Pantograph Detection Algorithm with Complex Background and External Disturbances.

As an important equipment for high-speed railway (HSR) to obtain electric power from outside, the state of the pantograph will directly affect the operation safety of HSR. In order to solve the problems that the current pantograph detection method is easily affected by the environment, cannot effectively deal with the interference of external scenes, has a low accuracy rate and can hardly meet the actual operation requirements of HSR, this study proposes a pantograph detection algorithm. The algorithm mainly includes three parts: the first is to use you only look once (YOLO) V4 to detect and locate the pantograph region in real-time; the second is the blur and dirt detection algorithm for the external interference directly affecting the high-speed camera (HSC), which leads to the pantograph not being detected; the last is the complex background detection algorithm for the external complex scene "overlapping" with the pantograph when imaging, which leads to the pantograph not being recognized effectively. The dirt and blur detection algorithm combined with blob detection and improved Brenner method can accurately evaluate the dirt or blur of HSC, and the complex background detection algorithm based on grayscale and vertical projection can greatly reduce the external scene interference during HSR operation. The algorithm proposed in this study was analyzed and studied on a large number of video samples of HSR operation, and the precision on three different test samples reached 99.92%, 99.90% and 99.98%, respectively. Experimental results show that the algorithm proposed in this study has strong environmental adaptability and can effectively overcome the effects of complex background and external interference on pantograph detection, and has high practical application value.

Race, Natriuretic Peptides, and High-Carbohydrate Challenge: A Clinical Trial.

RATIONALE: Lower NP (natriuretic peptide) levels may contribute to the development of cardiometabolic diseases. Blacks have lower NP levels than middle-aged and older white adults. A high-carbohydrate challenge causes an upregulation of a negative ANP regulator microRNA-425 (miR-425), which reduces ANP (atrial-NP) levels in whites. OBJECTIVES: We designed a prospective trial to study racial differences in (1) NP levels among young adults, (2) NP response to a high-carbohydrate challenge, and (3) explore underlying mechanisms for race-based differences. METHODS AND RESULTS: Healthy self-identified blacks and whites received 3 days of study diet followed by a high-carbohydrate challenge. Gene expression from whole blood RNA was assessed in the trial participants. Additionally, atrial and ventricular tissue samples from the Myocardial Applied Genomics Network repository were examined for NP system gene expression. Among 72 healthy participants, we found that B-type-NP, NT-proBNP (N-terminal-pro-B-type NP), and MRproANP (midregional-pro-ANP) levels were 30%, 47%, and 18% lower in blacks compared with whites (P≤0.01), respectively. The decrease in MRproANP levels in response to a high-carbohydrate challenge differed by race (blacks 23% [95% CI, 19%-27%] versus whites 34% [95% CI, 31%-38]; Pinteraction<0.001), with no change in NT-proBNP levels. We did not observe any racial differences in expression of genes encoding for NPs (NPPA/NPPB) or NP signaling (NPR1) in atrial and ventricular tissues. NP processing (corin), clearance (NPR3), and regulation (miR-425) genes were ≈3.5-, ≈2.5-, and ≈2-fold higher in blacks than whites in atrial tissues, respectively. We also found a 2-and 8-fold higher whole blood RNA expression of gene encoding for Neprilysin (MME) and miR-425 among blacks than whites. CONCLUSIONS: Racial differences in NP levels are evident in young, healthy adults suggesting a state of NP deficiency exists in blacks. Impaired NP processing and clearance may contribute to race-based NP differences. Higher miR-425 levels in blacks motivate additional studies to understand differences in NP downregulation after physiological perturbations. CLINICAL TRIAL REGISTRATION: URL: https://clinicaltrials.gov/ct2/show/NCT03072602. Unique identifier: NCT03072602.

In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa.

Reliable genome editing via Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 may provide a means to correct inherited diseases in patients. As proof of principle, we show that CRISPR/Cas9 can be used in vivo to selectively ablate the rhodopsin gene carrying the dominant S334ter mutation (Rho(S334)) in rats that model severe autosomal dominant retinitis pigmentosa. A single subretinal injection of guide RNA/Cas9 plasmid in combination with electroporation generated allele-specific disruption of Rho(S334), which prevented retinal degeneration and improved visual function.

BRCA1/2 Mutations and Cardiovascular Function in Breast Cancer Survivors.

OBJECTIVE: Animal models suggest that BRCA1/2 mutations increase doxorubicin-induced cardiotoxicity risk but data in humans are limited. We aimed to determine whether germline BRCA1/2 mutations are associated with cardiac dysfunction in breast cancer survivors. METHODS: In a single-center cross-sectional study, stage I-III breast cancer survivors were enrolled according to three groups: (1) BRCA1/2 mutation carriers treated with doxorubicin; (2) BRCA1/2 mutation non-carriers treated with doxorubicin; and (3) BRCA1/2 mutation carriers treated with non-doxorubicin cancer therapy. In age-adjusted analysis, core-lab quantitated measures of echocardiography-derived cardiac function and cardiopulmonary exercise testing (CPET) were compared across the groups. A complementary in vitro study was performed to assess the impact of BRCA1 loss of function on human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) survival following doxorubicin exposure. RESULTS: Sixty-seven women with mean (standard deviation) age of 50 (11) years were included. Age-adjusted left ventricular ejection fraction (LVEF) was lower in participants receiving doxorubicin regardless of BRCA1/2 mutation status (p = 0.03). In doxorubicin-treated BRCA1/2 mutation carriers and non-carriers, LVEF was lower by 5.4% (95% CI; -9.3, -1.5) and 4.8% (95% CI; -9.1, -0.5), respectively compared to carriers without doxorubicin exposure. No significant differences in VO2max were observed across the three groups (poverall = 0.07). Doxorubicin caused a dose-dependent reduction in viability of iPSC-CMs in vitro without differences between BRCA1 mutant and wild type controls (p > 0.05). CONCLUSIONS: BRCA1/2 mutation status was not associated with differences in measures of cardiovascular function or fitness. Our findings do not support a role for increased cardiotoxicity risk with BRCA1/2 mutations in women with breast cancer.

Individual-specific functional epigenomics reveals genetic determinants of adverse metabolic effects of glucocorticoids.

Glucocorticoids (GCs) are widely used as anti-inflammatory drugs, but their long-term use has severe metabolic side effects. Here, by treating multiple individual adipose stem cell-derived adipocytes and induced pluripotent stem cell-derived hepatocytes with the potent GC dexamethasone (Dex), we uncovered cell-type-specific and individual-specific GC-dependent transcriptomes and glucocorticoid receptor (GR) cistromes. Individual-specific GR binding could be traced to single-nucleotide polymorphisms (SNPs) that altered the binding motifs of GR or its cooperating factors. We also discovered another set of genetic variants that modulated Dex response through affecting chromatin accessibility or chromatin architecture. Several SNPs that altered Dex-regulated GR binding and gene expression controlled Dex-driven metabolic perturbations. Remarkably, these genetic variations were highly associated with increases in serum glucose, lipids, and body mass in subjects on GC therapy. Knowledge of the genetic variants that predispose individuals to metabolic side effects allows for a precision medicine approach to the use of clinically relevant GCs.

Pathogenic LMNA variants disrupt cardiac lamina-chromatin interactions and de-repress alternative fate genes.

Pathogenic mutations in LAMIN A/C (LMNA) cause abnormal nuclear structure and laminopathies. These diseases have myriad tissue-specific phenotypes, including dilated cardiomyopathy (DCM), but how LMNA mutations result in tissue-restricted disease phenotypes remains unclear. We introduced LMNA mutations from individuals with DCM into human induced pluripotent stem cells (hiPSCs) and found that hiPSC-derived cardiomyocytes, in contrast to hepatocytes or adipocytes, exhibit aberrant nuclear morphology and specific disruptions in peripheral chromatin. Disrupted regions were enriched for transcriptionally active genes and regions with lower LAMIN B1 contact frequency. The lamina-chromatin interactions disrupted in mutant cardiomyocytes were enriched for genes associated with non-myocyte lineages and correlated with higher expression of those genes. Myocardium from individuals with LMNA variants similarly showed aberrant expression of non-myocyte pathways. We propose that the lamina network safeguards cellular identity and that pathogenic LMNA variants disrupt peripheral chromatin with specific epigenetic and molecular characteristics, causing misexpression of genes normally expressed in other cell types.

Investigation of a dilated cardiomyopathy-associated variant in BAG3 using genome-edited iPSC-derived cardiomyocytes.

Mutations in B cell lymphoma 2-associated athanogene 3 (BAG3) are recurrently associated with dilated cardiomyopathy (DCM) and muscular dystrophy. Using isogenic genome-edited human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we examined how a DCM-causing BAG3 mutation (R477H), as well as complete loss of BAG3 (KO), impacts myofibrillar organization and chaperone networks. Although unchanged at baseline, fiber length and alignment declined markedly in R477H and KO iPSC-CMs following proteasome inhibition. RNA sequencing revealed extensive baseline changes in chaperone- and stress response protein-encoding genes, and protein levels of key BAG3 binding partners were perturbed. Molecular dynamics simulations of the BAG3-HSC70 complex predicted a partial disengagement by the R477H mutation. In line with this, BAG3-R477H bound less HSC70 than BAG3-WT in coimmunoprecipitation assays. Finally, myofibrillar disarray triggered by proteasome inhibition in R477H cells was mitigated by overexpression of the stress response protein heat shock factor 1 (HSF1). These studies reveal the importance of BAG3 in coordinating protein quality control subsystem usage within the cardiomyocyte and suggest that augmenting HSF1 activity might be beneficial as a means to mitigate proteostatic stress in the context of BAG3-associated DCM.

Identification of WSB1 gene as an important regulator in the development of zebrafish embryo during midblastula transition.

To uncover novel genes potentially involved in embryo development, especially at the midblastula transition (MBT) phase in the developing embryo of zebrafish, Affymetrix zebrafish GeneChip microarray analysis was carried out on the expression of 14,900 gene transcripts. The results of the analysis showed that 360 genes were clearly up-regulated and 119 genes were markedly down-regulated. Many of these genes were involved in transcription factor activity, nucleic acid binding, and cell growth. The present study showed that significant changes in transcript abundance occurred during the MBT phase. The expression of eight of these 479 genes was identified by reverse transcription-polymerase chain reaction analysis, confirming the microarray results. The WSB1 gene, found to be down-regulated by the microarray and reverse transcription-polymerase chain reaction analyses, was selected for further study. Sequence analysis of the WSB1 gene showed that it encodes a protein with 75% identity to the corresponding active human orthologs. In addition, WSB1 gene expression was detected at a higher level at 2 h post fertilization and at a lower level at 4 h post fertilization, consistent with the chip results. Overexpression of the WSB1 gene can result in the formation of abnormalities in embryos, as determined by fluorescence-activated cell sorting. The present study showed unequivocally that the occurrence of WSB1 expression is an important event during the MBT phase in the development of zebrafish embryos.

In utero CRISPR-mediated therapeutic editing of metabolic genes.

In utero gene editing has the potential to prenatally treat genetic diseases that result in significant morbidity and mortality before or shortly after birth. We assessed the viral vector-mediated delivery of CRISPR-Cas9 or base editor 3 in utero, seeking therapeutic modification of Pcsk9 or Hpd in wild-type mice or the murine model of hereditary tyrosinemia type 1, respectively. We observed long-term postnatal persistence of edited cells in both models, with reduction of plasma PCSK9 and cholesterol levels following in utero Pcsk9 targeting and rescue of the lethal phenotype of hereditary tyrosinemia type 1 following in utero Hpd targeting. The results of this proof-of-concept work demonstrate the possibility of efficiently performing gene editing before birth, pointing to a potential new therapeutic approach for selected congenital genetic disorders.

Opposing Roles of Acetylation and Phosphorylation in LIFR-Dependent Self-Renewal Growth Signaling in Mouse Embryonic Stem Cells.

LIF promotes self-renewal of mouse embryonic stem cells (mESCs), and in its absence, the cells differentiate. LIF binds to the LIF receptor (LIFR) and activates the JAK-STAT3 pathway, but it remains unknown how the receptor complex triggers differentiation or self-renewal. Here, we report that the LIFR cytoplasmic domain contains a self-renewal domain within the juxtamembrane region and a differentiation domain within the C-terminal region. The differentiation domain contains four SPXX repeats that are phosphorylated by MAPK to restrict STAT3 activation; the self-renewal domain is characterized by a 3K motif that is acetylated by p300. In mESCs, acetyl-LIFR undergoes homodimerization, leading to STAT3 hypo- or hyper-activation depending on the presence or absence of gp130. LIFR-activated STAT3 restricts differentiation via cytokine induction. Thus, LIFR acetylation and serine phosphorylation differentially promote stem cell self-renewal and differentiation.

Large, Diverse Population Cohorts of hiPSCs and Derived Hepatocyte-like Cells Reveal Functional Genetic Variation at Blood Lipid-Associated Loci.

Genome-wide association studies have struggled to identify functional genes and variants underlying complex phenotypes. We recruited a multi-ethnic cohort of healthy volunteers (n = 91) and used their tissue to generate induced pluripotent stem cells (iPSCs) and hepatocyte-like cells (HLCs) for genome-wide mapping of expression quantitative trait loci (eQTLs) and allele-specific expression (ASE). We identified many eQTL genes (eGenes) not observed in the comparably sized Genotype-Tissue Expression project's human liver cohort (n = 96). Focusing on blood lipid-associated loci, we performed massively parallel reporter assays to screen candidate functional variants and used genome-edited stem cells, CRISPR interference, and mouse modeling to establish rs2277862-CPNE1, rs10889356-DOCK7, rs10889356-ANGPTL3, and rs10872142-FRK as functional SNP-gene sets. We demonstrated HLC eGenes CPNE1, VKORC1, UBE2L3, and ANGPTL3 and HLC ASE gene ACAA2 to be lipid-functional genes in mouse models. These findings endorse an iPSC-based experimental framework to discover functional variants and genes contributing to complex human traits.

Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation.

Induced pluripotent stem (iPS) cells generated using Yamanaka factors have great potential for use in autologous cell therapy. However, genomic abnormalities exist in human iPS cells, and most mouse iPS cells are not fully pluripotent, as evaluated by the tetraploid complementation assay (TCA); this is most likely associated with the DNA damage response (DDR) occurred in early reprogramming induced by Yamanaka factors. In contrast, nuclear transfer can faithfully reprogram somatic cells into embryonic stem (ES) cells that satisfy the TCA. We thus hypothesized that factors involved in oocyte-induced reprogramming may stabilize the somatic genome during reprogramming, and improve the quality of the resultant iPS cells. To test this hypothesis, we screened for factors that could decrease DDR signals during iPS cell induction. We determined that Zscan4, in combination with the Yamanaka factors, not only remarkably reduced the DDR but also markedly promoted the efficiency of iPS cell generation. The inclusion of Zscan4 stabilized the genomic DNA, resulting in p53 downregulation. Furthermore, Zscan4 also enhanced telomere lengthening as early as 3 days post-infection through a telomere recombination-based mechanism. As a result, iPS cells generated with addition of Zscan4 exhibited longer telomeres than classical iPS cells. Strikingly, more than 50% of iPS cell lines (11/19) produced via this "Zscan4 protocol" gave rise to live-borne all-iPS cell mice as determined by TCA, compared to 1/12 for lines produced using the classical Yamanaka factors. Our findings provide the first demonstration that maintaining genomic stability during reprogramming promotes the generation of high quality iPS cells.