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Pancreatic ductal adenocarcinoma (PDA), a leading cause of cancer-related death in the United States, has a high metastatic rate, and is associated with persistent immune suppression. AXL, a member of the TAM (TYRO3, AXL, MERTK) receptor tyrosine kinase family, is a driver of metastasis and immune suppression in multiple cancer types. Here we use single-cell RNA-sequencing to reveal that AXL is expressed highly in tumor cells that have a mesenchymal-like phenotype and that AXL expression correlates with classic markers of epithelial-to-mesenchymal transition. We demonstrate that AXL deficiency extends survival, reduces primary and metastatic burden, and enhances sensitivity to gemcitabine in an autochthonous model of PDA. PDA in AXL-deficient mice displayed a more differentiated histology, higher nucleoside transporter expression, and a more active immune microenvironment compared with PDA in wild-type mice. Finally, we demonstrate that AXL-positive poorly differentiated tumor cells are critical for PDA progression and metastasis, emphasizing the potential of AXL as a therapeutic target in PDA.Implications:These studies implicate AXL as a marker of undifferentiated PDA cells and a target for therapy.

S1. Gene Ontology (GO) analysis of gene clusters from AXL-positive cancer cell populations based on scRNA-Seq data from Hosein et al (35). S2. Expression of Axl in tumors from WT and AXLLacZ/LacZ (AAKIC) KIC mice. S3. Tumor tissues from KIC and AxlLacZ/LacZ KIC mice were evaluated for indicated targets. S4. Flow cytometry of tumor-associated myeloid and lymphoid cells from KIC (n=11) and AxlLacZ/LacZ KIC (n=9). CD11b+ myeloid cells, CD4/CD3, and CD8/CD3 ratio were analyzed. 5. A) Cell proliferation was investigated in three different WT and AXL KO KPfC cells using MTS assay with eight technical replicates. B) Cell migration was investigated in three different WT and AXL KO KPfC cells by a "scratch" assay with three technical replicates. Monolayers of the indicated cells were wounded with a pipet tip. The cells were incubated in 1% serum media. Wound closure was monitored at 12, 24, and 36 hours and is reported as % wound closure. ***P < 0.001; ****P < 0.0001; by t-test. C) Colony formation (colonies/hpf) for three different WT and AXL KO KPfC cells with three technical replicates is shown by box plot (min to max). **P < 0.01; by t-test. S6. Expression of E-cadherin in WT and AXL KO KPfC cells. S7. A) WT and AxlLacZ/LacZ C57BL/6 mice orthotopically injected with 500,000 cells/mouse WT and AXL KO Pan02 cells were enrolled in a survival study. Groups were as follows: WT Pan02 cells in WT C57BL/6 mice (n=9); WT KPfC cells in AxlLacZ/LacZ C57BL/6 mice (n=7); AXL KO KPfC cells in WT C57BL/6 mice (n=6). B) 500,000 cells/mouse WT and AXL KO Pan02 cells were splenically injected into WT and AxlLacZ/LacZ C57BL/6 mice. Liver metastases were investigated 7 weeks after injection. Groups were as follows: WT Pan02 cells in WT C57BL/6 mice (n=8); WT KPfC cells in AxlLacZ/LacZ C57BL/6 mice (n=7); AXL KO KPfC cells in WT C57BL/6 mice (n=10); AXL KO KPfC cells in AxlLacZ/LacZ C57BL/6 mice (n=9). H&E and Alcian Blue stain were conducted in the liver tissues to confirm metastasis. Magnification, 10X. C) 1,000,000 cells/mouse WT and AXL KO Panc1 cells were orthotopically injected into NON SCID mice. Tumor weight were investigated 11 weeks after injection.

With the evolution of Web technologies, various services have become available in a pervasive network environment. Combining simple atomic services into sophisticated applications with a quality-of-service (QoS) guarantee has become a widely studied problem. Given the increasing number of QoS attributes to be considered, conventional service composition approaches with manual workflows and massive computational burdens are no longer effective. Therefore, this article first suggests an efficient many-objective (with four or more objectives) automatic service composition approach named MaSC. In particular, this article introduces a temporal goal decomposition mechanism based on a temporal model to divide an unwieldy problem into several fine-grained subproblems. Viewing this model as an individual representation, we employ an evolutionary process with a novel fitness function to explore the composition solution. The experimental results on the benchmarks show that our approach can simultaneously optimize up to six objectives and achieve a better trade-off between the computation cost and the QoS than two recently proposed automatic composition approaches.

Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene, is a lethal neuromuscular disease. Correction of DMD mutations in animal models has been achieved by CRISPR/Cas9 genome editing using Streptococcus pyogenes Cas9 (SpCas9) delivered by adeno-associated virus (AAV). However, due to the limited viral packaging capacity of AAV, two AAV vectors are required to deliver the SpCas9 nuclease and its single guide RNA (sgRNA), impeding its therapeutic application. We devised an efficient single-cut gene-editing method using a compact Staphylococcus aureus Cas9 (SaCas9) to restore the open reading frame of exon 51, the most commonly affected out-of-frame exon in DMD. Editing of exon 51 in cardiomyocytes derived from human induced pluripotent stem cells revealed a strong preference for exon reframing via a two-nucleotide deletion. We adapted this system to express SaCas9 and sgRNA from a single AAV9 vector. Systemic delivery of this All-In-One AAV9 system restored dystrophin expression and improved muscle contractility in a mouse model of DMD with exon 50 deletion. These findings demonstrate the effectiveness of CRISPR/SaCas9 delivered by a consolidated AAV delivery system in the correction of DMD in vivo, representing a promising therapeutic approach to correct the genetic causes of DMD., Graphical abstract, CRISPR/Cas-mediated gene editing is being used for therapeutic purposes. In this study, Zhang et al. developed a consolidated AAV delivery system to package both SaCas9 nuclease and sgRNA in a single AAV vector and correct Duchenne muscular dystrophy mutations in human cardiomyocytes and mice.

The human brain contains an extraordinarily diverse set of neuronal and glial cell types. Recent advances in single cell transcriptomics have begun to delineate the cellular heterogeneity in different brain regions, but the transcriptional regulatory programs responsible for the identity and function of each brain cell type remain to be defined. Here, we carried out single nucleus ATAC-seq analysis to probe the open chromatin landscape from over 1.1 million cells in 42 brain regions of three neurotypical adult donors. Integrative analysis of the resulting data identified 107 distinct cell types and revealed the cell-type-specific usage of 544,735 candidate cis-regulatory DNA elements (cCREs) in the human genome. Nearly 1/3 of them displayed sequence conservation as well as chromatin accessibility in the mouse brain. On the other hand, nearly 40% cCREs were human specific, with chromatin accessibility associated with species-restricted gene expression. Interestingly, these human specific cCREs were enriched for distinct families of retrotransposable elements, which displayed cell-type-specific chromatin accessibility. We uncovered strong associations between specific brain cell types and neuropsychiatric disorders. We futher developed deep learning models to predict regulatory function of non-coding disease risk variants.

Duchenne muscular dystrophy (DMD) is a fatal muscle disorder characterized by cycles of degeneration and regeneration of multinucleated myofibers and pathological activation of a variety of other muscle-associated cell types. The extent to which different nuclei within the shared cytoplasm of a myofiber may display transcriptional diversity and whether individual nuclei within a multinucleated myofiber might respond differentially to DMD pathogenesis is unknown. Similarly, the potential transcriptional diversity among nonmuscle cell types within dystrophic muscle has not been explored. Here, we describe the creation of a mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent disease-causing mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with myofiber nuclei, as well as other mononucleated cell types that contribute to the muscle pathology associated with DMD, we performed single-nucleus transcriptomics of skeletal muscle of mice with dystrophin exon 51 deletion. Our results reveal distinctive and previously unrecognized myonuclear subtypes within dystrophic myofibers and uncover degenerative and regenerative transcriptional pathways underlying DMD pathogenesis. Our findings provide insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.

The present study aims to acquire deeper understanding of the runner blade channel vortices phenomenon by numerical simulations of the whole turbine passage. Several operating points along different specific discharge were calculated by a hybrid turbulence model, Very Large Eddy Simulation, which was proved that it was able to capture the blade channel vortices similarly. The results showed that the blade channel vortices structure and intensity were various for different runners at incipient point. The flow pattern revealed that the direct relationship of a backflow region and the blade attacking angle near the hub had a distinct variation in vortex structure and intensity. Furthermore, this paper referred a new method to evaluate the strength of blade channel vortex quantitatively, basing on the evolution of specific energy gradient over the stream-wise location. Therefore, this new method enabled the researchers to understand deeply the flow characteristics associated with the blade channel vortices formation and carry out efficiently the hydraulic optimization design.

Hepatitis B is caused by hepatitis B virus (HBV), and persistent HBV infection is a global public health problem, with 257 million people as HBV chronic carriers. Viral covalently closed circular DNA (cccDNA) is a key factor to establish persistent infection in infected hepatocytes. Current antiviral therapies have no direct impact on pre-existing cccDNA reservoir, which can be assembled into minichromosome by hijacking host factors. Understanding the mechanisms of epigenetic regulation in cccDNA minichromosome is crucial to develop new therapy on cccDNA, an attractive target for HBV cure. This review summarizes the current advances in epigenetic regulation of cccDNA minichromosome, which might provide clues to novel druggable targets to cure hepatitis B by either silencing or eliminating cccDNA reservoir.

A precessing vortex core in the draft tube of Francis turbine is considered as unstable flow conditions and the cause of many undesirable system instabilities for hydraulic power stations. The feature of vortex rope, pressure fluctuations and runner outlet velocity at part-load conditions are investigated experimentally and numerically in this paper. An obvious vortex rope could be observed at 0.88Q11BEP point and produces the most intensive periodic pressure fluctuation at the vortex rotation frequency fv along the whole passage of Francis turbine. At other conditions away from this point, the vortex rope becomes unobvious or even invisible, and the associated pressure fluctuation amplitude also reduces. This trend could be well represented by the new parameters G and Vs, which could be used to determine the strength of helical vortex field based on the gradient of time-averaged velocity on horizontal section of the draft tube cone. Transient simulations using VLES turbulence model could obtain satisfying results by comparing with experimental data, especially in terms of the time-averaged axial velocity profile resolution. Therefore, this new method enables the designers to evaluate vortex strength in engineering applications.

Noncompaction cardiomyopathy is a common congenital cardiac disorder associated with abnormal ventricular cardiomyocyte trabeculation and impaired pump function. The genetic basis and underlying mechanisms of this disorder remain elusive. We show that the genetic deletion of RNA-binding protein with multiple splicing (Rbpms), an uncharacterized RNA-binding factor, causes perinatal lethality in mice due to congenital cardiovascular defects. The loss of Rbpms causes premature onset of cardiomyocyte binucleation and cell cycle arrest during development. Human iPSC-derived cardiomyocytes with RBPMS gene deletion have a similar blockade to cytokinesis. Sequencing analysis revealed that RBPMS plays a role in RNA splicing and influences RNAs involved in cytoskeletal signaling pathways. We found that RBPMS mediates the isoform switching of the heart-enriched LIM domain protein Pdlim5. The loss of Rbpms leads to an abnormal accumulation of Pdlim5-short isoforms, disrupting cardiomyocyte cytokinesis. Our findings connect premature cardiomyocyte binucleation to noncompaction cardiomyopathy and highlight the role of RBPMS in this process.

Rationale: Cilp1 (cartilage intermediate layer protein 1) is a secreted extracellular matrix protein normally associated with bone and cartilage development. Its function and mechanism of action in adult heart disease remain elusive. Objective: To establish the function and mechanism of action of Cilp1 in postmyocardial infarction (MI) cardiac remodeling. Methods and Results: We investigated the expression of Cilp1 in mouse models of pathological cardiac remodeling and human heart failure patients. Cilp1 was expressed predominantly in cardiac fibroblasts and upregulated in response to cardiac injury and in the heart and blood of patients with heart failure. We generated Cilp1 knock out (KO) and transgenic (Tg) mice with NCilp1 (N-terminal half of the protein) overexpressed in myofibroblasts. Cilp1 KO mice had better cardiac function, reduced number of immune cells and myofibroblasts, and enhanced microvascular survival after MI compared with wild-type littermates. Conversely, NCilp1-transgenic mice had augmented loss of cardiac function, increased number of myofibroblasts and infarct size after the MI injury. RNA-seq and gene ontology analysis indicated that cell proliferation and mTORC1 (mammalian Target of Rapamycin Complex 1) signaling were downregulated in KO hearts compared with wild-type hearts. In vivo BrdU labeling and immunofluorescence staining showed that myofibroblast proliferation in the Cilp1 KO heart was downregulated. Biaxial mechanical testing and extracellular matrix gene expression analysis indicated that while MI caused significant stiffness in wild-type hearts it had little effect on KO hearts. Upregulation of collagen expression after MI injury was attenuated in KO hearts. Recombinant CILP1 protein or NCilp1-conditioned medium promoted proliferation of neonatal rat ventricular cardiac fibroblasts via the mTORC1 signaling pathway. Conclusions: Our studies established a pathological role of Cilp1 in promoting post-MI remodeling, identified a novel function of Cilp1 in promoting myofibroblast proliferation, and suggested that Cilp1 may serve as a potential biomarker for pathological cardiac remodeling and target for fibrotic heart disease.

To identify non-cell-autonomous effectors of cardiomyocyte mitosis, we analyzed a transcriptomic screen of regenerating and non-regenerating neonatal hearts for differentially-expressed secreted proteins – which we hypothesized could include candidate mitogens. We identified and validated IGFBP3, which has a Janus-like stabilizing and sequestering effect on IGF growth factors, as a neonatal injury-associated secreted protein. IGFBP3 is expressed by and secreted from vascular cells in the neonatal heart after cardiac injury, notably in the infarct border zone. We found that global deletion of IGFBP3 blunted neonatal regeneration, while gain-of-function experiments using recombinant IGFBP3 and a transgenic mouse model uncovered a pro-mitotic effect of IGFBP3 on cardiomyocytes in vitro and in the adult heart. We show that site-specific expression of an IGFBP3 protease (PAPP-A2) and its inhibitor (STC2) coordinate the spatial release of IGF2 in the infarct zone to regio-selectively activate the INSR-ERK-AKT cell growth pathways in cardiomyocytes. Collectively, our work highlights the spatiotemporal orchestration of endothelial-cardiomyocyte interactions that are required for neonatal cardiac regeneration.

Recent studies have identified a unique cancer-associated fibroblast (CAF) population termed antigen-presenting CAFs (apCAFs), characterized by the expression of major histocompatibility complex class II molecules, suggesting a function in regulating tumor immunity. Here, by integrating multiple single-cell RNA-sequencing studies and performing robust lineage-tracing assays, we find that apCAFs are derived from mesothelial cells. During pancreatic cancer progression, mesothelial cells form apCAFs by downregulating mesothelial features and gaining fibroblastic features, a process induced by interleukin-1 and transforming growth factor β. apCAFs directly ligate and induce naive CD4+ T cells into regulatory T cells (Tregs) in an antigen-specific manner. Moreover, treatment with an antibody targeting the mesothelial cell marker mesothelin can effectively inhibit mesothelial cell to apCAF transition and Treg formation induced by apCAFs. Taken together, our study elucidates how mesothelial cells may contribute to immune evasion in pancreatic cancer and provides insight on strategies to enhance cancer immune therapy. Citation Format: Huocong Huang, Zhaoning Wang, Yuqing Zhang, Rachana N. Pradhan, Debolina Ganguly, Raghav Chandra, Gilbert Murimwa, Steven Wright, Xiaowu Gu, Ravikanth Maddipati, Sören Müller, Shannon J. Turley, Rolf A. Brekken. Mesothelial cell-derived antigen-presenting cancer-associated fibroblasts induce expansion of regulatory T cells in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C078.

Rationale: Absence of dystrophin in Duchenne muscular dystrophy (DMD) results in the degeneration of skeletal and cardiac muscles. Owing to advances in respiratory management of patients with DMD, cardiomyopathy has become a significant aspect of the disease. While CRISPR/Cas9 genome editing technology holds great potential as a novel therapeutic avenue for DMD, little is known about the potential of DMD correction using CRISPR/Cas9 technology to mitigate cardiac abnormalities in DMD. Objective: To define the effects of CRISPR/Cas9 genome editing on structural, functional, and transcriptional abnormalities in DMD-associated cardiac disease. Methods and Results: We generated induced pluripotent stem cells from a patient with a deletion of exon 44 of the DMD gene (ΔEx44) and his healthy brother. We targeted exon 45 of the DMD gene by CRISPR/Cas9 genome editing to generate corrected DMD induced pluripotent stem cell lines, wherein the DMD open reading frame was restored via reframing or exon skipping. While DMD cardiomyocytes demonstrated morphological, structural, and functional deficits compared with control cardiomyocytes, cardiomyocytes from both corrected DMD lines were similar to control cardiomyocytes. Bulk RNA-sequencing of DMD cardiomyocytes showed transcriptional dysregulation consistent with dilated cardiomyopathy, which was mitigated in corrected DMD cardiomyocytes. We then corrected dysfunctional DMD cardiomyocytes by adenoviral delivery of Cas9/gRNA and showed that correction of DMD cardiomyocytes postdifferentiation reduces their arrhythmogenic potential. Single-nucleus RNA-sequencing of hearts of DMD mice showed transcriptional dysregulation in cardiomyocytes and fibroblasts, which in corrected mice was reduced to similar levels as wild-type mice. Conclusions: We show that CRISPR/Cas9-mediated correction of DMD ΔEx44 mitigates structural, functional, and transcriptional abnormalities consistent with dilated cardiomyopathy irrespective of how the protein reading frame is restored. We show that these effects extend to postnatal editing in induced pluripotent stem cell-derived cardiomyocytes and mice. These findings provide key insights into the utility of genome editing as a novel therapeutic for DMD-associated cardiomyopathy.

The runner blade channel vortex could induce cavitation erosion, noise and vibration, when Francis turbines operate at part load condition. It's usually difficult to simulate the channel vortex exactly in runner passage, this paper attempts to predict the runner channel vortex in some operating conditions with different discharge and speed factors.The results show that the structure and intensity of runner channel vortex are various between the vortex incipient and development line. The velocity distribution indicates that there is a relationship between the channel vortex and backflow region near the hub with different specific discharge factors, while the flow incident angle has a distinct variation with different specific speed factors. Furthermore, this work enables the researchers to understand the flow field and carry out optimization design in runner hydraulic research.