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Rationale: IgA can induce activation of neutrophils which are the most abundant cell type in blood, but the development of IgA as therapeutic has been confounded by its short half-life and a weak ability to recruit NK cells as effector cells. Therefore, we generated an X-shaped antibody (X-body) based on the principle of molecular self-assembly that combines the activities of both IgG and IgA, which can effectively recruit and activate NK cells, macrophages, and neutrophils to kill tumor cells. Methods: X-body was generated by using a self-assembly strategy. The affinity of the X-body with the antigen and Fc receptors was tested by surface plasmon resonance. The shape of X-body was examined using negative staining transmission electron microscopy. The tumor cell killing activity of X-body was assessed in vitro and in multiple syngeneic mouse models. To explore the mechanism of X-body, tumor-infiltrating immune cells were analyzed by single-cell RNA-seq and flow cytometry. The dependence of neutrophil, macrophage, and NK cells for the X-body efficacy was confirmed by in vivo depletion of immune cell subsets. Results: The X-body versions of rituximab and trastuzumab combined the full spectrum activity of IgG and IgA and recruited NK cells, macrophages, and neutrophils as effector cells for eradication of tumor cells. Treatment with anti-hCD20 and anti-hHER2 X-bodies leads to a greater reduction in tumor burden in tumor-bearing mice compared with the IgA or IgG counterpart, and no obvious adverse effect is observed upon X-body treatment. Moreover, the X-body has a serum half-life and drug stability comparable to IgG. Conclusions: The X-body, as a myeloid-cell-centered therapeutic strategy, holds promise for the development of more effective cancer-targeting therapies than the current state of the art., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Pollen tube guidance regulates the growth direction and ovule targeting of pollen tubes in pistils, which is crucial for the completion of sexual reproduction in flowering plants. The Arabidopsis (Arabidopsis thaliana) pollen-specific receptor kinase (PRK) family members PRK3 and PRK6 are specifically tip-localized and essential for pollen tube growth and guidance. However, the mechanisms controlling the polar localization of PRKs at the pollen tube tip are unclear. The Arabidopsis P4-ATPase ALA3 helps establish the polar localization of apical phosphatidylserine (PS) in pollen tubes. Here, we discovered that loss of ALA3 function caused pollen tube defects in growth and ovule targeting and significantly affected the polar localization pattern of PRK3 and PRK6. Both PRK3 and PRK6 contain two polybasic clusters in the intracellular juxtamembrane domain, and they bound to PS in vitro. PRK3 and PRK6 with polybasic cluster mutations showed reduced or abolished binding to PS and altered polar localization patterns, and they failed to effectively complement the pollen tube-related phenotypes of prk mutants. These results suggest that ALA3 influences the precise localization of PRK3, PRK6, and other PRKs by regulating the distribution of PS, which plays a key role in regulating pollen tube growth and guidance., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Chronic inflammation is a key pathological process in atherosclerosis. RNA binding proteins (RBPs) have been reported to play an important role in atherosclerotic plaque formation, and they could regulate the expression of inflammatory factors by phosphorylation modification. Y-box binding protein 1 (YB1) is an RBP that has participated in many inflammatory diseases. Here, we found an increased expression of phosphorylated YB1 (pYB1) in atherosclerotic plaques and demonstrated that YB1 dephosphorylation reduced lipid accumulation and lesion area in the aorta in vivo . Additionally, we found that inflammatory cytokines were downregulated in the presence of YB1 dephosphorylation, particularly CCL2, which participates in the pathogenesis of atherosclerosis. Furthermore, we demonstrated that CCL2 mRNA rapid degradation was mediated by the glucocorticoid receptor-mediated mRNA decay (GMD) process during YB1 dephosphorylation, which resulted in the downregulation of CCL2 expression. In conclusion, YB1 phosphorylation affects the development of atherosclerosis through modulating inflammation, and targeting YB1 phosphorylation could be a potential strategy for the treatment of atherosclerosis by anti-inflammation., Competing Interests: Author BL was employed by the company Jilin Zhongtai Biotechnology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tang, Li, Yang, Yang, Geng, Liu, Zhang, Liu, Xue, Zhang, Wang and Zhao.)

Type 2 diabetes mellitus (T2DM) is associated with a high prevalence of diastolic dysfunction and congestive heart failure. A potential contributing factor is the accelerated accumulation of diffuse myocardial fibrosis and stiffness. Novel cardiac magnetic resonance (CMR) imaging techniques can identify both myocardial fibrosis and contractility quantitatively. This study aimed to investigate the dynamic characteristics of the myocardial strain and altered extracellular volume (ECV) fraction as determined by 7.0 T CMR in T2DM mice. C57Bl/6J mice were randomly divided into T2DM (fed a high-fat diet) and control (fed a normal diet) groups. They were scanned on 7.0 T MRI every 4 weeks until the end of week 24. The CMR protocol included multi-slice cine imaging to assess left ventricle strain and strain rate, and pre- and post-contrast T1 mapping images to quantify ECV. The ECV in the T2DM mice was significantly higher (p < 0.05) than that in the control group since week 12 with significantly impaired myocardial strain (p < 0.05). A significant linear correlation was established between myocardial strain and ECV (p < 0.001) and left ventricular-ejection fraction and ECV (p = 0.003). The results suggested that CMR feature tracking-derived myocardial strain analysis can assess functional abnormalities that may be associated with ECM alterations in diabetic cardiomyopathy, contributing to the study of diabetic therapy effects.

Urban agglomerations are an important symbol in the development of modernization. In this paper, we utilize the National Urban Agglomeration (NUA) policy as a quasi-natural experiment in the Chinese context. Adopting data from 280 cities from 2005 to 2019 as research samples, we use difference-in-differences (DID) and spatial difference-in-differences models (SDID) to examine the effect and mechanism of the implementation of the NUA policy on economic development and environmental pollution in China. The result shows that the NUA policy can achieve urban economic and environmental gains, which still holds after the robustness test. The heterogeneity analysis showed that the effects of the NUA policy are more evident in large and medium-sized cities. The curbing effect of the NUA policy on pollution emissions is apparent in the eastern region. Considering spatial heterogeneity, the expected economic and environmental benefits of the NUA policy are partially borne out. In addition to the green technology innovation, the NUA policy also influences regional economic development and environmental pollution through industrial agglomeration and the upgrading of industrial structures, respectively. It is essential to strengthen regional cooperation and establish the development concept of community interests between cities.

Objectives: To assess volumetric DCE-MRI radiomics nomogram in predicting response to neoadjuvant chemotherapy (nCT) in EC patients., Methods: This retrospective analysis of a prospective study enrolled EC patients with stage cT1N + M0 or cT2-4aN0-3M0 who received DCE-MRI within 7 days before chemotherapy, followed by surgery. Response assessment was graded from 1 to 5 according to the tumor regression grade (TRG). Patients were stratified into responders (TRG1 + 2) and non-responders (TRG3 + 4 + 5). 72 radiomics features and vascular permeability parameters were extracted from DCE-MRI. The discriminating performance was assessed with ROC. Decision curve analysis (DCA) was used for comparing three different models., Results: This cohort included 82 patients, and 72 tumor radiomics features and vascular permeability parameters acquired from DCE-MRI. mRMR and LASSO were performed to choose the optimized subset of radiomics features, and 3 features were selected to create the radiomics signature that were significantly associated with response (P < 0.001). AUC of combining radiomics signature and DCE-MRI performance in the training (n = 41) and validation (n = 41) cohort was 0.84 (95% CI 0.57-1) and 0.86 (95% CI 0.74-0.97), respectively. This combined model showed the best discrimination between responders and non-responders, and showed the highest positive and positive predictive value in both training set and test set., Conclusions: The radiomics features are useful for nCT response prediction in EC patients., (© 2022. The Author(s).)

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant cancers worldwide. Despite the promising outcome of immune checkpoint inhibitors and agonist antibody therapies in different malignancies, PDAC exhibits high resistance due to its immunosuppressive tumor microenvironment (TME). Ameliorating the TME is thus a rational strategy for PDAC therapy. The intratumoral application of oncolytic herpes simplex virus-1 (oHSV) upregulates pro-inflammatory macrophages and lymphocytes in TME, and enhances the responsiveness of PDAC to immunotherapy. However, the antitumor activity of oHSV remains to be maximized. The aim of this study is to investigate the effect of the CD40L armed oHSV on the tumor immune microenvironment, and ultimately prolong the survival of the PDAC mouse model., Methods: The membrane-bound form of murine CD40L was engineered into oHSV by CRISPR/Cas9-based gene editing. oHSV-CD40L induced cytopathic effect and immunogenic cell death were determined by microscopy and flow cytometry. The expression and function of oHSV-CD40L was assessed by reporter cell assay. The oHSV-CD40L was administrated intratumorally to the immune competent syngeneic PDAC mouse model, and the leukocytes in TME and tumor-draining lymph node were analyzed by multicolor flow cytometry. Intratumoral cytokines were determined by ELISA., Results: Intratumoral application of oHSV-CD40L efficiently restrained the tumor growth and prolonged the survival of the PDAC mouse model. In TME, oHSV-CD40L-treated tumor accommodated more maturated dendritic cells (DCs), which in turn activated T helper 1 and cytotoxic CD8 + T cells in an interferon-γ-dependent and interleukin-12-dependent manner. In contrast, the regulatory T cells were significantly reduced in TME by oHSV-CD40L treatment. Repeated dosing and combinational therapy extended the lifespan of PDAC mice., Conclusion: CD40L-armed oncolytic therapy endues TME with increased DCs maturation and DC-dependent activation of cytotoxic T cells, and significantly prolongs the survival of the model mice. This study may lead to the understanding and development of oHSV-CD40L as a therapy for PDAC in synergy with immune checkpoint blockade., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

How the avian sex chromosomes first evolved from autosomes remains elusive as 100 million years (My) of divergence and degeneration obscure their evolutionary history. The Sylvioidea group of songbirds is interesting for understanding avian sex chromosome evolution because a chromosome fusion event ∼24 Ma formed "neo-sex chromosomes" consisting of an added (new) and an ancestral (old) part. Here, we report the complete female genome (ZW) of one Sylvioidea species, the great reed warbler (Acrocephalus arundinaceus). Our long-read assembly shows that the added region has been translocated to both Z and W, and whereas the added-Z has retained its gene order the added-W part has been heavily rearranged. Phylogenetic analyses show that recombination between the homologous added-Z and -W regions continued after the fusion event, and that recombination suppression across this region took several million years to be completed. Moreover, recombination suppression was initiated across multiple positions over the added-Z, which is not consistent with a simple linear progression starting from the fusion point. As expected following recombination suppression, the added-W show signs of degeneration including repeat accumulation and gene loss. Finally, we present evidence for nonrandom maintenance of slowly evolving and dosage-sensitive genes on both ancestral- and added-W, a process causing correlated evolution among orthologous genes across broad taxonomic groups, regardless of sex linkage., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

LangChuangHeJi (LCHJ) decoction has been used as a supplementary therapy to reduce the dose of prednisone and improve the therapeutic effects in systemic lupus erythematosus (SLE) maintenance. We aimed to explore the underlying mechanisms of the therapeutic effects of LCHJ. Spleen and lymph node weight, renal tissue histology, anti-dsDNA and anti-nuclear antibody levels in serum, and urinary protein levels were measured to evaluate the therapeutic effects. We further measured serum levels of multiple cytokines and antibody subsets, and performed flow cytometry analysis to observe effects of LCHJ on the frequency and activation of T cells and T cell subsets, as well as accumulation of plasma cells in splenocytes of MRL/lpr mice. LCHJ exhibited significant therapeutic effects on MRL/lpr mice. LCHJ significantly controlled the in vivo inflammation and dramatically prevented the accumulation of DN T and plasma cells in MRL/lpr mice. Moreover, LCHJ significantly suppressed the accumulation of CD138+ T cells in MRL/lpr mice, which led to the decreased production of the anti-dsDNA antibody in vivo. LCHJ significantly decreased CD4+CD138+ T cells originated from CD4+CD138- T cells, which subsequently prevented the accumulation of CD138+ T cells in MRL/lpr mice. Our results indicated that LCHJ alleviated renal injuries and prevented the enlargement of the spleen and lymph nodes by suppressing DN T cell accumulation, and reduced anti-dsDNA antibody secretion by preventing the accumulation of CD138+ T cells., Competing Interests: None., (AJTR Copyright © 2021.)

Development of CAR-T therapy led to immediate success in the treatment of B cell leukemia. Manufacturing of therapy-competent functional CAR-T cells needs robust protocols for ex vivo/in vitro expansion of modified T-cells. This step is challenging, especially if non-viral low-efficiency delivery protocols are used to generate CAR-T cells. Modern protocols for CAR-T cell expansion are imperfect since non-specific stimulation results in rapid outgrowth of CAR-negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. To develop a specific and improved protocol for CAR-T cell expansion, cell-derived membrane vesicles are taken advantage of, and the simple structural demands of the CAR-antigen interaction. This novel approach is to make antigenic microcytospheres from common cell lines stably expressing surface-bound CAR antigens, and then use them for stimulation and expansion of CAR-T cells. The data presented in this article clearly demonstrate that this protocol produced antigen-specific vesicles with the capacity to induce stronger stimulation, proliferation, and functional activity of CAR-T cells than is possible with existing protocols. It is predicted that this new methodology will significantly advance the ability to obtain improved populations of functional CAR-T cells for therapy., (© 2021 The Authors. Small published by Wiley-VCH GmbH.)

Objective: To explore the feasibility of the whole tumor histogram analysis parameters derived from dynamic contrast-enhanced MRI (DCE-MRI) based on stack-of stars (StarVIBE) to predict T and N staging of resectable gastric cancer (GC)., Methods: Eighty-seven patients confirmed as GC by histopathology were enrolled in this prospective study. DCE-MRI were performed before surgery, and quantitative DCE parameters (K trans , K ep , V e ) and histogram metrics (Skewness, Kurtosis and Entropy) were measured by Omni-Kinetics software. Intraclass correlation coefficient (ICC) testing was used to determine the consistency of K trans , K ep and V e values and histogram metrics values between two radiologists using Bland-Altman analysis. The quantitative DCE parameters or histogram metrics values between T stage or N stage were compared using ANOVA or Kruskal-Wallis testing. Receiver operating characteristic (ROC) analyses was performed to find out the best parameters for identifying T and N staging., Results: There was statistical difference in K trans , K ep , V e and entropy to identify T staging (P=0.015, 0.033, <0.001, and 0.007, respectively), and in pairwise comparisons of V e values showed statistically difference between T1+2 and T3 group (P<0.001), T1+2 and T4 group (P<0.001). There were statistical differences in V e to identify N staging (P=0.041). In ROC analysis, V e was the best parameter for identifying T staging (AUC: 0.788, the sensitivity and specificity was 0.929 and 0.578, respectively) and N staging (AUC: 0.590, the sensitivity and specificity was 0.714 and 0.899, respectively)., Conclusion: The whole tumor histogram analysis parameters derived from StarVIBE DCE-MRI may be able to quantitatively evaluate T and N staging of GC, so as to help clinical treatment decision optimization., Competing Interests: The authors report no conflicts of interest in this work., (© 2021 Yan et al.)

Maize is an important crop worldwide, as well as a valuable model with vast genetic diversity. Accurate genome and annotation information for a wide range of inbred lines would provide valuable resources for crop improvement and pan-genome characterization. In this study, we generated a high-quality de novo genome assembly (contig N50 of 15.43 Mb) of the Chinese elite inbred line RP125 using Nanopore long-read sequencing and Hi-C scaffolding, which yield highly contiguous, chromosome-length scaffolds. Global comparison of the RP125 genome with those of B73, W22, and Mo17 revealed a large number of structural variations. To create new germplasm for maize research and crop improvement, we carried out an EMS mutagenesis screen on RP125. In total, we obtained 5818 independent M2 families, with 946 mutants showing heritable phenotypes. Taking advantage of the high-quality RP125 genome, we successfully cloned 10 mutants from the EMS library, including the novel kernel mutant qk1 (quekou: "missing a small part" in Chinese), which exhibited partial loss of endosperm and a starch accumulation defect. QK1 encodes a predicted metal tolerance protein, which is specifically required for Fe transport. Increased accumulation of Fe and reactive oxygen species as well as ferroptosis-like cell death were detected in qk1 endosperm. Our study provides the community with a high-quality genome sequence and a large collection of mutant germplasm., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Viral myocarditis (VMC) is a cardiac disease associated with myocardial inflammation and injury induced by virus infection. Cardiomyocytes have recently been regarded as key players in eliciting and modulating inflammation within the myocardium. Kruppel-like factor 10 (KLF10) is a crucial regulator of various pathological processes and plays different roles in a variety of diseases. However, its role in VMC induced by coxsackievirus B3 (CVB3) infection remains unknown. In this study, we report that cardiac KLF10 confers enhanced protection against viral myocarditis. We found that KLF10 expression was downregulated upon CVB3 infection. KLF10 deficiency enhanced cardiac viral replication and aggravated VMC progress. Bone marrow chimera experiments indicated that KLF10 expression in nonhematopoietic cells was involved in the pathogenesis of VMC. We further identified MCP-1 as a novel target of KLF10 in cardiomyocytes, and KLF10 cooperated with histone deacetylase 1 (HDAC1) to negatively regulate MCP-1 expression by binding its promoter, leading to activation of MCP-1 transcription and recruitment of Ly6C high monocytes/macrophages into the myocardium. This novel mechanism of MCP-1 regulation by KLF10 might provide new insights into the pathogenesis of VMC and a potential therapeutic target for VMC., (© 2020. CSI and USTC.)

Currently, high-throughput approaches are lacking in the isolation of antibodies with functional readouts beyond simple binding. This situation has impeded the next generation of cancer immunotherapeutics, such as bispecific T cell engager (BiTE) antibodies or agonist antibodies against costimulatory receptors, from reaching their full potential. Here, we developed a highly efficient droplet-based microfluidic platform combining a lentivirus transduction system that enables functional screening of millions of antibodies to identify potential hits with desired functionalities. To showcase the capacity of this system, functional antibodies for CD40 agonism with low frequency (<0.02%) were identified with two rounds of screening. Furthermore, the versatility of the system was demonstrated by combining an anti-Her2 × anti-CD3 BiTE antibody library with functional screening, which enabled efficient identification of active anti-Her2 × anti-CD3 BiTE antibodies. The platform could revolutionize next-generation cancer immunotherapy drug development and advance medical research., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

COVID-19 pandemic caused by SARS-CoV-2 constitutes a global public health crisis with enormous economic consequences. Monoclonal antibodies against SARS-CoV-2 can provide an important treatment option to fight COVID-19, especially for the most vulnerable populations. In this work, potent antibodies binding to SARS-CoV-2 Spike protein were identified from COVID-19 convalescent patients. Among them, P4A1 interacts directly with and covers majority of the Receptor Binding Motif of the Spike Receptor-Binding Domain, shown by high-resolution complex structure analysis. We further demonstrate the binding and neutralizing activities of P4A1 against wild type and mutant Spike proteins or pseudoviruses. P4A1 was subsequently engineered to reduce the potential risk for Antibody-Dependent Enhancement of infection and to extend its half-life. The engineered antibody exhibits an optimized pharmacokinetic and safety profile, and it results in complete viral clearance in a rhesus monkey model of COVID-19 following a single injection. These data suggest its potential against SARS-CoV-2 related diseases.

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Y.G., Z.R., L.L., J.W., Z.L., D.F., X.W. and C.N. are inventors in a pending patent application filed on the reported antibodies. L.L., J.W., Z.L., X.W., C.N., C.D., L.Z., G.G. and N.S. are employees of Harbour Biomed (Suzhou) Co. Ltd. Other authors declare no competing interests. All reagents and information presented in this study are available from corresponding authors upon reasonable request.

Pancreatic ductal adenocarcinoma (PDAC) is the major type of pancreatic malignancy with very poor prognosis. Despite the promising results of immune checkpoint inhibitors (ICIs) in some solid tumors, immunotherapy is less effective for PDAC due to its immunosuppressive tumor microenvironment (TME). In this report, we established an immunocompetent syngeneic PDAC model and investigated the effect of oncolytic herpes simplex virus-1 (oHSV) on the composition of TME immune cells. The oHSV treatment significantly reduced tumor burden and prolonged the survival of tumor-bearing mice. Further, by single cell RNA sequencing (scRNA-seq) and multicolor fluorescence-activated cell sorting (FACS) analysis, we demonstrated that oHSV administration downregulated tumor-associated macrophages (TAMs), especially the anti-inflammatory macrophages, and increased the percentage of tumor-infiltrating lymphocytes, including activated cytotoxic CD8 + T cells and T helper (Th)1 cells. Besides, the combination of oHSV and immune checkpoint modulators extended the lifespan of the tumor-bearing mice. Overall, our data suggested that oHSV reshapes the TME of PDAC by boosting the immune activity and leads to improved responsiveness of PDAC to immunotherapy., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Dual-energy CT (DECT) permits the simultaneous operation of two different kV levels, providing a potential method toward the assessment of diffuse myocardial fibrosis. The purpose of this study was to determine the accuracy of DECT for evaluation of the myocardial extracellular volume (ECV) fraction in comparison with single-energy CT (SECT)., Methods: Myocardial ECV was quantified in fifteen dogs using DECT and dynamic equilibrium SECT before and after doxorubicin administration. Cardiac magnetic resonance imaging (CMRI) was used to assess myocardial function. The histological collagen volume fraction (CVF) was calculated as the gold standard. The Bland-Altman analysis was performed to compare the agreement between DECT-ECV and SECT-ECV. The association among ECV values derived from DECT and SECT, CVF, and left ventricular ejection fraction (LVEF) were determined by correlation analysis. The variations of these values were evaluated using repeated ANOVA., Results: The DECT- and SECT-ECV were increased with the elongation of modeling time (pre-modeling vs . 16-week models vs . 24-week models: DECT-ECV 24.1%±1.1%, 35.1%±1.3% and 37.6%±1.4%; SECT-ECV 22.9%±0.8%, 33.6%±1.2% and 36.3%±1.0%; n=30 in per-subject analysis, all P<0.05). Both ECV values of DECT and SECT correlated well with the histological CVF results (R=0.935 and 0.952 for the DECT-ECV and SECT-ECV; all P<0.001; n=13). Bland-Altman plots showed no significant differences between DECT- and SECT-ECV., Conclusions: DECT-ECV correlated well with both SECT-ECV and histology, showing the feasibility of DECT in evaluating doxorubicin-induced diffuse myocardial interstitial fibrosis., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (Available at http://dx.doi.org/10.21037/cdt-20-798). SW disclosed the personal employment relationship with GE Healthcare Chin. The other authors have no conflicts of interest to declare., (2021 Cardiovascular Diagnosis and Therapy. All rights reserved.)

Follistatin-like 1 (FSTL1) is a matricellular protein that is upregulated during development and disease, including idiopathic pulmonary fibrosis (IPF), keloid, and arthritis. The profibrotic and pro-inflammatory roles of FSTL1 have been intensively studied during the last several years, as well as in this report. We screened and identified epitope-specific monoclonal neutralizing antibodies (nAbs) to functionally block FSTL1. FSTL1 nAbs attenuated bleomycin-induced pulmonary and dermal fibrosis in vivo and transforming growth factor (TGF)-β1-induced dermal fibrosis ex vivo in human skin. In addition, FSTL1 nAbs significantly reduced existing lung fibrosis and skin fibrosis in experimental models. FSTL1 nAbs exerted their potent antifibrotic effects via reduced TGF-β1 responsiveness and subsequent myofibroblast activation and extracellular matrix production. We also observed that FSTL1 nAbs attenuated the severity of collagen-induced arthritis in mice, which was accompanied by reduced inflammatory responses in vitro. Our findings suggest that FSTL1 nAbs are a promising new therapeutic strategy for the treatment of multiple organ fibrosis and systemic autoimmune diseases., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Rationale: Fc engineering has become the focus of antibody drug development. The current mutagenesis and in silico protein design methods are confined by the limited throughput and high cost, while the high-throughput phage display and yeast display technologies are not suitable for screening glycosylated Fc variants. Here we developed a mammalian cell display-based Fc engineering platform. Methods: By using mammalian cell display and next generation sequencing, we screened millions of Fc variants for optimized affinity and specificity for FcγRIIIa or FcγRIIb. The identified Fc variants with improved binding to FcγRIIIa were substituted into trastuzumab and rituximab and the effector function of antibodies were examined in the PBMC-based assay. On the other hand, the identified Fc variants with selectively enhanced FcγRIIb binding were applied to CD40 agonist antibody and the activities of the antibodies were measured on different cell assays. The immunostimulatory activity of CD40 antibodies was also evaluated by OVA-specific CD8 + T cell response model in FcγR/CD40-humanized mice. Results: Using this approach, we screened millions of Fc variant and successfully identified several novel Fc variants with enhanced FcγRIIIa or FcγRIIb binding. These identified Fc variants displayed a dramatic increase in antibody-dependent cellular cytotoxicity in PBMC-based assay. Novel variants with selectively enhanced FcγRIIb binding were also identified. CD40 agonist antibodies substituted with these Fc variants displayed activity more potent than the parental antibody in the in vitro and in vivo models . Conclusions: This approach increased the throughput of Fc variant screening from thousands to millions magnitude, enabled screening variants containing multiple mutations and could be integrated with glycoengineering technology, represents an ideal platform for Fc engineering. The initial efforts demonstrated the capability of the platform and the novel Fc variants could be substituted into nearly any antibody for the next generation of antibody therapeutics., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Tamoxifen (TAMR) resistance remains a massive obstacle for breast cancer (BC) management. The precise parts of long non-coding RNA ataxin 8 opposite strand (ATXN8OS) in BC TAMR resistance have not been defined., Methods: The levels of ATXN8OS, vasodilator-stimulated phosphoprotein (VASP), and miR-16-5p were assessed by quantitative real-time polymerase chain reaction or western blot. Colony formation and cell viability were analyzed by MTT and colony formation assays, respectively. Targeted interactions among miR-16-5p, ATXN8OS, and VASP were confirmed by dual-luciferase reporter assay. Animal studies were performed to observe the role of ATXN8OS in TAMR sensitivity in vivo ., Results: ATXN8OS expression was increased in BC tissues and cells. ATXN8OS depletion promoted BC cell sensitivity to TAMR. ATXN8OS sequestered miR-16-5p by directly binding to miR-16-5p. The promotional effect of ATXN8OS knockdown on BC cell TAMR sensitivity was mediated by miR-16-5p. VASP was a direct target of miR-16-5p, and miR-16-5p overexpression enhanced TAMR sensitivity by VASP. Moreover, ATXN8OS regulated VASP expression by acting as a miR-16-5p sponge. In addition, ATXN8OS knockdown augmented BC TAMR sensitivity in vivo ., Conclusion: ATXN8OS knockdown enhanced BC TAMR sensitivity partially through the miR-16-5p/VASP axis, highlighting a potential therapeutic target for improving the clinical benefits of TAMR treatment in BC patients., Competing Interests: Competing interests: The authors declare that they have no competing interests., (© 2021 Hongkai Zhang et al., published by De Gruyter.)

Background: Patients with esophageal cancer (EC) undergo endoscopic ultrasound and CT based cancer staging. Recent technical developments allow improved MRI quality with diminished motion artifact that may allow MRI to compare favorable to CT for noninvasive staging. Hence the purpose of the study was to assess image quality and diagnostic accuracy of 3 T MRI versus CT and EUS for preoperative T-staging of potentially resectable esophageal cancer., Methods: Between October-2014 and December-2017, esophageal cancer patients with T-staging by EUS were enrolled in this prospective study. Post-operative histopathologic T-staging was the reference standard. All participants underwent MRI [T2- multi-shot turbo spin echo sequence (msTSE), diffusion-weighted imaging (DWI), and 3D gradient-echo based sequence (3D-GRE)] and CT [non-contrast and multiphase contrast-enhanced CT scanning] 5.6 + 3.6 days after endoscopy. Surgery was performed within 3.6 + 3.5 days after imaging. Two blinded endoscopists (reader 1 and 2) and radiologists (reader 3 and 4) independently evaluated EUS and CT/MRI, respectively. Considering the clinical relevance, patients were dichotomized into early (T1 and T2) vs late (T3 and T4) stage cancer before assessment. For statistical purpose, the binary decision was defined as the ability of the imaging technique to diagnose early stage/not early stage esophageal cancer. Diagnostic performance of EUS, MRI and CT was compared using McNemar's test with Bonferroni correction; kappa values were assessed for reader performance., Results: 74 study participants (60 ± 8 yrs.; 56 men) with esophageal cancer were evaluated, of whom 85%(63/74) had squamous cell carcinoma, 61%(45/74) were at early stage and 39%(29/74) were at late stage cancer, as determined by histopathology. Intra- and Inter-reader agreement for pre-operative vs post-operative T-staging was excellent for all imaging modalities. Compared to CT, MRI showed significantly higher accuracy for both the readers (reader3: 96% vs 82%, p = 0.0038, reader4: 95% vs 80%, p = 0.0076, for MRI vs CT, respectively). Further, MRI outperformed EUS with higher specificity (reader 1 vs 3: 59% vs 93%, p = 0.0015, reader 2 vs 4: 66% vs 93%, p = 0.0081, for EUS vs MRI respectively), and accuracy (reader 1 vs 3: 81% vs 96%, p = 0.0022, reader 2 vs 4: 85% vs 95%, p = 0.057, for EUS vs MRI, respectively)., Conclusion: For resectable esophageal cancer, MRI had better diagnostic performance for tumor staging compared to CT and EUS., Trial Registration: ChiCTR, ChiCTR-DOD, Registered 2nd October 2014, http://www.chictr.org.cn/showproj.aspx?proj=9620.

Background: We aimed to investigate the association of lesion-specific epicardial adipose tissue (EAT) volume and density with the presence of myocardial ischemia., Methods: We enrolled 45 patients (55 lesions) with known or suspected coronary artery disease who underwent coronary computed tomography angiography (CTA) followed by invasive fractional flow reserve (FFR) assessment within 30 days. EAT volume (index) and density in patient-, vessel- and lesion-level were measured on CTA images. Lesion-specific ischemia was defined as a lesion with stenosis diameter > 90% or FFR ≤0.80. Multivariate analysis determined the independent association of EAT parameters with lesion-specific ischemia., Results: Mean age of the patients was 60 years, and 75% were male. Overall, 55.6% of patients had ischemic lesions and a mean FFR baseline value of 0.82 ± 0.10. Total EAT volume index was significantly higher in patients with functionally or anatomically significant stenosis. Specifically, peri-lesion EAT volume index, not the density, was positively correlated with lesion-specific ischemia independent of luminal stenosis and plaque characteristics (hazard ratio 1.56, 95% confidence interval 1.04-2.33, P = 0.032; per 0.1 ml/m 2 increase). Moreover, peri-lesion EAT volume was negatively correlated with lesion FFR values, whereas total EAT volume was positively correlated with fat accumulation and glucose metabolism. In addition, there was no association of EAT volume or density with myocardial ischemia in vessel-level analysis., Conclusions: Lesion-specific EAT volume index, but not density, seems positively and independently associated with myocardial ischemia, while its incremental diagnostic value of lesion-specific ischemia should be further investigated.

Objective: The identification of DNA polymerase epsilon (POLE) mutation subtypes in endometrial cancer is critical for molecular classification. The mutation of the POLE gene could only be detected by sequencing until now. We propose to validate and develop the feasibility of using BaseScope, an in situ hybridization (ISH) assay, for the detection of POLE mutations in high-grade endometrioid carcinomas (EC). Methods: Among 51 paraffin-embedded samples of high-grade EC, BaseScope-ISH assays were used to detect the RNA mutation status of the POLE gene, mainly focusing on two hotspot mutations of P286R and V411L. The number of positive signals in the cytoplasm was counted, setting the positive threshold and determining the in situ hybridization results. The sensitivity and specificity of BaseScope-ISH assay were compared with that of the Sanger sequencing results. Results: Based on the BaseScope assay, there were 19 positive samples and 32 negative samples in a total of 51 samples. Of the 19 positive samples, 10 samples showed P286R site mutations in the POLE gene, while the other nine samples were V411L site mutations. Only one sample with the V411L site mutation identified by Sanger sequencing showed negative signal value. The remaining 31 cases without the P286R site mutation or V411L site mutations all showed negative signal. This analysis result showed the sensitivity was 95% and the specificity was 100% for the BaseScope assay detecting POLE mutants in high-grade EC. Conclusion: In the case of high-grade EC, combined with morphological characteristics, the BaseScope assay can effectively and specifically identify POLE mutation cases, providing a reliable foundation for the application of clinical diagnosis and molecular classification.

Rationale: Thymoma is the most common primary tumor in the anterior mediastinum. All major subtypes of thymoma can be clinically aggressive. However, type A thymoma is usually benign and rarely invasive, let alone invasive to the trachea. There are no published reports of thymoma diagnosed in a "coughed up' tissue". Here, we report an 80-year-old man who coughed up a piece of tissue which was histopathologically diagnosed as type A thymoma., Patient Concerns: An 80-year-old man who had a history of thymoma for 8 years was admitted in our hospital with his severe cough and dyspnea and his "coughed up" something. He felt much better after coughing up the piece of tissue. He saved the tissue and sent it to the pathology department., Diagnoses: The clinical diagnosis was lung cancer. But the final pathological diagnosis was type A thymoma., Interventions: The patient refused to get chemotherapy, or surgery, and only would like to get the traditional Chinese medicine., Outcomes: The patient is stable for 19 months after he coughed up some of the tumor tissue., Lessons: The type A thymoma could be slowly aggressive. If left untreated, it would also invade into the trachea. When treat a patient with history of even a benign tumor, we should always bear the rare possibility in mind to avoid misdiagnosis. For an elderly patient, a "wait and see" policy may be acceptable.

Bcl-2 family proteins control a decisive apoptotic event: mitochondrial outer membrane permeabilization (MOMP). To discover MOMP-regulating proteins, we expressed a library of intracellular single-chain variable fragments (scFvs) ("intrabodies") and selected for those rescuing cells from apoptosis induced by BimS (the short isoform of Bim). One anti-apoptotic intrabody, intrabody 5 (IB5), recognized pyruvate kinase M2 (PKM2), which is expressed in cancer cells. PKM2 deletion ablated this clonogenic rescue; thus, IB5 activated a latent cytoprotective function of PKM2. This resulted not from pyruvate kinase activity per se but rather from the formation of an active tetrameric conformation of PKM2. A stably tetrameric PKM2 mutant, K422R, promoted cell survival even in the absence of IB5, and IB5 further increased survival. Mitochondria isolated from IB5-expressing cells were relatively resistant to MOMP in vitro. In cells, IB5 expression up-regulated Mitofusin-1 (Mfn1) and increased mitochondrial length. Importantly, Mfn1 deficiency abrogated IB5's cytoprotective effect. PKM2's anti-apoptotic function could help explain its preferential expression in human cancer., Competing Interests: The authors have declared that no competing interests exist.

Objective: The purpose of this study was to explore the value of 3-T MRI for evaluating the preoperative T staging of esophageal cancer (EC) treated with neoadjuvant chemotherapy (NAC), with histopathologic confirmation., Subjects and Methods: This prospective study enrolled patients for whom endoscopic biopsy showed EC and pretreatment CT showed stage cT1N+M0 or cT2-T4aN0-N3M0. All patients received two cycles of NAC (paclitaxel and nedaplatin protocol) followed by 3-T MRI and surgical resection. Readers assigned a T category on MRI, and postoperative pathologic confirmation was considered the reference standard. Interreader agreement, the diagnostic accuracy of T staging on T2-weighted turbo spin-echo (TSE) BLADE (Siemens Healthcare), contrast-enhanced StarVIBE (Siemens Healthcare), high-resolution delayed phase StarVIBE, and the combination of the three sequences were analyzed and compared with postoperative pathologic T staging., Results: The study included 79 patients. Mean time between NAC and MRI was 23 days. Interreader agreements of T category assignment were excellent for T2-weighted TSE BLADE (κ = 0.810, p < 0.0001), contrast-enhanced StarVIBE (κ = 0.845, p < 0.0001), high-resolution delayed phase StarVIBE (κ = 0.897, p < 0.0001), and the combination of the three sequences (κ = 0.880, p < 0.0001). The highest accuracy for T0, T1, T2, and T4a lesions was on high-resolution delayed phase StarVIBE (96.2%, 92.4%, 91.1%, and 91.1% for reader 1; 94.9%, 89.9%, 91.1%, and 94.9% for reader 2), and the highest accuracy for T3 lesions was on T2-weighted TSE BLADE (92.4% and 94.9% for reader 1 and reader 2, respectively). Diagnostic accuracy of the combination of the three sequences was not improved compared with individual sequences., Conclusion: High-resolution delayed phase StarVIBE had the highest diagnostic accuracy in staging EC after NAC for all T categories except T3, for which T2-weighted TSE BLADE had the highest accuracy. Combining all three sequences did not improve diagnostic accuracy.

Background: We developed an evidence-based medical record for doctor-patient relationship building through an integrated therapy of traditional Chinese and Western medicine (DPEBMR) to assess the efficacy in patients with digestive system diseases. The instrument was categorized into a doctor section and a patient section. In this article, our main goal was to test the validity and reliability of the patient's section for DPEBMR (DPEBMR-P)., Methods: One hundred patients were recruited. Doctors and patients used a predefined format to jointly record the illness. Cronbach's α and factor analysis were used to evaluate the reliability and structure validity, respectively., Results: Cronbach's α of all 12 items from DPEBMR-P was 0.906, which demonstrated high reliability. The Kaiser-Meyer-Olkin value was 0.811 and Bartlett's spherical test value was 452.2 (P<0.05), which meant it was suitable for performing factor analysis. A total of 3 items were identified as factors, and each had high loading: items of mental status (0.584 to 0.833), items of therapeutic effect (0.518 to 0.797), and items of appetite (0.857 to 0.882)., Conclusions: The DPEBMR-P indicated substantial reliability and structure validity., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

We report the development of a novel platform to enhance the efficacy and safety of follicular lymphoma (FL) treatment. Since lymphoma is a clonal malignancy of a diversity system, every tumor has a different antibody on its cell surface. Combinatorial autocrine-based selection is used to rapidly identify specific ligands for these B cell receptors on the surface of FL tumor cells. The selected ligands are used in a chimeric antigen receptor T cell (CAR-T) format for redirection of human cytotoxic T lymphocytes. Essentially, the format is the inverse of the usual CAR-T protocol. Instead of being a guide molecule, the antibody itself is the target. Thus, these studies raise the possibility of personalized treatment of lymphomas using a private antibody binding ligand that can be obtained in a few weeks.

Background: Carcinosarcoma of the liver is a very rare tumor composed of a mixture of carcinomatous and sarcomatous elements. Less than 25 adequately documented cases have been reported, with inadequate description of imaging features. In order to improve the awareness of this rare tumor, this study aimed to analyze the clinicopathologic and imaging features of six cases of hepatic carcinosarcoma (HCS) confirmed by surgical pathologic evaluation., Methods: We retrospectively studied the clinicopathologic and imaging features of six cases of HCS (matching the World Health Organization definition) and discussed the differential diagnosis on the basis of imaging findings. The patients, including five men and one woman, were 38 to 69 years of age. Five patients underwent CT scans, one underwent MRI scans., Results: While 3 patients were positive for hepatitis-B surface antigen, 2 had cirrhosis. The largest tumor diameter ranged from 5.0 to 21.0 cm. Satellite nodules, venous thrombi, and organ invasion (gastric wall, gallbladder, and right adrenal gland) were identified. Pathologically, the carcinomatous components corresponded to hepatocellular carcinoma in three cases, cholangiocellular carcinoma in one case, and adenocarcinoma in two cases. The sarcomatous components exhibited complex features, with undifferentiated spindle cells in five cases and a leiomyosarcoma in one. All tumors showed heterogeneous density/intensity with extensive cystic change and necrosis; spot calcification was observed in one case. Capsule was not identified. While four tumors showed heterogeneous hypervascular enhancement, two showed hypovascular enhancement. All patients underwent surgical resection. The follow-up period ranged from 2 to 18 months. Four patients died from recurrence and metastasis., Conclusions: The clinical and imaging features of HCS are heterogeneous. Due to the heterogenous nature and very low morbidity of HCS, combination of careful analysis of imaging findings and clinical features might be useful for a more accurate diagnosis of HCS.

Purpose: To compare the T staging of potentially resectable esophageal cancer using free-breathing radial VIBE (r-VIBE) and breath-hold Cartesian VIBE (C-VIBE), with pathologic confirmation of the T stage., Materials and Methods: Fifty patients with endoscopically proven esophageal cancer and indeterminate T1/T2/T3 stage by CT scan were examined on a 3-T scanner. The MRI protocol included C-VIBE at 150 seconds post-IV contrast, immediately followed by a work-in-progress r-VIBE with identical spatial resolution (1.1 mm × 1.1 mm × 3.0 mm). Two independent readers assigned a T stage on MRI according to the 7th edition of UICC-AJCC TNM Classification, and postoperative pathologic confirmation was considered the gold standard. Interreader agreement was also calculated., Results: The T staging agreement between both VIBE techniques and postoperative pathologic T staging was 52% (26/50) for C-VIBE, 80% (40/50) for r-VIBE for reader 1, and 50% (25/50), 82% (41/50) for reader 2, respectively. For the esophageal cancer with invading lamina propria, muscularis mucosae, or submucosa (T1 stage), r-VIBE achieved 86% (12/14) agreement for both readers 1 and 2. For invasion of muscularis propria (T2 stage), r-VIBE achieved 83% (25/30) for both readers 1 and 2, whereas for the invasion of adventitia (T3 stage), r-VIBE could only achieve agreement in 50% (3/6) and 67% (4/6) for readers 1 and 2, respectively., Conclusion: Contrast-enhanced free-breathing r-VIBE is superior to breath-hold CVIBE in T staging of potentially resectable esophageal cancer, especially for T1 and T2., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To evaluate lesion enhancement performance of Multi-Arterial CAIPIRINHA-Dixon-TWIST-Volume-Interpolated Breath-Hold Examination (MA-CDT-VIBE) for the detection of hepatic metastases., Materials and Methods: Thirty-one patients with suspicious hepatic metastases were enrolled in this retrospective study. Two independent radiologists scored visualization of each lesion on a scale of 1 (poor visualization) to 11 (excellent visualization) on 11 sets of images. These included 6 hepatic arterial sub-phases acquired in one breath-hold, 1 series of the mean of 6 hepatic arterial sub-phases, 3 subtracted arterial sub-phases, and 1 portal venous phase. The phases with good (score 8-10) and excellent (score 11) lesion visualization were identified, and the number of lesions seen on each of these phases was compared to the number of lesions that was seen best on the equivalent-to-conventional single arterial phase as well as to those that were see best on the mean of 6 hepatic arterial sub-phases. Inter-reader agreement was also calculated., Results: The MA-CDT-VIBE was successfully acquired in 25 patients with hypervascular metastases (96 lesions) and 6 patients with hypovascular metastases (13 lesions). In case of hypervascular metastases, the 6th/6 arterial sub-phase had excellent lesion visualization (sore of 11) in 56 and 44 lesions for the 2 readers, respectively. Good lesion visualization (score of 8-10) was recorded in 5th/6 arterial subphases, in 81 and 67 lesions for the 2 readers, respectively. In case of hypovascular metastases, the portal venous phase had excellent lesion visualization (sore of 11) in all 13 lesions for the 2 readers. Good lesion visualization (score of 8-10) was recorded in 12 and 13 lesions on the 5th/6 and 6th/6 arterial subphases, respectively. More hypervascular lesions scored good (score of 8-10) and excellent (score of 11) on the 5th/6 and 6th/6 phases of MA-CDT-VIBE compared with the equivalent-to-conventional single arterial phase (3rd/6) and the set with mean of 6 hepatic arterial sub-phases. The results were statistically significant (t test, P<.0001). Inter-reader agreement was good for hypervascular lesions (kappa=0.627, P<.0001) and excellent for hypovascular lesions (kappa=1.0, P<.0001), respectively., Conclusions: The MA-CDT-VIBE improves lesion conspicuity by providing a wide observation window for hypervascular lesions. For hypovascular lesions, the advantage of multiple arterial sub-phases over the portal venous phase is not apparent., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

There is a need for a general reporter system to study the interactions between a ligand and membrane protein in living cells. Here, we demonstrate a method that allows identification of undiscovered ligand partners of a membrane protein in its natural milieu. This assay will be of importance especially for multiple pass transmembrane proteins such as ion channels and GPCRs.

Animal venoms represent a rich source of pharmacologically active peptides that interact with ion channels. However, a challenge to discovering drugs remains because of the slow pace at which venom peptides are discovered and refined. An efficient autocrine-based high-throughput selection system was developed to discover and refine venom peptides that target ion channels. The utility of this system was demonstrated by the discovery of novel Kv1.3 channel blockers from a natural venom peptide library that was formatted for autocrine-based selection. We also engineered a Kv1.3 blocker peptide (ShK) derived from sea anemone to generate a subtype-selective Kv1.3 blocker with a long half-life in vivo., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Until today, there is no consistency about the prognosis and the diagnostic criteria of the "malignant" pancreatic solid pseudopapillary neoplasms (m-SPNs). We here made a retrospective study of 26 such cases and try to give a comprehensive description of their pathological characteristics and clinical course. We found out that among those malignant cases, the most common involved extrapancreatic organ was the duodenum, followed by the spleen and the portal vein. The lymph node and the liver metastasis were also seen in 19 % cases, respectively. Most of the patients were female (22/26). Calcification, foamy cytoplasm, and bizarre nuclei of tumor cells were more common in male patients. Every patient underwent surgical resection and had excellent prognosis. There were two patients who had metastasis to the liver 6 years after excision. The recurrence status correlated with the family history of malignant tumor. No patient died of the disease directly during the mean follow-up time of 73.9 months (21-135 months). Our results supported the idea that the prognosis of the advanced stage SPNs was excellent. The surgical resection seemed effectively enough for these patients. However, all the patients need close follow-ups, especially those who had family history of malignant tumors.

An attractive, but as yet generally unrealized, approach to cancer therapy concerns discovering agents that change the state of differentiation of the cancer cells. Recently, we discovered a phenomenon that we call "receptor pleiotropism" in which agonist antibodies against known receptors induce cell fates that are very different from those induced by the natural agonist to the same receptor. Here, we show that one can take advantage of this phenomenon to convert acute myeloblastic leukemic cells into natural killer cells. Upon induction with the antibody, these leukemic cells enter into a differentiation cascade in which as many as 80% of the starting leukemic cells can be differentiated. The antibody-induced killer cells make large amounts of perforin, IFN-γ, and granzyme B and attack and kill other members of the leukemic cell population. Importantly, induction of killer cells is confined to transformed cells, in that normal bone marrow cells are not induced to form killer cells. Thus, it seems possible to use agonist antibodies to change the differentiation state of cancer cells into those that attack and kill other members of the malignant clone from which they originate.

AKT2 is a crucial mediator in the tumorigenesis and thought to be an ideal target for the treatment of malignancies. Increasing evidence has also shown that microRNAs (miRNAs) could regulate or be regulated by mRNAs and they also might serve as therapeutic agencies or targets. MiR-29s (miR-29a, miR-29b and miR-29c) had been approved that they decreased in gastric cancers (GC) by targeting CCND2, MMP2 and p42.3. However, whether miR-29s would target AKT2 have not been investigated in GC. Here, we explored the relationship between miR-29s and AKT2 and found that in GC cell lines (HGC-27 and MGC-803) and GC clinical samples the decreased levels of miR-29s accompanied by increased AKT2 expression. Introduction of miR-29s into GC cells resulted in decreased AKT2 expression and decreased the ability of cancer cells invasion, so did the siRNA-AKT2. Our studies revealed that miR-29s expression is downregulated in GC and they could repress the AKT2 expression and the inactivation of AKT and GSK3beta leading to inhibit the GC cells invasion. Taken together, our findings suggested that AKT2 may be one of the targets of miR29s in gastric cancer. By increasing the expression of miR-29s or decreasing AKT2 expression may be promising in combating with GC.

When combinatorial antibody libraries are rendered infectious for eukaryotic cells, the integrated antibody genotype and cellular phenotype become permanently linked and each cell becomes a selection system unto itself. These systems should be ideal for the identification of proteins and pathways that regulate differentiation so long as selection systems can be devised. Here we use a selection system based on the ability of secreted antibodies to alter the morphology of colonies expressing them when grown in soft agar. Importantly, this approach is different from all previous studies in that it used a pure discovery format where unbiased libraries that were not preselected against any known protein were used as probes. As such, the strategy is analogous to classical forward genetic approaches except that it operates directly at the protein level. This approach led to the identification of integrin-binding agonist antibodies that efficiently converted human stem cells to dendritic cells.

We describe a system for direct selection of antibodies that are receptor agonists. Combinatorial antibody libraries in lentiviruses are used to infect eukaryotic cells that contain a fluorescent reporter system coupled to the receptor for which receptor agonist antibodies are sought. In this embodiment of the method, very large numbers of candidate antibodies expressing lentivirus and eukaryotic reporter cells are packaged together in a format where each is capable of replication, thereby forging a direct link between genotype and phenotype. Following infection, cells that fluoresce are sorted and the integrated genes encoding the agonist antibodies recovered. We validated the system by illustrating its ability to generate rapidly potent antibody agonists that are complete thrombopoietin phenocopies. The system should be generalizable to any pathway where its activation can be linked to production of a selectable phenotype., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We report here the generation of antibody agonists from intracellular combinatorial libraries that transdifferentiate human stem cells. Antibodies that are agonists for the granulocyte colony stimulating factor receptor were selected from intracellular libraries on the basis of their ability to activate signaling pathways in reporter cells. We used a specialized "near neighbor" approach in which the entire antibody library and its target receptor are cointegrated into the plasma membranes of a population of reporter cells. This format favors unusual interactions between receptors and their protein ligands and ensures that the antibody acts in an autocrine manner on the cells that produce it. Unlike the natural granulocyte-colony stimulating factor that activates cells to differentiate along a predetermined pathway, the isolated agonist antibodies transdifferentiated human myeloid lineage CD34+ bone marrow cells into neural progenitors. This transdifferentiation by agonist antibodies is different from more commonly used methods because initiation is agenetic. Antibodies that act at the plasma membrane may have therapeutic potential as agents that transdifferentiate autologous cells.

A method is presented that uses combinatorial antibody libraries to endow cells with new binding energy landscapes for the purpose of regulating their phenotypes. Antibodies that are expressed in cells infected with a lentiviral combinatorial antibody library are selected directly for function rather than only for binding. The potential diversity space can be very large because more than one lentivirus can infect a single cell. Thus, the initial combinatorial diversity of ~1.0 × 10(11) members generated by the random association of antibody heavy and light chains is greatly increased by the reassortment of the antibody Fv domains themselves inside cells. The power of the system is illustrated by its ability to select unusual antibodies. Here, the selected antibodies are potent erythropoietin agonists whose ontogeny depends on recombination at the protein level of pairs of antibodies expressed in the same cell to generate heterodimeric bispecific antibodies. The obligate synergy between the different binding specificities of the antibody's monomeric subunits appears to replicate the asymmetric binding mechanism of authentic erythropoietin.

Objectives: This study consisted of two parts. One part was to analyze the survival rates of adenoid cystic carcinoma (ACC) in Chinese and explain the difference between our data and the literature. The other was to analyze the relationship between the expression of CD117 and the histological grade and the prognosis., Methods: A retrospective study of 80 ACC patients was performed. Clinical data were collected, and p63, CD117 were detected by immunohistochemical staining., Results: Eighty patients received follow-ups 3 to 216 months after initial diagnosis. ACC occurred in the lacrimal gland (26.3%, n = 21), nasal cavity and parasinus (33.8%, n = 27) and other sites (40.0%, n = 33). The 5-year and 10-year survival rates were 66.41% and 10.16%, respectively. Over expression of CD117 was detected in p63-negative cells in 94.3% of cases and in p63-positive cells in 45.8%. The expression of CD117 in p63-positive cells was significantly associated with the histological grade (P<0.001) and prognosis (P = 0.037) in patients in the advanced stage., Conclusions: ACC had a good 5-year survival but poor 10-year survival in Chinese, which differed from the occidental data. More p63+/CD117+ cells were associated with a higher histological grade and poorer outcome., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1701457278762097.

The occurrence of two primary tumors simultaneously is relatively common. However, the occurrence of three kinds of gynecological tumors simultaneously is extremely rare. Here we presented a 79-year-old woman who had simultaneously three primary gynecological tumors which were relatively uncommon. Those tumors were myxoid leiomyosarcoma and endometrial mucinous adenocarcinoma in the uterus and a Leydig cell tumor in the right ovary. Uterine myxoid leiomyosarcoma is a very rare and aggressive variant of uterine sarcoma despite of its minimal even no atypia and a little mitotic activity. Mucinous endometrial adenocarcinoma is also a rare subtype which comprise only 0.6-5% of uterine cancers. Leydig cell tumor is a very rare ovarian tumor which is composed entirely or predominantly of Leydig cell.

Use of large combinatorial antibody libraries and next-generation sequencing of nucleic acids are two of the most powerful methods in modern molecular biology. The libraries are screened using the principles of evolutionary selection, albeit in real time, to enrich for members with a particular phenotype. This selective process necessarily results in the loss of information about less-fit molecules. On the other hand, sequencing of the library, by itself, gives information that is mostly unrelated to phenotype. If the two methods could be combined, the full potential of very large molecular libraries could be realized. Here we report the implementation of a phenotype-information-phenotype cycle that integrates information and gene recovery. After selection for phage-encoded antibodies that bind to targets expressed on the surface of Escherichia coli, the information content of the selected pool is obtained by pyrosequencing. Sequences that encode specific antibodies are identified by a bioinformatic analysis and recovered by a stringent affinity method that is uniquely suited for gene isolation from a highly degenerate collection of nucleic acids. This approach can be generalized for selection of antibodies against targets that are present as minor components of complex systems.

Detection of metastatic cells is clinically demanded to diagnose metastasis in the early stage and access the therapeutic response to anticancer drugs. We applied phage display technology to cultured cells with different metastasis potentials and obtained four metastasis-associated peptides. The association between peptides and metastatic cells was validated by ELISA as well as biosensor studies. The selected phage-peptides not only bound SW620, the metastatic cell against which the peptides were screened, but were also able to capture breast cancer cells of high metastasis. The phage-peptide-modified LAPS (light-addressable potentiometric sensor) was able to distinguish metastatic cells from non-metastatic cells and detect as few as 100 metastatic cells per ml of blood. Thus LAPS modified with specific phage-peptides may be developed to provide a new diagnostic approach that can aid the treatment of cancer.