A Chromosome-Level Genome Assembly of the Reef Stonefish (Synanceia verrucosa) Provides Novel Insights into Stonustoxin (sntx) Genes.

Reef stonefish (Synanceia verrucosa) is one of the most venomous fishes, but its biomedical study has been restricted to molecular cloning and purification of its toxins, instead of high-throughput genetic research on related toxin genes. In this study, we constructed a chromosome-level haplotypic genome assembly for the reef stonefish. The genome was assembled into 24 pseudo-chromosomes, and the length totaled 689.74 Mb, reaching a contig N50 of 11.97 Mb and containing 97.8% of complete BUSCOs. A total of 24,050 protein-coding genes were annotated, of which metalloproteinases, C-type lectins, and stonustoxins (sntx) were the most abundant putative toxin genes. Multitissue transcriptomic and venom proteomic data showed that sntx genes, especially those clustered within a 50-kb region on the chromosome 2, had higher transcription levels than other types of toxins as well as those sntx genes scatteringly distributed on other chromosomes. Further comparative genomic analysis predicted an expansion of sntx-like genes in the Percomorpha lineage including nonvenomous fishes, but Scorpaenoidei species experienced extra independent sntx duplication events, marking the clear-cut origin of authentic toxic stonustoxins. In summary, this high-quality genome assembly and related comparative analysis of toxin genes highlight valuable genetic differences for potential involvement in the evolution of venoms among Scorpaeniformes fishes.

Venomics Reveals the Venom Complexity of Sea Anemone Heteractis magnifica.

The venoms of various sea anemones are rich in diverse toxins, which usually play a dual role in capturing prey and deterring predators. However, the complex components of such venoms have not been well known yet. Here, venomics of integrating transcriptomic and proteomic technologies was applied for the first time to identify putative protein and peptide toxins from different tissues of the representative sea anemone, Heteractis magnifica. The transcriptomic analysis of H. magnifica identified 728 putative toxin sequences, including 442 and 381 from the tentacles and the column, respectively, and they were assigned to 68 gene superfamilies. The proteomic analysis confirmed 101 protein and peptide toxins in the venom, including 91 in the tentacles and 39 in the column. The integrated venomics also confirmed that some toxins such as the ShK-like peptides and defensins are co-expressed in both the tentacles and the column. Meanwhile, a homology analysis was conducted to predict the three-dimensional structures and potential activity of seven representative toxins. Altogether, this venomics study revealed the venom complexity of H. magnifica, which will help deepen our understanding of cnidarian toxins, thereby supporting the in-depth development of valuable marine drugs.

Chemical Synthesis and Insecticidal Activity Research Based on α-Conotoxins.

The escalating resistance of agricultural pests to chemical insecticides necessitates the development of novel, efficient, and safe biological insecticides. Conus quercinus, a vermivorous cone snail, yields a crude venom rich in peptides for marine worm predation. This study screened six α-conotoxins with insecticidal potential from a previously constructed transcriptome database of C. quercinus, characterized by two disulfide bonds. These conotoxins were derived via solid-phase peptide synthesis (SPPS) and folded using two-step iodine oxidation for further insecticidal activity validation, such as CCK-8 assay and insect bioassay. The final results confirmed the insecticidal activities of the six α-conotoxins, with Qc1.15 and Qc1.18 exhibiting high insecticidal activity. In addition, structural analysis via homology modeling and functional insights from molecular docking offer a preliminary look into their potential insecticidal mechanisms. In summary, this study provides essential references and foundations for developing novel insecticides.

Response to "Malondialdehyde-Induced Post-Translational Modification of Human Hemoglobin".

Although malondialdehyde and methylglyoxal have the same molecular formula, they have different chemistry in forming protein adducts. The major lysine adduct of malondialdehyde in hemoglobin is the N-propenal type, while that of methylglyoxal is N6-(1-carboxyethyl)lysine. This Letter provides evidence that the "methylglyoxal-like" hemoglobin adducts are not derived from malondialdehyde. This Letter also discusses the quantification of malondialdehyde-induced post-translational modifications in human hemoglobin by different mass spectrometry-based methods.

Identification of exosomal hsa-miR-483-5p as a potential biomarker for hepatocellular carcinoma via microRNA expression profiling of tumor-derived exosomes.

To date, most studies of exosomes related to hepatocellular carcinoma (HCC) have used commercial cancer cell lines or patient plasma as source material. In this study, we isolated exosomes directly from HCC tissues to investigate the potential of exosomal contents as biomarkers for HCC. Exosomes were identified and verified using transmission electron microscopy, nano-flow cytometry analysis, and western blotting. Tissue-derived exosomal miRNA expression was profiled by high-throughput sequencing, and differential expression of miRNAs was validated by quantitative real-time polymerase chain reaction analysis. The diagnostic performance of differentially expressed exosomal miRNAs for HCC was evaluated by receiver operating characteristic curve analysis. Target genes of these miRNAs were verified using luciferase reporter assays, and their functions were studied through in vitro and rescue assays. In total, 225 differentially expressed exosomal miRNAs were identified in HCC samples compared with adjacent liver tissues, and some were associated with HCC tumorigenesis and progression. Comparison of the expression profiles of tissue-derived and plasma-derived exosomal miRNAs identified hsa-miR-483-5p as the only differentially expressed miRNA detected in both HCC tissue and plasma, and this was in a validation group of HCC patients. Analysis of the diagnostic performance of plasma exosomal hsa-miR-483-5p or plasma hsa-miR-483-5p found that both could differentiate HCC and non-HCC cases. In vitro ectopic miR-483-5p expression promoted HCC cell proliferation. CDK15 was confirmed to bind with miR-483-5p directly, and thus, miR-483-5p may function by downregulating CDK15. Hsa-miR-483-5p represents a potential specific and sensitive biomarker for HCC diagnosis.

Recognition and management of hemorrhaging in combination with emerging enterogenic sepsis during a hepatectomy: a case report.

BACKGROUND: Patients with hemorrhagic shock may develop emerging enterogenic sepsis due to damage to the intestinal mucosal barrier and translocation of intestinal bacteria and endotoxins caused by ischemic injury. Because of the dual effects of anesthesia state and hemorrhagic shock, perioperative emerging enterogenic sepsis is even more rare and insidious. CASE PRESENTATION: We reported a case of 56-year-old man who underwent right hepatectomy for intrahepatic bile duct stones. Severe hemorrhage occurred during the procedure and the hemodynamics neither improved nor worsened after rehydration therapy and vasoactive drug administration. Based on the patient's history and clinical presentation, a possible enterogenic sepsis was considered. After anti-infective treatment and hormone supplementation, the patient's circulation improved significantly and he had an uneventful recovery. CONCLUSION: The possibility of emerging enterogenic sepsis in hemorrhagic shock must always be taken into consideration. Familiarity with the risk factors and pathophysiological alterations of enterogenic sepsis is a prerequisite for early recognition and sound clinical decision making.

Neurotoxicity and the potential molecular mechanisms of mono-2-ethylhexyl phthalic acid (MEHP) in zebrafish.

Mono-2-ethylhexyl phthalic acid (MEHP) is the most toxic metabolite of plasticizer di-2-ethylhexyl phthalic acid (DEHP), and there is limited information available on the effects of MEHP on neurotoxicity. This study aims to examine the neurotoxicity of MEHP and preliminarily explore its potential molecular mechanisms. We found that MEHP impeded the growth of zebrafish embryos and the neurodevelopmental-related gene expression at environmentally relevant concentrations. MEHP exposure also induces oxidative stress response and brain cell apoptosis accompanied by a decrease in acetylcholinesterase (AChE) activity in zebrafish larvae. RNA-Seq and bioinformatics analysis showed that MEHP treatment altered the nervous system, neurogenic diseases, and visual perception pathways. The locomotor activity in dark-to-light cycles and phototaxis test confirmed the abnormal neural behavior of zebrafish larvae. Besides, the immune system has produced a large number of differentially expressed genes related to neural regulation. Inflammatory factor IL1ß and IL-17 signaling pathways highly respond to MEHP, indicating that inflammation caused by immune system imbalance is a potential mechanism of MEHP-induced neurotoxicity. This study expands the understanding of the toxicity and molecular mechanisms of MEHP, providing a new perspective for in-depth neurotoxicity exploration of similar compounds.

Comparative transcriptome analysis of abnormal cap and healthy fruiting bodies of the edible mushroom Lentinula edodes.

Lentinula edodes, a commercially important mushroom, is cultivated worldwide. Artificially cultivated L. edodes often present with abnormal symptoms in the fruiting body, which affect their commercial value and reduce production efficiency. In this study, we carried out a comparative transcriptome analysis of normal fruiting body pileus (LeNP), normal margin in abnormal fruiting body pileus (LeAPNM), and abnormal margin in abnormal fruiting body pileus (LeAPAM). Metabolic pathways such as those involved in transmembrane transport, ribosome production, tryptophan metabolism, arginine and proline metabolism, and the metabolism of other amino acids were significantly enriched in LeAPAM. F-box, short-chain dehydrogenases/reductases, the major facilitator superfamily, and the FMN_red superfamily are related to malformation in L. edodes. Genes encoding heat shock proteins, G protein, and ß-1,3-glucanase in the GH5 family showed different expression patterns, suggesting that these genes are involved in the development of L. edodes fruiting bodies. In particular, CAZymes, which are involved in the development of cell walls in L. edodes, were highly expressed in LeAPAM. According to TEM observation, the cell wall of LeAPAM samples showed significant thickening compared to the other samples. These results suggested that cell wall anabolism in LeAPAM samples was more active than that in normal fruiting bodies, enhancing the environmental adaptability of the fungus. This study provides preliminary data for future research aimed at solving the phenomenon of abnormal fruiting bodies of L. edodes.

Exposed CendR Domain in Homing Peptide Yields Skin-Targeted Therapeutic in Epidermolysis Bullosa.

Systemic skin-selective therapeutics would be a major advancement in the treatment of diseases affecting the entire skin, such as recessive dystrophic epidermolysis bullosa (RDEB), which is caused by mutations in the COL7A1 gene and manifests in transforming growth factor-ß (TGF-ß)-driven fibrosis and malignant transformation. Homing peptides containing a C-terminal R/KXXR/K motif (C-end rule [CendR] sequence) activate an extravasation and tissue penetration pathway for tumor-specific drug delivery. We have previously described a homing peptide CRKDKC (CRK) that contains a cryptic CendR motif and homes to angiogenic blood vessels in wounds and tumors, but it cannot penetrate cells or tissues. In this study, we demonstrate that removal of the cysteine from CRK to expose the CendR sequence confers the peptide novel ability to home to normal skin. Fusion of the truncated CRK (tCRK) peptide to the C terminus of an extracellular matrix protein decorin (DCN), a natural TGF-ß inhibitor, resulted in a skin-homing therapeutic molecule (DCN-tCRK). Systemic DCN-tCRK administration in RDEB mice led to inhibition of TGF-ß signaling in the skin and significant improvement in the survival of RDEB mice. These results suggest that DCN-tCRK has the potential to be utilized as a novel therapeutic compound for the treatment of dermatological diseases such as RDEB.

Trained Innate Immunity by Repeated Low-Dose Lipopolysaccharide Injections Displays Long-Term Neuroprotective Effects.

Disrupted immune response is an important feature of many neurodegenerative conditions, including sepsis-associated cognitive impairment. Accumulating evidence has demonstrated that immune memory occurs in microglia, which has a significant impact on pathological hallmarks of neurological diseases. However, it remains unclear whether immune memory can cause subsequent alterations in the brain immune response and affect neurobehavioral outcomes in sepsis survivors. In the present study, mice received daily intraperitoneal injection of low-dose lipopolysaccharide (LPS, 0.1 mg/kg) for three consecutive days to induce immune memory (immune tolerance) and then were subjected to sham operation or cecal ligation and puncture (CLP) 9 months later, followed by a battery of neurobehavioral and biochemical studies. Here, we showed that repeated low-dose LPS injection-induced immune memory protected mice from sepsis-induced cognitive and affective impairments, which were accompanied by significantly decreased brain proinflammatory cytokines and immune response. In conclusion, our study suggests that modulation of brain immune responses by repeated LPS injections confers neuroprotective effects by preventing overactivated immune response in response to subsequent septic insult.

Cord Blood-Derived Stem Cells Suppress Fibrosis and May Prevent Malignant Progression in Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by mutations in the Col7a1 gene. Patients with RDEB suffer from recurrent erosions in skin and mucous membranes and have a high risk for developing cutaneous squamous cell carcinoma (cSCCs). TGFß signaling has been associated with fibrosis and malignancy in RDEB. In this study, the activation of TGFß signaling was demonstrated in col7a1-/- mice as early as a week after birth starting in the interdigital folds of the paws, accompanied by increased deposition of collagen fibrils and elevated dermal expression of matrix metalloproteinase (MMP)-9 and MMP-13. Furthermore, human cord blood-derived unrestricted somatic stem cells (USSCs) that we previously demonstrated to significantly improve wound healing and prolong the survival of col7a1-/- mice showed the ability to suppress TGFß signaling and MMP-9 and MMP-13 expression meanwhile upregulating anti-fibrotic TGFß3 and decorin. In parallel, we cocultured USSCs in a transwell with RDEB patient-derived fibroblasts, keratinocytes, and cSCC, respectively. The patient-derived cells were constitutively active for STAT, but not TGFß signaling. Moreover, the levels of MMP-9 and MMP-13 were significantly elevated in the patient derived-keratinocytes and cSCCs. Although USSC coculture did not inhibit STAT signaling, it significantly suppressed the secretion of MMP-9 and MMP-13, and interferon (IFN)-γ from RDEB patient-derived cells. Since epithelial expression of these MMPs is a biomarker of malignant transformation and correlates with the degree of tumor invasion, these results suggest a potential role for USSCs in mitigating epithelial malignancy, in addition to their anti-inflammatory and anti-fibrotic functions. Stem Cells 2018;36:1839-12.

Rescue of the mucocutaneous manifestations by human cord blood derived nonhematopoietic stem cells in a mouse model of recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering disease caused by mutations in COL7A1-encoding type VII collagen (C7). Currently, there is no curative therapy for patients with RDEB. Our previous studies demonstrated that human umbilical cord blood (HUCB) derived unrestricted somatic stem cells (USSCs) express C7 and facilitate wound healing in a murine wounding model. The primary objective of this study is to investigate the therapeutic functions of USSCs in the C7 null (Col7a1(-/-) ) C57BL6/J mice, a murine model of RDEB. We demonstrated that intrahepatic administration of USSCs significantly improved the blistering phenotype and enhanced the life span in the recipients. The injected USSCs trafficked to the sites of blistering and were incorporated in short-term in the recipients' skin and gastrointestinal tract. Consistent with an overall histological improvement in the epidermal-dermal adherence following USSC treatment, the expression of C7 at the basement membrane zone was detected and the previously disorganized integrin α6 distribution was normalized. We also demonstrated that USSCs treatment induced an infiltration of macrophages with a regenerative "M2" phenotype. Our data suggest that HUCB-derived USSCs improved the RDEB phenotype through multiple mechanisms. This study has warranted future clinical investigation of USSCs as a novel and universal allogeneic stem cell donor source in selected patients with RDEB.

PPARß/δ-ANGPTL4 axis mediates the promotion of mono-2-ethylhexyl phthalic acid on MYCN-amplified neuroblastoma development.

Di-2-ethylhexyl phthalic acid (DEHP) is one of the most widely used plasticizers in the industry, which can improve the flexibility and durability of plastics. It is prone to migrate from various daily plastic products through wear and leaching into the surrounding environment and decompose into the more toxic metabolite mono-2-ethylhexyl phthalic acid (MEHP) after entering the human body. However, the impacts and mechanisms of MEHP on neuroblastoma are unclear. We exposed MYCN-amplified neuroblastoma SK-N-BE(2)C cells to an environmentally related concentration of MEHP and found that MEHP increased the proliferation and migration ability of tumor cells. The peroxisome proliferator-activated receptor (PPAR) ß/δ pathway was identified as a pivotal signaling pathway in neuroblastoma, mediating the effects of MEHP through transcriptional sequencing analysis. Because MEHP can bind to the PPARß/δ protein and initiate the expression of the downstream gene angiopoietin-like 4 (ANGPTL4), the PPARß/δ-specific agonist GW501516 and antagonist GSK3787, the recombinant human ANGPTL4 protein, and the knockdown of gene expression confirmed the regulation of the PPARß/δ-ANGPTL4 axis on the malignant phenotype of neuroblastoma. Based on the critical role of PPARß/δ and ANGPTL4 in the metabolic process, a non-targeted metabolomics analysis revealed that MEHP altered multiple metabolic pathways, particularly lipid metabolites involving fatty acyls, glycerophospholipids, and sterol lipids, which may also be potential factors promoting tumor progression. We have demonstrated for the first time that MEHP can target binding to PPARß/δ and affect the progression of neuroblastoma by activating the PPARß/δ-ANGPTL4 axis. This mechanism confirms the health risks of plasticizers as tumor promoters and provides new data support for targeted prevention and treatment of neuroblastoma.

Diversity analysis of sea anemone peptide toxins in different tissues of Heteractis crispa based on transcriptomics.

Peptide toxins found in sea anemones venom have diverse properties that make them important research subjects in the fields of pharmacology, neuroscience and biotechnology. This study used high-throughput sequencing technology to systematically analyze the venom components of the tentacles, column, and mesenterial filaments of sea anemone Heteractis crispa, revealing the diversity and complexity of sea anemone toxins in different tissues. A total of 1049 transcripts were identified and categorized into 60 families, of which 91.0% were proteins and 9.0% were peptides. Of those 1049 transcripts, 416, 291, and 307 putative proteins and peptide precursors were identified from tentacles, column, and mesenterial filaments respectively, while 428 were identified when the datasets were combined. Of these putative toxin sequences, 42 were detected in all three tissues, including 33 proteins and 9 peptides, with the majority of peptides being ShKT domain, ß-defensin, and Kunitz-type. In addition, this study applied bioinformatics approaches to predict the family classification, 3D structures, and functional annotation of these representative peptides, as well as the evolutionary relationships between peptides, laying the foundation for the next step of peptide pharmacological activity research.

The addition of peripheral nerve blocks to routine spinal or general anesthesia was associated with decreased risks of major adverse events after total hip or knee arthroplasty: A retrospective, propensity score-matched cohort study.

Background: Although total joint arthroplasty is the most effective procedures for end-stage arthritis, the incidence of postoperative death and complications remains high. The association of additional peripheral nerve blocks (PNBs) to routine spinal or general anesthesia with major adverse events (including mortality and complication rates) in elective total hip arthroplasty (THA) or total knee arthroplasty (TKA) has been subject to inconclusive findings. Methods: This retrospective observational single institution study included all patients ≧ 18 years undergoing their first elective THA or TKA from January 1, 2012 to December 31, 2021. A 1:2 propensity score matching (PSM) was performed to account for the baseline differences between two groups that were accepted to PNB or not. Kaplan-Meier curves were employed to estimate the effects of PNB on mortality. The associations of PNB and the complications were assessed by logistic regression models. Results: We identified 1328 patients, among whom 197 had PNB and 1131 had not. The 90-day all-cause mortality was significantly reduced in patients with PNBs (0 % vs 2.79 %, P = 0.041) after THA or TKA, when compared to the non-PNB group. PNB was also associated with a lower risk of pulmonary complications (odds ratio [OR], 0.430; 95%confidence interval [CI],0.216-0.857) and deep vein thrombosis (OR, 0.103; 95%CI, 0.011-0.954). Interpretation: The results of this observational, propensity score-matched cohort study suggested a strong association between the addition of PNBs to routine spinal or general anesthesia and decreased risks of major adverse events.

Circumventing resistance within the Ewing sarcoma microenvironment by combinatorial innate immunotherapy.

BACKGROUND: Pediatric patients with recurrent/metastatic Ewing sarcoma (ES) have a dismal 5-year survival. Novel therapeutic approaches are desperately needed. Natural killer (NK) cell number and function are low in ES patient tumors, in large part due to the immunosuppressive tumor microenvironment (TME). Melanoma cell adhesion molecule (MCAM) is highly expressed on ES and associated with ES metastasis. NKTR-255 is a polymer-conjugated recombinant human interleukin-15 (IL-15) agonist improving NK cell activity and persistence. Magrolimab (MAG) is a CD47 blockade that reactivates the phagocytic activity of macrophages. METHODS: Transcriptome profiling coupled with CIBERSORT analyses in both ES mouse xenografts and human patient tumors were performed to identify mechanisms of NK resistance in ES TME. A chimeric antigen receptor (CAR) NK cell targeting MCAM was engineered by CAR mRNA electroporation into ex vivo expanded NK cells. In vitro cytotoxicity assays were performed to investigate the efficacy of anti-MCAM-CAR-NK cell alone or combined with NKTR-255 against ES cells. Interferon-γ and perforin levels were measured by ELISA. The effect of MAG on macrophage phagocytosis of ES cells was evaluated by in vitro phagocytosis assays. Cell-based and patient-derived xenograft (PDX)-based xenograft mouse models of ES were used to investigate the antitumor efficacy of CAR-NK alone and combined with NKTR-255 and MAG in vivo. RESULTS: We found that NK cell infiltration and activity were negatively regulated by tumor-associated macrophages (TAM) in ES TME. Expression of anti-MCAM CAR significantly and specifically enhanced NK cytotoxic activity against MCAMhigh but not MCAM-knockout ES cells in vitro, and significantly reduced lung metastasis and extended animal survival in vivo. NKTR-255 and MAG significantly enhanced in vitro CAR-NK cytotoxicity and macrophage phagocytic activity against ES cells, respectively. By combining with NKTR-255 and MAG, the anti-MCAM-CAR-NK cell significantly decreased primary tumor growth and prolonged animal survival in both cell- and PDX-based ES xenograft mouse models. CONCLUSIONS: Our preclinical studies demonstrate that immunotherapy via the innate immune system by combining tumor-targeting CAR-NK cells with an IL-15 agonist and a CD47 blockade is a promising novel therapeutic approach to targeting MCAMhigh malignant metastatic ES.

Efficiently targeting neuroblastoma with the combination of anti-ROR1 CAR NK cells and N-803 in vitro and in vivo in NB xenografts.

The prognosis for children with recurrent and/or refractory neuroblastoma (NB) is dismal. The receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is highly expressed on the surface of NB cells, provides a potential target for novel immunotherapeutics. Anti-ROR1 chimeric antigen receptor engineered ex vivo expanded peripheral blood natural killer (anti-ROR1 CAR exPBNK) cells represent this approach. N-803 is an IL-15 superagonist with enhanced biological activity. In this study, we investigated the in vitro and in vivo anti-tumor effects of anti-ROR1 CAR exPBNK cells with or without N-803 against ROR1+ NB models. Compared to mock exPBNK cells, anti-ROR1 CAR exPBNK cells had significantly enhanced cytotoxicity against ROR1+ NB cells, and N-803 further increased cytotoxicity. High-dimensional analysis revealed that N-803 enhanced Stat5 phosphorylation and Ki67 levels in both exPBNK and anti-ROR1 CAR exPBNK cells with or without NB cells. In vivo, anti-ROR1 CAR exPBNK plus N-803 significantly (p < 0.05) enhanced survival in human ROR1+ NB xenografted NSG mice compared to anti-ROR1 CAR exPBNK alone. Our results provide the rationale for further development of anti-ROR1 CAR exPBNK cells plus N-803 as a novel combination immunotherapeutic for patients with recurrent and/or refractory ROR1+ NB.

Combinatorial macrophage induced innate immunotherapy against Ewing sarcoma: Turning "Two Keys" simultaneously.

BACKGROUND: Macrophages play important roles in phagocytosing tumor cells. However, tumors escape macrophage phagocytosis in part through the expression of anti-phagocytic signals, most commonly CD47. In Ewing sarcoma (ES), we found that tumor cells utilize dual mechanisms to evade macrophage clearance by simultaneously over-expressing CD47 and down-regulating cell surface calreticulin (csCRT), the pro-phagocytic signal. Here, we investigate the combination of a CD47 blockade (magrolimab, MAG) to inhibit the anti-phagocytic signal and a chemotherapy regimen (doxorubicin, DOX) to enhance the pro-phagocytic signal to induce macrophage phagocytosis of ES cells in vitro and inhibit tumor growth and metastasis in vivo. METHODS: Macrophages were derived from human peripheral blood monocytes by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). Flow cytometry- and microscopy-based in-vitro phagocytosis assays were performed to evaluate macrophage phagocytosis of ES cells. Annexin-V assay was performed to evaluate apoptosis. CD47 was knocked out by CRISPR/Cas9 approach. ES cell-based and patient-derived-xenograft (PDX)-based mouse models were utilized to assess the effects of MAG and/or DOX on ES tumor development and animal survival. RNA-Seq combined with CIBERSORTx analysis was utilized to identify changes in tumor cell transcriptome and tumor infiltrating immune cell profiling in MAG and/or DOX treated xenograft tumors. RESULTS: We found that MAG significantly increased macrophage phagocytosis of ES cells in vitro (p < 0.01) and had significant effect on reducing tumor burden (p < 0.01) and increasing survival in NSG mouse model (p < 0.001). The csCRT level on ES cells was significantly enhanced by DOX in a dose- and time-dependent manner (p < 0.01). Importantly, DOX combined with MAG significantly enhanced macrophage phagocytosis of ES cells in vitro (p < 0.01) and significantly decreased tumor burden (p < 0.01) and lung metastasis (p < 0.0001) and extended animal survival in vivo in two different mouse models of ES (p < 0.0001). Furthermore, we identified CD38, CD209, CD163 and CD206 as potential markers for ES-phagocytic macrophages. Moreover, we found increased M2 macrophage infiltration and decreased expression of Cd209 in the tumor microenvironment of MAG and DOX combinatorial therapy treated tumors. CONCLUSIONS: By turning "two keys" simultaneously to reactivate macrophage phagocytic activity, our data demonstrated an effective and highly translatable alternative therapeutic approach utilizing innate (tumor associated macrophages) immunotherapy against high-risk metastatic ES.

A humanized orthotopic mouse model for preclinical evaluation of immunotherapy in Ewing sarcoma.

The advent of novel cancer immunotherapy approaches is revolutionizing the treatment for cancer. Current small animal models for most cancers are syngeneic or genetically engineered mouse models or xenograft models based on immunodeficient mouse strains. These models have been limited in evaluating immunotherapy regimens due to the lack of functional human immune system. Development of animal models for bone cancer faces another challenge in the accessibility of tumor engraftment sites. Here, we describe a protocol to develop an orthotopic humanized mouse model for a bone and soft tissue sarcoma, Ewing sarcoma, by transplanting fresh human cord blood CD34+ hematopoietic stem cells into young NSG-SGM3 mice combined with subsequent Ewing sarcoma patient derived cell engraftment in the tibia of the humanized mice. We demonstrated early and robust reconstitution of human CD45+ leukocytes including T cells, B cells, natural killer cells and monocytes. Ewing sarcoma xenograft tumors successfully orthotopically engrafted in the humanized mice with minimal invasive procedures. We validated the translational utility of this orthotopic humanized model by evaluating the safety and efficacy of an immunotherapy antibody, magrolimab. Treatment with magrolimab induces CD47 blockade resulting in significantly decreased primary tumor growth, decreased lung metastasis and prolonged animal survival in the established humanized model. Furthermore, the humanized model recapitulated the dose dependent toxicity associated with the CD47 blockade as observed in patients in clinical trials. In conclusion, this orthotopic humanized mouse model of Ewing sarcoma represents an improved platform for evaluating immunotherapy in bone and soft tissue sarcoma, such as Ewing sarcoma. With careful design and optimization, this model is generalizable for other bone malignancies.

N6-methyladenosine demethylase FTO regulates synaptic and cognitive impairment by destabilizing PTEN mRNA in hypoxic-ischemic neonatal rats.

Hypoxic-ischemic brain damage (HIBD) can result in significant global rates of neonatal death or permanent neurological disability. N6-methyladenosine (m6A) modification of RNA influences fundamental aspects of RNA metabolism, and m6A dysregulation is implicated in various neurological diseases. However, the biological roles and clinical significance of m6A in HIBD remain unclear. We currently evaluated the effect of HIBD on cerebral m6A methylation in RNAs in neonatal rats. The m6A dot blot assay showed a global augmentation in RNA m6A methylation post-HI. Herein, we also report on demethylase FTO, which is markedly downregulated in the hippocampus and is the main factor involved with aberrant m6A modification following HI. By conducting a comprehensive analysis of RNA-seq data and m6A microarray results, we found that transcripts with m6A modifications were more highly expressed overall than transcripts without m6A modifications. The overexpression of FTO resulted in the promotion of Akt/mTOR pathway hyperactivation, while simultaneously inhibiting autophagic function. This is carried out by the demethylation activity of FTO, which selectively demethylates transcripts of phosphatase and tensin homolog (PTEN), thus promoting its degradation and reduced protein expression after HI. Moreover, the synaptic and neurocognitive disorders induced by HI were effectively reversed through the overexpression of FTO in the hippocampus. Cumulatively, these findings demonstrate the functional importance of FTO-dependent hippocampal m6A methylome in cognitive function and provides novel mechanistic insights into the therapeutic potentials of FTO in neonatal HIBD.