IL-17 signaling in primary sclerosing cholangitis patient-derived organoids.

BACKGROUND: The pathogenesis of primary sclerosing cholangitis (PSC) is unclear, although studies implicate IL-17A as an inflammatory mediator in this disease. However, a direct assessment of IL-17 signaling in PSC cholangiocytes is lacking. In this study, we aimed to investigate and characterize the response of PSC extrahepatic cholangiocyte organoids (ECO) to IL-17A stimulation. METHODS: Cholangiocytes obtained from patients with PSC and without PSC by endoscopic retrograde cholangiography were cultured as ECO. The ECO were treated with vehicle or IL-17A and assessed by transcriptomics, secretome analysis, and genome sequencing. RESULTS: Unsupervised clustering of all integrated single-cell RNA sequencing data identified 8 cholangiocyte clusters that did not differ between PSC and non-PSC ECO. However, PSC ECO cells demonstrated a robust response to IL-17 treatment, as noted by an increased number of differentially expressed genes by transcriptomics and more abundant chemokine and cytokine expression and secretion. After rigorous filtering, genome sequencing identified candidate somatic variants shared among PSC ECO from unrelated individuals. However, no candidate rare variants in genes regulating the IL-17 pathway were identified, but rare variants regulating the MAPK signaling pathway were present in all PSC ECO. CONCLUSIONS: PSC and non-PSC patient-derived ECO respond differently to IL-17 stimulation, implicating this pathway in the pathogenesis of PSC.

Syngeneic murine models with distinct immune microenvironments represent subsets of human intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a poorly immunogenic malignancy associated with limited survival. Syngeneic immunocompetent mouse models of CCA are an essential tool to elucidate the tumor immune microenvironment (TIME), understand mechanisms of tumor immune evasion, and test novel immunotherapeutic strategies. The scope of this study was to develop and characterize immunocompetent CCA models with distinct genetic drivers, and correlate tumor genomics, immunobiology, and therapeutic response. METHODS: A multifaceted approach including scRNA-seq, CITE-seq, whole exome and bulk RNA sequencing was employed. FDA-approved PD-1/PD-L1 antibodies were tested in humanized PD-1/PD-L1 mice (HuPD-H1). RESULTS: A genetic mouse model of intrahepatic CCA (iCCA) driven by intrabiliary transduction of Fbxw7ΔF/Akt that mimics human iCCA was generated. From the Fbxw7ΔF/Akt tumors, a murine cell line (FAC) and syngeneic model with genetic and phenotypic characteristics of human iCCA were developed. Established SB1 (YAPS127A/Akt) and KPPC (KrasG12Dp53L/L) models were compared to the FAC model. Although the models had transcriptomic similarities, they had substantial differences as well. Mutation patterns of FAC, SB1, and KPPC cells matched different mutational signatures in Western and Japanese CCA patient cohorts. KPPC tumors had a high tumor mutation burden. FAC tumors had a T cell-infiltrated TIME, while SB1 tumors had a preponderance of suppressive myeloid cells. FAC, SB1, and KPPC tumors matched different immune signatures in human iCCA cohorts. Moreover, FAC, SB1, and KPPC tumor-bearing HuPD-H1 mice displayed differential responses to nivolumab or durvalumab. CONCLUSIONS: Syngeneic iCCA models display a correlation between tumor genotype and TIME phenotype, with differential responses to FDA-approved immunotherapies. This study underscores the importance of leveraging multiple preclinical models to understand responses to immunotherapy in different genetic subsets of human CCA. IMPACT AND IMPLICATIONS: Understanding the relationship between tumor genotype and the phenotype of the immune microenvironment is an unmet need in cholangiocarcinoma (CCA). Herein, we use syngeneic murine models of intrahepatic CCA with different genetic drivers to demonstrate a correlation between tumor genotype and immune microenvironment phenotype in murine models, which is associated with differential responses to FDA-approved immunotherapies. This information will help guide other preclinical studies. Additionally, it emphasizes that immune checkpoint inhibition in patients with CCA is not a "one-size-fits-all" approach. Our observations suggest that, as for targeted therapies, patients should be stratified and selected for treatment according to their tumor genetics.

Sulfation on polysaccharides from Zizania latifolia extracted using ultrasound: Characterization, antioxidant and anti-non-small cell lung cancer activities.

Zizania latifolia is a highly nutritious vegetable being praised as "Ginseng in Water". Polysaccharides are the main bioactive ingredients in Z. latifolia, but there have been no reports on the yield- and activity-guided ultrasonic-assisted extraction (UAE), sulfation and anti-non-small cell lung cancer (NSCLC) activity. In this study, Z. latifolia polysaccharides (ZLP) were extracted using UAE under an optimized power, followed by sulfation to give three derivatives (SZLP-1 âˆ¼ 3). After characterization, the antioxidant and anti-NSCLC activities were evaluated. The optimal ultrasonic power for ZLP extraction was screened out to be 300 W, under which the yield was 16.9 ± 2.10 %, and the scavenging rate against 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical was 63.3 ± 5.71 %, significantly higher than those of other powers and hot-water extraction. A series of characterizations fully confirmed the sulfated modification of ZLP. Sulfation improved the antioxidation of ZLP and was positively proportional to the degree of substitution (DS), of which SZLP-2 with a DS of 15.1 ± 2.50 elicited strong hydroxyl and DPPH radicals-scavenging capacities. Meanwhile, SZLP-2 also exerted promising anti-NSCLC potency via inhibiting A549 cell proliferation, with a median inhibition concentration (IC50) of 0.57 ± 0.01 mg/mL at 72 h, markedly smaller than that of unmodified ZLP (0.78 ± 0.04 mg/mL). In summary, the yield- and activity-guided UAE led to the ZLP with high yield and strong antioxidation. Further sulfation enhanced the bioactivities and produced the promising SZLP-2, which showed great potential in the development of novel antioxidant and anti-NSCLC drug.

Noncanonical TRAIL Signaling Promotes Myeloid-Derived Suppressor Cell Abundance and Tumor Growth in Cholangiocarcinoma.

BACKGROUND & AIMS: Proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling as a cause of cancer cell death is a well-established mechanism. However, TRAIL-receptor (TRAIL-R) agonists have had very limited anticancer activity in human beings, challenging the concept of TRAIL as a potent anticancer agent. Herein, we aimed to define mechanisms by which TRAIL+ cancer cells can leverage noncanonical TRAIL signaling in myeloid-derived suppressor cells (MDSCs) promoting their abundance in murine cholangiocarcinoma (CCA). METHODS: Multiple immunocompetent syngeneic, orthotopic models of CCA were used. Single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing of CD45+ cells in murine tumors from the different CCA models was conducted. RESULTS: In multiple immunocompetent murine models of CCA, implantation of TRAIL+ murine cancer cells into Trail-r-/- mice resulted in a significant reduction in tumor volumes compared with wild-type mice. Tumor-bearing Trail-r-/- mice had a significant decrease in the abundance of MDSCs owing to attenuation of MDSC proliferation. Noncanonical TRAIL signaling with consequent nuclear factor-κB activation in MDSCs facilitated enhanced MDSC proliferation. Single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing of immune cells from murine tumors showed enrichment of a nuclear factor-κB activation signature in MDSCs. Moreover, MDSCs were resistant to TRAIL-mediated apoptosis owing to enhanced expression of cellular FLICE inhibitory protein, an inhibitor of proapoptotic TRAIL signaling. Accordingly, cellular FLICE inhibitory protein knockdown sensitized murine MDSCs to TRAIL-mediated apoptosis. Finally, cancer cell-restricted deletion of Trail significantly reduced MDSC abundance and murine tumor burden. CONCLUSIONS: Our findings highlight the therapeutic potential of targeting TRAIL+ cancer cells for treatment of a poorly immunogenic cancer.

Polysaccharides from waste Zingiber mioga leaves: Ultrasonic-microwave-assisted extraction, characterization, antioxidant and anticoagulant potentials.

Zingiber mioga is a highly economic crop that is used to produce vegetables, spices and herbal pharmaceuticals. Its edible flower bud contributes most to the economic value, but the big leaves were discarded as agricultural waste, which urgently needs to be exploited. In this work, polysaccharides from waste Z. mioga leaves (PWZMLs) were extracted using ultrasonic-microwave-assisted extraction (UMAE). After purification and characterization, the antioxidation and anticoagulation of PWZMLs were evaluated to appraise the potential in cardiovascular protection. Under the liquid-solid ratio of 26: 1 mL/g, after ultrasonication at 495 W for 10 min, followed by microwaving at 490 W for 5 min, the yield of PWZMLs achieved to 6.22 ± 0.14 %, notably higher (P < 0.01) than other methods, and ultrasound contributed more to the yield than microwave. Various analyses confirmed that PWZMLs were negatively charged polysaccharides with galacturonic acid the dominant uronic acid. PWZMLs exerted excellent antioxidant capacity, especially for scavenging 1, 1-diphenyl-2-picrylhydrazyl radical. PWZMLs also elicited promising anticoagulant property, particularly for prolonging activated partial thromboplastin time and lowering fibrinogen, which were almost equivalent to heparin at the same concentration. PWZMLs contained two polysaccharide fractions (199.53 and 275.42 kDa) that could synergistically contribute to the pronounced antioxidant and anticoagulant activities. The PWZMLs extracted with optimized UMAE have great potential in cardiovascular protection.

IL-17 Signaling in Primary Sclerosing Cholangitis Patient-Derived Organoids.

The pathogenesis of primary sclerosing cholangitis (PSC) is unclear, although studies implicate IL-17A as an inflammatory mediator in this disease. However, a direct assessment of IL-17 signaling in PSC cholangiocytes is lacking. In this study we aimed to investigate the response of PSC extrahepatic cholangiocyte organoids (ECO) to IL-17A stimulation. Cholangiocytes obtained from PSC and non-PSC patients by endoscopic retrograde cholangiography (ERC) were cultured as ECO. The ECO were treated with vehicle or IL-17A and assessed by transcriptomics, secretome analysis, and genome sequencing (GS). Unsupervised clustering of all integrated scRNA-seq data identified 8 cholangiocyte clusters which did not differ between PSC and non-PSC ECO. However, PSC ECO cells demonstrated a robust response to IL-17 treatment, noted by an increased number of differentially expressed genes (DEG) by transcriptomics, and more abundant chemokine and cytokine expression and secretion. After rigorous filtering, GS identified candidate somatic variants shared among PSC ECO from unrelated individuals. However, no candidate rare variants in genes regulating the IL-17 pathway were identified, but rare variants regulating the MAPK signaling pathway were present in all PSC ECO. In conclusion, PSC and non-PSC patient derived ECO respond differently to IL-17 stimulation implicating this pathway in the pathogenesis of PSC.

Noncanonical TRAIL Signaling Promotes Myeloid-Derived Suppressor Cell Abundance and Tumor Progression in Cholangiocarcinoma.

Proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling as a cause of cancer cell death is a well-established mechanism. However, TRAIL-receptor (TRAIL-R) agonists have had very limited anticancer activity in humans, challenging the concept of TRAIL as a potent anticancer agent. Herein, we demonstrate that TRAIL + cancer cells can leverage noncanonical TRAIL signaling in myeloid-derived suppressor cells (MDSCs) promoting their abundance in murine cholangiocarcinoma (CCA). In multiple immunocompetent syngeneic, orthotopic murine models of CCA, implantation of TRAIL + murine cancer cells into Trail-r -/- mice resulted in a significant reduction in tumor volumes compared to wild type mice. Tumor bearing Trail-r -/- mice had a significant decrease in the abundance of MDSCs due to attenuation of MDSC proliferation. Noncanonical TRAIL signaling with consequent NF-κB activation in MDSCs facilitated enhanced MDSC proliferation. Single cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) of CD45 + cells in murine tumors from three distinct immunocompetent CCA models demonstrated a significant enrichment of an NF-κB activation signature in MDSCs. Moreover, MDSCs were resistant to TRAIL-mediated apoptosis due to enhanced expression of cellular FLICE inhibitory protein (cFLIP), an inhibitor of proapoptotic TRAIL signaling. Accordingly, cFLIP knockdown sensitized murine MDSCs to TRAIL-mediated apoptosis. Finally, cancer cell-restricted deletion of Trail significantly reduced MDSC abundance and murine tumor burden. In summary, our findings define a noncanonical TRAIL signal in MDSCs and highlight the therapeutic potential of targeting TRAIL + cancer cells for the treatment of a poorly immunogenic cancer.

SCCNet: Self-correction boundary preservation with a dynamic class prior filter for high-variability ultrasound image segmentation.

The highly ambiguous nature of boundaries and similar objects is difficult to address in some ultrasound image segmentation tasks, such as neck muscle segmentation, leading to unsatisfactory performance. Thus, this paper proposes a two-stage network called SCCNet (self-correction context network) using a self-correction boundary preservation module and class-context filter to alleviate these problems. The proposed self-correction boundary preservation module uses a dynamic key boundary point (KBP) map to increase the capability of iteratively discriminating ambiguous boundary points segments, and the predicted segmentation map from one stage is used to obtain a dynamic class prior filter to improve the segmentation performance at Stage 2. Finally, three datasets, Neck Muscle, CAMUS and Thyroid, are used to demonstrate that our proposed SCCNet outperforms other state-of-the art methods, such as BPBnet, DSNnet, and RAGCnet. Our proposed network shows at least a 1.2-3.7% improvement on the three datasets, Neck Muscle, Thyroid, and CAMUS. The source code is available at https://github.com/lijixing0425/SCCNet.

Genetic therapy in a mitochondrial disease model suggests a critical role for liver dysfunction in mortality.

The clinical and largely unpredictable heterogeneity of phenotypes in patients with mitochondrial disorders demonstrates the ongoing challenges in the understanding of this semi-autonomous organelle in biology and disease. Previously, we used the gene-breaking transposon to create 1200 transgenic zebrafish strains tagging protein-coding genes (Ichino et al., 2020), including the lrpprc locus. Here, we present and characterize a new genetic revertible animal model that recapitulates components of Leigh Syndrome French Canadian Type (LSFC), a mitochondrial disorder that includes diagnostic liver dysfunction. LSFC is caused by allelic variations in the LRPPRC gene, involved in mitochondrial mRNA polyadenylation and translation. lrpprc zebrafish homozygous mutants displayed biochemical and mitochondrial phenotypes similar to clinical manifestations observed in patients, including dysfunction in lipid homeostasis. We were able to rescue these phenotypes in the disease model using a liver-specific genetic model therapy, functionally demonstrating a previously under-recognized critical role for the liver in the pathophysiology of this disease.

A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene.

Previously we showed the generation of a protein trap library made with the gene-break transposon (GBT) in zebrafish (Danio rerio) that could be used to facilitate novel functional genome annotation towards understanding molecular underpinnings of human diseases (Ichino et al, 2020). Here, we report a significant application of this library for discovering essential genes for heart rhythm disorders such as sick sinus syndrome (SSS). SSS is a group of heart rhythm disorders caused by malfunction of the sinus node, the heart's primary pacemaker. Partially owing to its aging-associated phenotypic manifestation and low expressivity, molecular mechanisms of SSS remain difficult to decipher. From 609 GBT lines screened, we generated a collection of 35 zebrafish insertional cardiac (ZIC) mutants in which each mutant traps a gene with cardiac expression. We further employed electrocardiographic measurements to screen these 35 ZIC lines and identified three GBT mutants with SSS-like phenotypes. More detailed functional studies on one of the arrhythmogenic mutants, GBT411, in both zebrafish and mouse models unveiled Dnajb6 as a novel SSS causative gene with a unique expression pattern within the subpopulation of sinus node pacemaker cells that partially overlaps with the expression of hyperpolarization activated cyclic nucleotide gated channel 4 (HCN4), supporting heterogeneity of the cardiac pacemaker cells.

Microwave-Assisted Enzymatic Extraction, Partial Characterization, and Antioxidant Potential of Polysaccharides from Sagittaria trifolia Tuber.

Sagittaria trifolia tuber is an aquatic vegetable. In this work, microwave-assisted enzymatic extraction (MEE) was used to extract S. trifolia tuber polysaccharides (STTPs). Optimum conditions were complex enzyme of 2 %, liquid-to-solid ratio of 43 : 1 mL g-1 , microwave power of 506 W, and time of 8 min, under which STTPs yield was 36.22±0.69 %, higher than those of other methods. STTPs were sulfated polysaccharides with sulfur valence of S6+ . STTPs comprised mannose, glucose, galactose, and arabinose at a mole ratio of 3.69 : 19.33 : 6.21 : 1.00, molecular weights of 3606 kDa and 149.6 kDa, particle size of 220 nm, and zeta potential of -5.02 mV. The surface of STTPs was full of bumps and holes, and abundant in O1s and non-functionalized C1s. STTPs would scavenge reactive oxygen species with advantage. It would provide an efficient MEE method to obtain antioxidant STTPs, also a clue for extracting polysaccharides from starch-rich crops.

Changes in ion channel expression and function associated with cardiac arrhythmogenic remodeling by Sorbs2.

The Sorbin and SH3 domain-containing protein 2 (Sorbs2) is an important component of cardiomyocyte sarcomere. It has been recently reported that loss of Sorbs2 is causally associated with arrhythmogenic cardiomyopathy in human. However, the ionic mechanisms leading to cardiac arrhythmogenesis by Sorbs2 deficiency are unknown. In this study, we hypothesized that Sorbs2 plays an important role in regulating cardiac ion channel expression and function. Using electrophysiological and molecular biological approaches, we found that the Sorbs2 knockout (KO) mice progressively developed cardiac structural and electrical remodeling as early as 1 to 2 months of age and died prematurely at 5 to 7 months of age. Electrocardiographic recordings showed that Sorbs2 KO mice had conduction delays, spontaneous ventricular extrasystoles and polymorphic ventricular tachyarrhythmia. Intracellular recordings revealed abnormal action potentials with depolarized resting potential, reduced upstroke velocity, prolonged repolarization, and effective refractory period in the ventricular preparations of Sorbs2 KO mice. Patch clamp experiments demonstrated that Sorbs2 KO mice displayed distinct abnormalities in the expression and function of cardiac ion channels, including those of the voltage-gated Na+ channels, L-type Ca2+ channels, the voltage-gated K+ channels and the inward-rectifier K+ channels. Moreover, Sorbs2 physically interacted with the RNAs and/or proteins of important cardiac ion channels and directly regulated their expression in vitro. Our results indicate that Sorbs2 plays a pivotal role in the regulation of cardiac channel physiology. Loss of Sorbs2 promotes cardiac ion channelopathies and life-threatening arrhythmias.

Knockout of SORBS2 Protein Disrupts the Structural Integrity of Intercalated Disc and Manifests Features of Arrhythmogenic Cardiomyopathy.

Background Sorbs2b (sorbin and SH3 domain-containing 2b) was recently identified as a cardiomyopathy gene from a zebrafish mutagenesis screen. However, cardiac functions of its mammalian ortholog remain elusive. Methods and Results We conducted a detailed expression and subcellular localization analysis of Sorbs2 ortholog in mice and a phenotypic characterization in Sorbs2 knockout mice. Sorbs2 is highly expressed in the mouse heart and encodes an adhesion junction/desmosome protein that is mainly localized to the intercalated disc. A mutation with near complete depletion of the Sorbs2 protein in mice results in phenotypes characteristic of human arrhythmogenic cardiomyopathy (ACM), including right ventricular dilation, right ventricular dysfunction, spontaneous ventricular tachycardia, and premature death. Sorbs2 is required to maintain the structural integrity of intercalated disc. Its absence resulted in profound cardiac electrical remodeling with impaired impulse conduction and action potential derangements. Targeted sequencing of human patients with ACM identified 2 rare splicing variants classified as likely pathogenic were in 2 unrelated individuals with ACM from a cohort of 59 patients with ACM. Conclusions The Sorbs2 knockout mouse manifests several key features reminiscent of human ACM. Although the candidacy of SORBS2 as a new ACM-susceptibility gene is supported by preliminary human genetics study, future validation in larger cohorts with ACM is needed.

Targeting tumor-associated macrophages and granulocytic myeloid-derived suppressor cells augments PD-1 blockade in cholangiocarcinoma.

Immune checkpoint blockade (ICB) has revolutionized cancer therapeutics. Desmoplastic malignancies, such as cholangiocarcinoma (CCA), have an abundant tumor immune microenvironment (TIME). However, to date, ICB monotherapy in such malignancies has been ineffective. Herein, we identify tumor-associated macrophages (TAMs) as the primary source of programmed death-ligand 1 (PD-L1) in human and murine CCA. In a murine model of CCA, recruited PD-L1+ TAMs facilitated CCA progression. However, TAM blockade failed to decrease tumor progression due to a compensatory emergence of granulocytic myeloid-derived suppressor cells (G-MDSCs) that mediated immune escape by impairing T cell response. Single-cell RNA sequencing (scRNA-Seq) of murine tumor G-MDSCs highlighted a unique ApoE G-MDSC subset enriched with TAM blockade; further analysis of a human scRNA-Seq data set demonstrated the presence of a similar G-MDSC subset in human CCA. Finally, dual inhibition of TAMs and G-MDSCs potentiated ICB. In summary, our findings highlight the therapeutic potential of coupling ICB with immunotherapies targeting immunosuppressive myeloid cells in CCA.

Phenotyping an adult zebrafish lamp2 cardiomyopathy model identifies mTOR inhibition as a candidate therapy.

Adult zebrafish is an emerging vertebrate model for studying genetic basis of cardiomyopathies; but whether the simple fish heart can model essential features of hypertrophic cardiomyopathy (HCM) remained unknown. Here, we report a comprehensive phenotyping of a lamp2 knockout (KO) mutant. LAMP2 encodes a lysosomal protein and is a causative gene of Danon disease that is characterized by HCM and massive autophagic vacuoles accumulation in the tissues. There is no effective therapy yet to treat this most lethal cardiomyopathy in the young. First, we did find the autophagic vacuoles accumulation in cardiac tissues from lamp2 KO. Next, through employing a set of emerging phenotyping tools, we revealed heart failure phenotypes in the lamp2 KO mutants, including decreased ventricular ejection fraction, reduced physical exercise capacity, blunted ß-adrenergic contractile response, and enlarged atrium. We also noted changes of the following indices suggesting cardiac hypertrophic remodeling in lamp2 KO: a rounded heart shape, increased end-systolic ventricular volume and density of ventricular myocardium, elevated actomyosin activation kinetics together with increased maximal isometric tension at the level of cardiac myofibrils. Lastly, we assessed the function of lysosomal-localized mTOR on the lamp2-associated Danon disease. We found that haploinsufficiency of mtor was able to normalize some characteristics of the lamp2 KO, including ejection fraction, ß-adrenergic response, and the actomyosin activation kinetics. In summary, we demonstrate the feasibility of modeling the inherited HCM in the adult zebrafish, which can be used to develop potential therapies.

The Value of Contrast-Enhanced Ultrasonography Combined with Real-Time Strain Elastography in the Early Diagnosis of Prostate Cancer.

To evaluate the performance of a combination of real-time strain elastography (RTSE) and contrast-enhanced transrectal ultrasound (CETRUS) for prostate cancer detection. Patients with serum prostate-specific antigen (PSA) levels of ≥4.0 ng/ml were prospectively enrolled between June 2014 and December 2016. 153 prostate nodules diagnosed by conventional ultrasound were prospectively enrolled and examined by CETRUS and RTSE before a biopsy. Multivariate logistic regression models were established for CETRUS, and CETRUS combined with RTSE to diagnose prostate malignancy. The diagnostic performances of CETRUS, RTSE, and their combined use were evaluated with the receiver operating characteristic (ROC) curve. The multivariate logistic regression for CETRUS combined with RTSE showed that enhanced strength, enhanced uniformity, and elasticity scores were the independent predictors of prostate malignancy. The area under the ROC curve of CETRUS combined with RTSE (0.921±0.023) was higher than that of CETRUS and RTSE (0.88±0.029 and 0.80±0.038, respectively; both p<0.05). Moreover, the sensitivity, accuracy and negative predictive value of CETRUS combined with RTSE were 92.1%, 86.2%, and 84.6%, respectively. The omission diagnostic rate of CETRUS combined with RTSE (7.9%) was reduced. And the diagnostic accuracy of CETRUS combined with RTSE was significantly higher than that of CETRUS and RTSE (p<0.05). While the diagnostic accuracy of CETRUS was close to the RTSE, the difference was not statistically significant (p>0.05). The combined RTSE with CETRUS approach significantly improved the sensitivity and overall accuracy for correctly identifying prostate cancer.

Real-Time Monitoring and Analysis of Zebrafish Electrocardiogram with Anomaly Detection.

Heart disease is the leading cause of mortality in the U.S. with approximately 610,000 people dying every year. Effective therapies for many cardiac diseases are lacking, largely due to an incomplete understanding of their genetic basis and underlying molecular mechanisms. Zebrafish (Danio rerio) are an excellent model system for studying heart disease as they enable a forward genetic approach to tackle this unmet medical need. In recent years, our team has been employing electrocardiogram (ECG) as an efficient tool to study the zebrafish heart along with conventional approaches, such as immunohistochemistry, DNA and protein analyses. We have overcome various challenges in the small size and aquatic environment of zebrafish in order to obtain ECG signals with favorable signal-to-noise ratio (SNR), and high spatial and temporal resolution. In this paper, we highlight our recent efforts in zebrafish ECG acquisition with a cost-effective simplified microelectrode array (MEA) membrane providing multi-channel recording, a novel multi-chamber apparatus for simultaneous screening, and a LabVIEW program to facilitate recording and processing. We also demonstrate the use of machine learning-based programs to recognize specific ECG patterns, yielding promising results with our current limited amount of zebrafish data. Our solutions hold promise to carry out numerous studies of heart diseases, drug screening, stem cell-based therapy validation, and regenerative medicine.

Modeling GATAD1-Associated Dilated Cardiomyopathy in Adult Zebrafish.

Animal models have played a critical role in validating human dilated cardiomyopathy (DCM) genes, particularly those that implicate novel mechanisms for heart failure. However, the disease phenotype may be delayed due to age-dependent penetrance. For this reason, we generated an adult zebrafish model, which is a simpler vertebrate model with higher throughput than rodents. Specifically, we studied the zebrafish homologue of GATAD1, a recently identified gene for adult-onset autosomal recessive DCM. We showed cardiac expression of gatad1 transcripts, by whole mount in situ hybridization in zebrafish embryos, and demonstrated nuclear and sarcomeric I-band subcellular localization of Gatad1 protein in cardiomyocytes, by injecting a Tol2 plasmid encoding fluorescently-tagged Gatad1. We next generated gatad1 knock-out fish lines by TALEN technology and a transgenic fish line that expresses the human DCM GATAD1-S102P mutation in cardiomyocytes. Under stress conditions, longitudinal studies uncovered heart failure (HF)-like phenotypes in stable KO mutants and a tendency toward HF phenotypes in transgenic lines. Based on these efforts of studying a gene-based inherited cardiomyopathy model, we discuss the strengths and bottlenecks of adult zebrafish as a new vertebrate model for assessing candidate cardiomyopathy genes.

Understanding cardiac sarcomere assembly with zebrafish genetics.

Mutations in sarcomere genes have been found in many inheritable human diseases, including hypertrophic cardiomyopathy. Elucidating the molecular mechanisms of sarcomere assembly shall facilitate understanding of the pathogenesis of sarcomere-based cardiac disease. Recently, biochemical and genomic studies have identified many new genes encoding proteins that localize to the sarcomere. However, their precise functions in sarcomere assembly and sarcomere-based cardiac disease are unknown. Here, we review zebrafish as an emerging vertebrate model for these studies. We summarize the techniques offered by this animal model to manipulate genes of interest, annotate gene expression, and describe the resulting phenotypes. We survey the sarcomere genes that have been investigated in zebrafish and discuss the potential of applying this in vivo model for larger-scale genetic studies.

Cessation of contraction induces cardiomyocyte remodeling during zebrafish cardiogenesis.

Contraction regulates heart development via a complex mechanotransduction process controlled by various mechanical forces. Here, we exploit zebrafish embryos as an in vivo animal model to discern the contribution from different mechanical forces and identify the underlying mechanotransductive signaling pathways of cardiogenesis. We treated 2 days postfertilization zebrafish embryos with Blebbistatin, a myosin II inhibitor, to stop cardiac contraction, which induces a response termed cessation of contraction-induced cardiomyocyte (CM) enlargement (CCE). Accompanying the CCE, lateral fusion of myofibrils was attenuated within CMs. The CCE can be blunted by loss of blood in tail-docked zebrafish but not in cloche mutant fish, suggesting that transmural pressure rather than shear stress is accountable for the chamber enlargement. By screening a panel of small molecule inhibitors, our data suggested essential functions of phosphoinositide 3-kinase signaling and protein synthesis in CCE, which are independent of the sarcomere integrity. In summary, we defined a unique CCE response in genetically tractable zebrafish embryos. A panel of assays was established to verify the contribution from extrinsic forces and interrogate underlying signaling pathways.