuronal cell types of the central nervous system (CNS) and highlight their potential use for the development of more complex in vitro models of neurodevelopment and disease. We present evidence from previous works in both rodents and humans of the importance of these cell types (oligodendrocytes, microglia, astrocytes) in neurological disease and highlight new hiPSC-based models that have sought to explore these relationships in vitro. Lastly, we summarize efforts toward conducting high-throughput screening experiments with hiPSCs and propose methods by which new screening platforms could be designed to better capture complex relationships between neural cell populations in health and disease.

Signaling mechanisms of the epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is an essential mechanism in embryonic development and tissue repair. EMT also contributes to the progression of disease, including organ fibrosis and cancer. EMT, as well as a similar transition occurring in vascular endothelial cells called endothelial-mesenchymal transition (EndMT), results from the induction of transcription factors that alter gene expression to promote loss of cell-cell adhesion, leading to a shift in cytoskeletal dynamics and a change from epithelial morphology and physiology to the mesenchymal phenotype. Transcription program switching in EMT is induced by signaling pathways mediated by transforming growth factor ß (TGF-ß) and bone morphogenetic protein (BMP), Wnt-ß-catenin, Notch, Hedgehog, and receptor tyrosine kinases. These pathways are activated by various dynamic stimuli from the local microenvironment, including growth factors and cytokines, hypoxia, and contact with the surrounding extracellular matrix (ECM). We discuss how these pathways crosstalk and respond to signals from the microenvironment to regulate the expression and function of EMT-inducing transcription factors in development, physiology, and disease. Understanding these mechanisms will enable the therapeutic control of EMT to promote tissue regeneration, treat fibrosis, and prevent cancer metastasis.

Failure and life cycle evaluation of watering valves.

Automated watering systems provide a reliable source of ad libitum water to animal cages. Our facility uses an automated water delivery system to support approximately 95% of the housed population (approximately 14,000 mouse cages). Drinking valve failure rates from 2002 through 2006 never exceeded the manufacturer standard of 0.1% total failure, based on monthly cage census and the number of floods. In 2007, we noted an increase in both flooding and cases of clinical dehydration in our mouse population. Using manufacturer's specifications for a water flow rate of 25 to 50 mL/min, we initiated a wide-scale screening of all valves used. During a 4-mo period, approximately 17,000 valves were assessed, of which 2200 failed according to scoring criteria (12.9% overall; 7.2% low flow; 1.6% no flow; 4.1% leaky). Factors leading to valve failures included residual metal shavings, silicone flash, introduced debris or bedding, and (most common) distortion of the autoclave-rated internal diaphragm and O-ring. Further evaluation revealed that despite normal autoclave conditions of heat, pressure, and steam, an extreme negative vacuum pull caused the valves' internal silicone components (diaphragm and O-ring) to become distorted and water-permeable. Normal flow rate often returned after a 'drying out' period, but components then reabsorbed water while on the animal rack or during subsequent autoclave cycles to revert to a variable flow condition. On the basis of our findings, we recalibrated autoclaves and initiated a preventative maintenance program to mitigate the risk of future valve failure.

Tetra-kis(4-cyano-pyridine)-palladium(II) bis-(trifluoro-methane-sulfonate).

The title salt, [Pd(C(6)H(4)N(2))(4)](CF(3)SO(3))(2), comprises Pd(4-cyano-pyridine)(4) dications balanced by two trifluoro-methane-sulfonate anions. The Pd(II) atom lies in a square-planar geometry defined by four N atoms which form equivalent Pd-N inter-actions. The 4-cyano-pyridine ligands are twisted out of the N(4) plane, forming dihedral angles ranging from 66.5 (2) to 89.9 (2)°. In the crystal packing, columns of edge-to-edge dications define channels in which reside the anions. A range of C-H⋯N and C-H⋯O hydrogen-bonding interactions stabilizes the crystal packing.

The bubble sign: an arthroscopic indicator of an intratendinous rotator cuff tear.

Intratendinous tears of the rotator cuff are, by definition, difficult to diagnose due to the absence of overt tendon disruption on both the bursal and articular surface of the rotator cuff. The authors describe the bubble sign, a bulging expansion of the rotator cuff tendon following injection of saline into the suspected lesion, as a useful indicator of an intratendinous tear of the rotator cuff.