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The prognosis and treatment outcomes of heart failure (HF) patients rely heavily on disease etiology, yet the majority of underlying signaling mechanisms are complex and not fully elucidated. Phosphorylation is a major point of protein regulation with rapid and profound effects on the function and activity of protein networks. Currently, there is a lack of comprehensive proteomic and phosphoproteomic studies examining cardiac tissue from HF patients with either dilated dilated cardiomyopathy (DCM) or ischemic cardiomyopathy (ICM). Here, we used a combined proteomic and phosphoproteomic approach to identify and quantify more than 5,000 total proteins with greater than 13,000 corresponding phosphorylation sites across explanted left ventricle (LV) tissue samples, including HF patients with DCM vs. nonfailing controls (NFC), and left ventricular infarct vs. noninfarct, and periinfarct vs. noninfarct regions of HF patients with ICM. Each pair-wise comparison revealed unique global proteomic and phosphoproteomic profiles with both shared and etiology-specific perturbations. With this approach, we identified a DCM-associated hyperphosphorylation cluster in the cardiomyocyte intercalated disc (ICD) protein, αT-catenin (CTNNA3). We demonstrate using both ex vivo isolated cardiomyocytes and in vivo using an AAV9-mediated overexpression mouse model, that CTNNA3 phosphorylation at these residues plays a key role in maintaining protein localization at the cardiomyocyte ICD to regulate conductance and cell--cell adhesion. Collectively, this integrative proteomic/phosphoproteomic approach identifies region- and etiology-associated signaling pathways in human HF and describes a role for CTNNA3 phosphorylation in the pathophysiology of DCM. [ABSTRACT FROM AUTHOR]

Cell-free DNA (cfDNA) fragmentation is nonrandom, at least partially mediated by various DNA nucleases, forming characteristic cfDNA end motifs. However, there is a paucity of tools for deciphering the relative contributions of cfDNA cleavage patterns related to underlying fragmentation factors. In this study, through non-negative matrix factorization algorithm, we used 256 5' 4-mer end motifs to identify distinct types of cfDNA cleavage patterns, referred to as "founder" end-motif profiles (F-profiles). F-profiles were associated with different DNA nucleases based on whether such patterns were disrupted in nuclease-knockout mouse models. Contributions of individual F-profiles in a cfDNA sample could be determined by deconvolutional analysis. We analyzed 93 murine cfDNA samples of different nuclease-deficient mice and identified six types of F-profiles. F-profiles I, II, and III were linked to deoxyribonuclease 1 like 3 (DNASE1L3), deoxyribonuclease 1 (DNASE1), and DNA fragmentation factor subunit beta (DFFB), respectively. We revealed that 42.9% of plasma cfDNA molecules were attributed to DNASE1L3-mediated fragmentation, whereas 43.4% of urinary cfDNA molecules involved DNASE1-mediated fragmentation. We further demonstrated that the relative contributions of F-profiles were useful to inform pathological states, such as autoimmune disorders and cancer. Among the six F-profiles, the use of F-profile I could inform the human patients with systemic lupus erythematosus. F-profile VI could be used to detect individuals with hepatocellular carcinoma, with an area under the receiver operating characteristic curve of 0.97. F-profile VI was more prominent in patients with nasopharyngeal carcinoma undergoing chemoradiotherapy. We proposed that this profile might be related to oxidative stress. [ABSTRACT FROM AUTHOR]

Cell-free DNA (cfDNA) fragmentation patterns contain important molecular information linked to tissues of origin. We explored the possibility of using fragmentation patterns to predict cytosine-phosphate-guanine (CpG) methylation of cfDNA, obviating the use of bisulfite treatment and associated risks of DNA degradation. This study investigated the cfDNA cleavage profile surrounding a CpG (i.e., within an 11-nucleotide [nt] window) to analyze cfDNA methylation. The cfDNA cleavage proportion across positions within the window appeared nonrandom and exhibited correlation with methylation status. The mean cleavage proportion was ~twofold higher at the cytosine of methylated CpGs than unmethylated ones in healthy controls. In contrast, the mean cleavage proportion rapidly decreased at the 1-nt position immediately preceding methylated CpGs. Such differential cleavages resulted in a characteristic change in relative presentations of CGN and NCG motifs at 5' ends, where N represented any nucleotide. CGN/NCG motif ratios were correlated with methylation levels at tissue-specific methylated CpGs (e.g., placenta or liver) (Pearson's absolute r > 0.86). cfDNA cleavage profiles were thus informative for cfDNA methylation and tissue-of-origin analyses. Using CG-containing end motifs, we achieved an area under a receiver operating characteristic curve (AUC) of 0.98 in differentiating patients with and without hepatocellular carcinoma and enhanced the positive predictive value of nasopharyngeal carcinoma screening (from 19.6 to 26.8%). Furthermore, we elucidated the feasibility of using cfDNA cleavage patterns to deduce CpG methylation at single CpG resolution using a deep learning algorithm and achieved an AUC of 0.93. FRAGmentomics-based Methylation Analysis (FRAGMA) presents many possibilities for noninvasive prenatal, cancer, and organ transplantation assessment. [ABSTRACT FROM AUTHOR]

Endocytosis is essential to all eukaryotes, but how cargoes are selected for internalization remains poorly characterized. Extracellular cargoes are thought to be selected by transmembrane receptors that bind intracellular adaptors proteins to initiate endocytosis. Here, we report a mechanism for clathrin-mediated endocytosis (CME) of extracellular lanthanum [La(III)] cargoes, which requires extracellular arabinogalactan proteins (AGPs) that are anchored on the outer face of the plasma membrane. AGPs were colocalized with La(III) on the cell surface and in La(III)- induced endocytic vesicles in Arabidopsis leaf cells. Superresolution imaging showed that La(III) triggered AGP movement across the plasma membrane. AGPs were then colocalized and physically associated with the μ subunit of the intracellular adaptor protein 2 (AP2) complexes. The AGP-AP2 interaction was independent of CME, whereas AGP's internalization required CME and AP2. Moreover, we show that AGP-dependent endocytosis in the presence of La(III) also occurred in human cells. These findings indicate that extracellular AGPs act as conserved CME cargo receptors, thus challenging the current paradigm about endocytosis of extracellular cargoes. [ABSTRACT FROM AUTHOR]

Systemic autoinflammatory diseases are caused by mutations in genes that function in innate immunity. Here, we report an autoinflammatory disease caused by loss-of-function mutations in OTULIN (FAM105B), encoding a deubiquitinase with linear linkage specificity. We identified two missense and one frameshift mutations in one Pakistani and two Turkish families with four affected patients. Patients presented with neonatal-onset fever, neutrophilic dermatitis/panniculitis, and failure to thrive, but without obvious primary immunodeficiency. HEK293 cells transfected with mutated OTULIN had decreased enzyme activity relative to cells transfected with WT OTULIN, and showed a substantial defect in the linear deubiquitination of target molecules. Stimulated patients' fibroblasts and peripheral blood mononuclear cells showed evidence for increased signaling in the canonical NF-κB pathway and accumulated linear ubiquitin aggregates. Levels of proinflammatory cytokines were significantly increased in the supernatants of stimulated primary cells and serum samples. This discovery adds to the emerging spectrum of human diseases caused by defects in the ubiquitin pathway and suggests a role for targeted cytokine therapies. [ABSTRACT FROM AUTHOR]

Cytokinesis in Trypanosoma brucei, an early branching protozoan, occurs along its longitudinal axis uni-directionally from the anterior tip of the new flagellum attachment zone filament toward the cell's posterior end. However, the underlying mechanisms remain elusive. Here we report that cytokinesis in T. brucei is regulated by a concerted action of Polo-like kinase, Aurora B kinase, and a trypanosome-specific protein CIF1. Phosphorylation of CIF1 by Polo-like kinase targets it to the anterior tip of the new flagellum attachment zone filament, where it subsequently recruits Aurora B kinase to initiate cytokinesis. Consistent with its role, CIF1 depletion inhibits cytokinesis initiation from the anterior end of the cell, but, surprisingly, triggers cytokinesis initiation from the posterior end of the cell, suggesting the activation of an alternative cytokinesis from the opposite cell end. Our results reveal the mechanistic roles of CIF1 and Polo-like kinase in cytokinesis initiation and elucidate the mechanism underlying the recruitment of Aurora B kinase to the cytokinesis initiation site at late anaphase. These findings also delineate a signaling cascade controlling cytokinesis initiation from the anterior end of the cell and uncover a backup cytokinesis that is initiated from the posterior end of the cell when the typical anterior-to-posterior cytokinesis is compromised. [ABSTRACT FROM AUTHOR]

It has long been observed that rare earth elements (REEs) regulate multiple facets of plant growth and development. However, the underlying mechanisms remain largely unclear. Here, using electron microscopic autoradiography, we show the life cycle of a light REE (lanthanum) and a heavy REE (terbium) in horseradish leaf cells. Our data indicate that REEs were first anchored on the plasma membrane in the form of nanoscale particles, and then entered the cells by endocytosis. Consistently, REEs activated endocytosis in plant cells, which may be the cellular basis of REE actions in plants. Moreover, we discovered that a portion of REEs was successively released into the cytoplasm, self-assembled to form nanoscale clusters, and finally deposited in horseradish leaf cells. Taken together, our data reveal the life cycle of REEs and their cellular behaviors in plant cells, which shed light on the cellular mechanisms of REE actions in living organisms. [ABSTRACT FROM AUTHOR]

The growing world population and shrinkage of arable land demand yield improvement of rice, one of the most important staple crops. To elucidate the genetic basis of yield and uncover its associated loci in rice, we resequenced the core recombinant inbred lines of Liang-You-Pei-Jiu, the widely cultivated super hybrid rice, and constructed a high-resolution linkage map. We detected 43 yield-associated quantitative trait loci, of which 20 are unique. Based on the high-density physical map, the genome sequences of paternal variety 93-11 and maternal cultivar PA64s of Liang-You-Pei-Jiu were significantly improved. The large recombinant inbred line population combined with plentiful high-quality single nucleotide polymorphisms and insertions/deletions between parental genomes allowed us to fine-map two quantitative trait loci, qSN8 and qSPB1, and to identify days to heading8 and lax panicle1 as candidate genes, respectively. The quantitative trait locus qSN8 was further confirmed to be days to heading8 by a compl mentation test. Our study provided an ideal platform for molecular breeding by targeting and dissecting yield-associated loci in rice. [ABSTRACT FROM AUTHOR]

Genome-wide association studies (GWAS) are a powerful means of identifying genes with disease-associated common variants, but they are not well-suited to detecting genes with disease-associated rare and low-frequency variants. In the current study of Behçet disease (BD), nonsynonymous variants (NSVs) identified by deep exonic resequencing of 10 genes found by GWAS (IL10, IL23R, CCR1, STAT4, KLRK1, KLRC1, KLRC2, KLRC3, KLRC4, and ERAP1) and 11 genes selected for their role in innate immunity (IL1B, IL1R1, IL1RN, NLRP3, MEFV, TNFRSF1A, PSTPIP1, CASP1, PYCARD, NOD2, and TLR4) were evaluated for BD association. A differential distribution of the rare and low-frequency NSVs of a gene in 2,461 BD cases compared with 2,458 controls indicated their collective association with disease. By stringent criteria requiring at least a single burden test with study-wide significance and a corroborating test with at least nominal significance, rare and low-frequency NSVs in one GWAS-identified gene, IL23R (P = 6.9 × 10-5), and one gene involved in innate immunity, TLR4 (P = 8.0 × 10-4), were associated with BD. In addition, damaging or rare damaging NOD2 variants were nominally significant across all three burden tests applied (P = 0.0063-0.045). Furthermore, carriage of the familial Mediterranean fever gene (MEFV) mutation Met694Val, which is known to cause recessively inherited familial Mediterranean fever, conferred BD risk in the Turkish population (OR, 2.65; P = 1.8 × 10-12). The disease-associated NSVs in MEFV and TLR4 implicate innate immune and bacterial sensing mechanisms in BD pathogenesis. [ABSTRACT FROM AUTHOR]

Mutations in the TNF family of proteins have been associated with inherited forms of immune deficiency. Using an array-based sequencing assay, we identified an autosomal-dominant deficiency in TNF-like weak inducer of apoptosis (TWEAK; TNFSF12) in a kindred with recurrent infection and impaired antibody responses to protein and polysaccharide vaccines. This mutation occurs in the sixth exon of TWEAK and results in the amino acid substitution R145C within the conserved TNF-homology domain of the full-length protein. TWEAK mutant protein formed high molecular weight aggregates under nonreducing conditions, suggesting an increased propensity for intermolecular interactions. As a result, mutant TWEAK associated with B-cell-activating factor (BAFF) protein and down-regulated the BAFF-mediated activation of the noncanonical NF-κB pathway through inhibition of p100 processing to p52, resulting in inhibition of BAFF-dependent B-cell survival and proliferation. As BAFF mediates T-cell-independent isotype switching and B-cell survival, our data implicate TWEAK as a disease-susceptibility gene for a humoral immunodeficiency. [ABSTRACT FROM AUTHOR]

Rapid electrical conduction in the His-Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification and morphogenesis of the His-Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction-forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. lrx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for lrx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart. [ABSTRACT FROM AUTHOR]

At the end of every heartbeat, cardiac myocytes must relax to allow filling of the heart. Impaired relaxation is a significant factor in heart failure, but all pathways regulating the cardiac relaxation apparatus are not known. Haploinsufficiency of the T-box transcription factor Tbx5 in mouse and man causes congenital heart defects (CHDs) as part of Holt-Oram syndrome (HOS). Here, we show that haploinsufficiency of Tbx5 in mouse results in cell-autonomous defects in ventricular relaxation. Tbx5 dosage modulates expression of the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2a encoded by Atp2a2 and Tbx5 haploinsufficiency in ventricular myocytes results in impaired Ca2+ uptake dynamics and Ca2+ transient prolongation. We also demonstrate that Tbx5 can activate the Atp2a2 promoter. Furthermore, we find that patients with HOS have significant diastolic filling abnormalities. These results reveal a direct genetic pathway that regulates cardiac diastolic function, implying that patients with structural CHDs may have clinically important underlying anomalies in heart function that merit treatment. [ABSTRACT FROM AUTHOR]

We analyze new and existing expression and transcription factor-binding data to characterize gene regulatory relations in mouse ES cells (ESC). In addition to confirming the key roles of Oct4, Sox2, and Nanog, our analysis identifies several genes, such as Esrrb, Stat3, Tcf7, Sall4, and LRH-1, as statistically significant coregulators. The regulatory interactions among 15 core regulators are used to construct a gene regulatory network in ESC. The network encapsulates extensive cross-regulations among the core regulators, highlights how they may control epigenetic processes, and reveals the surprising roles of nuclear receptors. Our analysis also provides information on the regulation of a large number of putative target genes of the network. [ABSTRACT FROM AUTHOR]

In mammals, meiosis is initiated at different time points in males and females, but the mechanism underlying this difference is unknown. Female germ cells begin meiosis during embryogenesis. In males, embryonic germ cells undergo G0/G1 mitotic cell cycle arrest, and meiosis begins after birth. In mice, the Stimulated by Retinoic Acid Gene 8 (Stra8) has been found to be required for the transition into meiosis in both female and male germ cells. Stra8 is expressed in embryonic ovaries just before meiotic initiation, whereas its expression in testes is first detected after birth. Here we examine the mechanism underlying the sex-specific timing of Stra8 expression and meiotic initiation in mice. Our work shows that signaling by retinoic acid (RA), an active derivative of vitamin A, is required for Stra8 expression and thereby meiotic initiation in embryonic ovaries. We also discovered that RA is sufficient to induce Stra8 expression in embryonic testes and in vitamin A-deficient adult testes in vivo. Finally, our results show that cytochrome p450 (CYP)-mediated RA metabolism prevents premature Stra8 expression in embryonic testes. Treatment with an inhibitor specific to RA-metabolizing enzymes indicates that a cytochrome p450 from the 26 family (CYP26) is responsible for delaying Stra8 expression in embryonic testes. Sex-specific regulation of RA signaling thus plays an essential role in meiotic initiation in embryonic ovaries and precludes its occurrence in embryonic testes. Because RA signaling regulates Stra8 expression in both embryonic ovaries and adult testes, this portion of the meiotic initiation pathway may be identical in both sexes. [ABSTRACT FROM AUTHOR]

Investigates the characteristic of genetic recombination on reciprocal and unilateral crossovers. Identification of parental phenotypes; Formation of the chromosomes; Observation of the nucleotide polymorphism.