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Background Current heart failure (HF) treatment is based on targeting symptoms and left ventricle dysfunction severity, relying on a common HF pathway paradigm to justify common treatments for HF patients. This common strategy may belie an incomplete understanding of heterogeneous underlying mechanisms and could be a barrier to more precise treatments. We hypothesized we could use RNA-sequencing (RNA-seq) in human heart tissue to delineate HF etiology-specific gene expression signatures. Results RNA-seq from 64 human left ventricular samples: 37 dilated (DCM), 13 ischemic (ICM), and 14 non-failing (NF). Using a multi-analytic approach including covariate adjustment for age and sex, differentially expressed genes (DEGs) were identified characterizing HF and disease-specific expression. Pathway analysis investigated enrichment for biologically relevant pathways and functions. DCM vs NF and ICM vs NF had shared HF-DEGs that were enriched for the fetal gene program and mitochondrial dysfunction. DCM-specific DEGs were enriched for cell-cell and cell-matrix adhesion pathways. ICM-specific DEGs were enriched for cytoskeletal and immune pathway activation. Using the ICM and DCM DEG signatures from our data we were able to correctly classify the phenotypes of 24/31 ICM and 32/36 DCM samples from publicly available replication datasets. Conclusions Our results demonstrate the commonality of mitochondrial dysfunction in end-stage HF but more importantly reveal key etiology-specific signatures. Dysfunctional cell-cell and cell-matrix adhesion signatures typified DCM whereas signals related to immune and fibrotic responses were seen in ICM. These findings suggest that transcriptome signatures may distinguish end-stage heart failure, shedding light on underlying biological differences between ICM and DCM.

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. Here, we show that the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNA-367/302s delivered as mature miRNA mimics greatly enhances the reprogramming of human primary fibroblasts into iPSCs. This synergistic activity is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to human primary fibroblasts. As a result, we can now generate up to 4,019 iPSC colonies from only 500 starting human primary neonatal fibroblasts and reprogram up to 90.7% of individually plated cells, producing multiple sister colonies. This methodology consistently generates clinically relevant, integration-free iPSCs from a variety of human patient’s fibroblasts under feeder-free conditions and can be applicable for the clinical translation of iPSCs and studying the biology of reprogramming. Induced pluripotent stem cells (iPSCs) have potential for regenerative medicine applications, but are generated with very low efficiency. Here, the authors show highly efficient reprogramming of human primary fibroblasts to iPSCs via the synergistic activity of synthetic modified mRNAs, mature miRNA mimics, and optimized culture methods.

Identification of microsatellites, or simple sequence repeats (SSRs), can be a time-consuming and costly investment requiring enrichment, cloning, and sequencing of candidate loci. Recently, however, high throughput sequencing (with or without prior enrichment for specific SSR loci) has been utilized to identify SSR loci. The direct \"Seq-to-SSR\" approach has an advantage over enrichment-based strategies in that it does not require a priori selection of particular motifs, or prior knowledge of genomic SSR content. It has been more expensive per SSR locus recovered, however, particularly for genomes with few SSR loci, such as bird genomes. The longer but relatively more expensive 454 reads have been preferred over less expensive Illumina reads. Here, we use Illumina paired-end sequence data to identify potentially amplifiable SSR loci (PALs) from a snake (the Burmese python, Python molurus bivittatus), and directly compare these results to those from 454 data. We also compare the python results to results from Illumina sequencing of two bird genomes (Gunnison Sage-grouse, Centrocercus minimus, and Clark's Nutcracker, Nucifraga columbiana), which have considerably fewer SSRs than the python. We show that direct Illumina Seq-to-SSR can identify and characterize thousands of potentially amplifiable SSR loci for as little as $10 per sample--a fraction of the cost of 454 sequencing. Given that Illumina Seq-to-SSR is effective, inexpensive, and reliable even for species such as birds that have few SSR loci, it seems that there are now few situations for which prior hybridization is justifiable.

Prenatal alcohol exposure is the foremost preventable etiology of intellectual disability and leads to a collection of diagnoses known as Fetal Alcohol Spectrum Disorders (FASD). Alcohol (EtOH) impacts diverse neural cell types and activity, but the precise functional pathophysiological effects on the human fetal cerebral cortex are unclear. Here, we used human cortical organoids to study the effects of EtOH on neurogenesis and validated our findings in primary human fetal neurons. EtOH exposure produced temporally dependent cellular effects on proliferation, cell cycle, and apoptosis. In addition, we identified EtOH-induced alterations in post-translational histone modifications and chromatin accessibility, leading to impairment of cAMP and calcium signaling, glutamatergic synaptic development, and astrocytic function. Proteomic spatial profiling of cortical organoids showed region-specific, EtOH-induced alterations linked to changes in cytoskeleton, gliogenesis, and impaired synaptogenesis. Finally, multi-electrode array electrophysiology recordings confirmed the deleterious impact of EtOH on neural network formation and activity in cortical organoids, which was validated in primary human fetal tissues. Our findings demonstrate progress in defining the human molecular and cellular phenotypic signatures of prenatal alcohol exposure on functional neurodevelopment, increasing our knowledge for potential therapeutic interventions targeting FASD symptoms.

In an experimental viral encephalitis mouse model in which mice are infected with reovirus, we show that IFN-γ induces blood-brain barrier leakage. We show that IFN-γ promotes Rho kinase activity, resulting in actin cytoskeletal contractions in the brain endothelium that lead to vascular junctional disorganization and cell-cell separations. These studies now provide insight into a previously unknown mechanism for how blood-brain barrier breakdown occurs in viral encephalitis and implicates IFN-γ-Rho kinase activity as major contributor to this phenomenon. By identifying this mechanism of blood-brain barrier breakdown, we now provide potential therapeutic targets in treating patients with viral causes of encephalitis with the hope of limiting damage to the central nervous system. Blood-brain barrier (BBB) breakdown is a hallmark of many diseases of the central nervous system (CNS). Loss of BBB integrity in CNS diseases such as viral encephalitis results in the loss of nutrient/oxygen delivery, rapid infiltration of immune cells, and brain swelling that can exacerbate neuronal injury. Despite this, the cellular and molecular mechanisms that underlie BBB breakdown in viral encephalitis are incompletely understood. We undertook a comprehensive analysis of the cellular and molecular signaling events that induce BBB breakdown in an experimental model of virus-induced encephalitis in which neonatal mice are infected with reovirus (serotype 3 strain Abney). We show that BBB leakage during reovirus infection correlates with morphological changes in the vasculature, reductions in pericytes (BBB supporting cells), and disorganization of vascular junctions. Pathway analysis on RNA sequencing from brain endothelial cells identified the activation of interferon (IFN) signaling within the brain vasculature following reovirus infection. Our in vitro and in vivo studies show that type II IFN mediated by IFN-γ, a well known antiviral signal, is a major contributor to BBB leakage during reovirus infection. We show that IFN-γ reduces barrier properties in cultured brain endothelial cells through Rho kinase (ROCK)-mediated cytoskeletal contractions, resulting in junctional disorganization and cell-cell separations. In vivo neutralization of IFN-γ during reovirus infection significantly improved BBB integrity, pericyte coverage, attenuated vascular ROCK activity, and junctional disorganization. Our work supports a model in which IFN-γ acts directly on the brain endothelium to induce BBB breakdown through a mechanism involving ROCK-induced junctional disorganization. IMPORTANCE In an experimental viral encephalitis mouse model in which mice are infected with reovirus, we show that IFN-γ induces blood-brain barrier leakage. We show that IFN-γ promotes Rho kinase activity, resulting in actin cytoskeletal contractions in the brain endothelium that lead to vascular junctional disorganization and cell-cell separations. These studies now provide insight into a previously unknown mechanism for how blood-brain barrier breakdown occurs in viral encephalitis and implicates IFN-γ-Rho kinase activity as major contributor to this phenomenon. By identifying this mechanism of blood-brain barrier breakdown, we now provide potential therapeutic targets in treating patients with viral causes of encephalitis with the hope of limiting damage to the central nervous system.

Interferon-gamma (IFN-γ) decreases infections in chronic granulomatous disease (CGD) with variably incomplete restoration of the fundamental CGD defect, converting oxygen to microbicidal oxidants during phagocytosis. We sought to understand other IFN-γ effects that could contribute to protection of CGD patients. We measured neutrophil function, gene expression, and biochemical parameters in nine CGD patients off IFN-γ and 10–12 hours after the first (1st) and fourth (4th) IFN-γ injection. Non-directed motility and bactericidal activity increased after IFN-γ administration; ingestion remained unchanged. Stimulated release of superoxide anion (O 2 - ) was minimally changed. Treatment decreased expression of 483 genes and increased 386. Expression of eleven genes associated with neutrophil activity was upregulated. Genes not routinely associated with neutrophil function also demonstrated increased expression, including MHCI & II proteins, guanylate-binding proteins, and an enzyme synthesizing a nitric-oxide (NO) synthetase cofactor. CD11b expression, F-actin assembly, and CD 274 antigen were increased after treatment as was NO in neutrophil lysates. Formation of neutrophil extracellular traps after ingestion of staphylococci was increased off IFN-γ compared with controls but moved toward normal after IFN-γ administration. IFN-γ protects against infection in CGD by several mechanisms that could potentially support others with compromised host defense.

The facial surface ectoderm is essential for normal development of the underlying cranial neural crest cell populations, providing signals that direct appropriate growth, patterning, and morphogenesis. Despite the importance of the ectoderm as a signaling center, the molecular cues and genetic programs implemented within this tissue are understudied. Here, we show that removal of two members of the AP-2 transcription factor family, AP-2α and AP-2ß, within the early embryonic ectoderm of the mouse leads to major alterations in the craniofacial complex. Significantly, there are clefts in both the upper face and mandible, accompanied by fusion of the upper and lower jaws in the hinge region. Comparison of ATAC-seq and RNA-seq analyses between controls and mutants revealed significant changes in chromatin accessibility and gene expression centered on multiple AP-2 binding motifs associated with enhancer elements within these ectodermal lineages. In particular, loss of these AP-2 proteins affects both skin differentiation as well as multiple signaling pathways, most notably the WNT pathway. We also determined that the mutant clefting phenotypes that correlated with reduced WNT signaling could be rescued by Wnt1 ligand overexpression in the ectoderm. Collectively, these findings highlight a conserved ancestral function for AP-2 transcription factors in ectodermal development and signaling, and provide a framework from which to understand the gene regulatory network operating within this tissue that directs vertebrate craniofacial development.

Anthropogenic changes are altering the environmental conditions and the biota of ecosystems worldwide. In many temperate grasslands, such as North American tallgrass prairie, these changes include alteration in historically important disturbance regimes (e.g., frequency of fires) and enhanced availability of potentially limiting nutrients, particularly nitrogen. Such anthropogenically-driven changes in the environment are known to elicit substantial changes in plant and consumer communities aboveground, but much less is known about their effects on soil microbial communities. Due to the high diversity of soil microbes and methodological challenges associated with assessing microbial community composition, relatively few studies have addressed specific taxonomic changes underlying microbial community-level responses to different fire regimes or nutrient amendments in tallgrass prairie. We used deep sequencing of the V3 region of the 16S rRNA gene to explore the effects of contrasting fire regimes and nutrient enrichment on soil bacterial communities in a long-term (20 yrs) experiment in native tallgrass prairie in the eastern Central Plains. We focused on responses to nutrient amendments coupled with two extreme fire regimes (annual prescribed spring burning and complete fire exclusion). The dominant bacterial phyla identified were Proteobacteria, Verrucomicrobia, Bacteriodetes, Acidobacteria, Firmicutes, and Actinobacteria and made up 80% of all taxa quantified. Chronic nitrogen enrichment significantly impacted bacterial community diversity and community structure varied according to nitrogen treatment, but not phosphorus enrichment or fire regime. We also found significant responses of individual bacterial groups including Nitrospira and Gammaproteobacteria to long-term nitrogen enrichment. Our results show that soil nitrogen enrichment can significantly alter bacterial community diversity, structure, and individual taxa abundance, which have important implications for both managed and natural grassland ecosystems.

The cytokine Interferon-γ (IFN-γ) exerts powerful immunoregulatory effects on the adaptive immune system and also enhances functions of the neutrophil (PMN). The clinical use of IFN-γ has been driven by the finding that its administration to patients with chronic granulomatous disease (CGD) results in decreased incidence and severity of infections. However, IFN-γ has no effect on the characteristic defect of CGD, the inability to convert oxygen to microbicidal metabolites including superoxide anion (O 2 - ) during the phagocytosis associated oxidative burst. We administered varying doses of IFN-γ to adult volunteers and studied the effects on plasma drug levels and response molecules and PMNs isolated from blood drawn at intervals over a 96- hour period. Plasma concentrations of IFN-γ, IP-10 and neopterin, and stimulated release of O 2 - from PMNs exhibited dose- and time-dependent increases after IFN-γ administration. Gene expression in PMNs was altered for 2775 genes; changes occurred rapidly after administration and returned to baseline in 24–36 hours. Several genes involved with neutrophil host defense were upregulated including those for components of the O 2 - generating NADPH oxidase; innate-immune and Fc receptors; proteins involved in MHCI and II; a regulator of circulating PMN number; guanylate binding proteins; and a key enzyme in synthesis of an essential NOS cofactor. Coordinate changes were detected in protein levels of representative products from several of these genes. Lysates from isolated neutrophils also demonstrated a spike in NO following IFN-γ administration. IFN-γ appears to increase non-oxygen dependent microbicidal functions of PMNs which could provide strategies to compensate for deficiencies, explain its clinical benefit for CGD patients and expand therapeutic applications of IFN-γ to other disorders. Trial registration: Protocol registered in ClinicalTrials.gov, NCT02609932 , Effect of IFN-γ on Innate Immune Cells.

Background Obesity increases breast cancer risk and breast cancer-specific mortality, particularly for people with estrogen receptor (ER)-positive tumors. Body mass index (BMI) is used to define obesity, but it may not be the best predictor of breast cancer risk or prognosis on an individual level. Adult weight gain is an independent indicator of breast cancer risk. Our previous work described a murine model of obesity, ER-positive breast cancer, and weight gain and identified fibroblast growth factor receptor (FGFR) as a potential driver of tumor progression. During adipose tissue expansion, the FGF1 ligand is produced by hypertrophic adipocytes as a stimulus to stromal preadipocytes that proliferate and differentiate to provide additional lipid storage capacity. In breast adipose tissue, FGF1 production may stimulate cancer cell proliferation and tumor progression. Methods We explored the effects of FGF1 on ER-positive endocrine-sensitive and resistant breast cancer and compared that to the effects of the canonical ER ligand, estradiol. We used untargeted proteomics, specific immunoblot assays, gene expression profiling, and functional metabolic assessments of breast cancer cells. The results were validated in tumors from obese mice and breast cancer datasets from women with obesity. Results FGF1 stimulated ER phosphorylation independently of estradiol in cells that grow in obese female mice after estrogen deprivation treatment. Phospho- and total proteomic, genomic, and functional analyses of endocrine-sensitive and resistant breast cancer cells show that FGF1 promoted a cellular phenotype characterized by glycolytic metabolism. In endocrine-sensitive but not endocrine-resistant breast cancer cells, mitochondrial metabolism was also regulated by FGF1. Comparison of gene expression profiles indicated that tumors from women with obesity shared hallmarks with endocrine-resistant breast cancer cells. Conclusions Collectively, our data suggest that one mechanism by which obesity and weight gain promote breast cancer progression is through estrogen-independent ER activation and cancer cell metabolic reprogramming, partly driven by FGF/FGFR. The first-line treatment for many patients with ER-positive breast cancer is inhibition of estrogen synthesis using aromatase inhibitors. In women with obesity who are experiencing weight gain, locally produced FGF1 may activate ER to promote cancer cell metabolic reprogramming and tumor progression independently of estrogen.