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Double-stranded break (DSB) repair of eukaryotic DNA is mainly accomplished by nonhomologous end joining and homologous recombination (HR). Providing exogenous templates during HR repair can result in the editing of target genes, which is the central mechanism of the well-established clustered regularly interspaced short palindromic repeats (CRISPR) gene editing system. Currently, exogenous templates are mainly DNA molecules, which can provoke a cellular immune response within the cell. In order to verify the feasibility of RNA molecules as repair templates for HR in mammalian cell genome editing, we fused RNA template molecules to the 3'-end of single guide RNA (sgRNA), so that the sgRNA and the homologous template RNA form a single RNA molecule. The results show this construct can be used as a repair template to achieve target gene editing in mammalian cells. In addition, the factors influencing HR mediated by RNA template molecules were investigated, and it was found that increasing the length of homologous arms and inducing an R-loop near the DSBcan effectively promote HR repair. Furthermore, intracellular homologous chromosomes may compete with exogenous RNA templates. The findings in this article provide a reference for the utilization of RNA template molecules to mediate target gene editing in eukaryotic cells, as well as a basis for the study of the mechanism by which RNA molecules mediate the repair of DSBs.

Objective: This study focuses on dose-response investigation using a codon-optimized and de novo -synthesized E-Selectin/AAV2 (E-Sel/AAV2) vector in preparation for Investigational New Drug (IND)-enabling of subsequent clinical studies., Background: Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia (CLTI). Understanding the dose for effective gene delivery is crucial for future IND-enabling studies., Methods: Expression of the codon-optimized E-Selectin gene was assessed by flow cytometry following in vitro cell transfection assay and RT-qPCR for murine limbs injected in vivo with AAV-m-E-Selectin (E-Sel/AAV2). Dose-response studies involved three cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly (IM) in divided aliquots, ranging from 2×10 9 VG to 2×10 11 VG, into ischemic limbs created by left femoral artery/vein ligation/excision and administration of nitric oxide synthase inhibitor, L-NAME. Limb perfusion, extent of gangrene free limb, functional limb recovery and therapeutic angiogenesis were assessed., Results: Codon-optimized E-Sel/AAV2 gene therapy exhibits superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo . Mice treated with a high dose (2×10 11 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber's scores, increased running stamina and neovascularization compared with lower doses tested with control groups, indicating a distinct dose-dependent response. No toxicity was detected in any of the animal groups studied., Conclusion: E-Sel/AAV2 Vascular Regeneration Gene Therapy (VRGT) holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2×10 11 VG aliquots injected IM., Competing Interests: Conflict of interest statement: Barry J. Byrne, Yulexi Y. Ortiz, Yan Li, Nga Le, Lucy Osafo, Antoine C. Ribieras, , Hongwei Shao, Carlos Theodore Huerta , Yuntao Wei, Andres Franco Bravo, Gustavo Falero-Diaz 1 , Roberta M Lassance-Soares, Roberto I Vazquez-Padron and Francesca Voza have no commercial or financial relationships that could be construed as a potential conflict of interest. Dr. Zhao-Jun Liu (Z.J.L.) and Dr. Omaida C. Velazquez (O.C.V.) declare the following potential conflicts of interest with respect to the research, authorship, and/or presentation and/or publication of some aspects of this work: the E-Selectin gene modification technologies were developed in our research laboratory and patented/licensed by the University of Miami. O.C.V. and Z.J.L. are co-inventors of this technology. This technology is fully owned by University of Miami and is Licensed with exclusivity to Ambulero Inc . This technology is currently under pre-clinical development by Ambulero Inc., a new incubator company spin out from the University of Miami that focuses on developing new vascular treatments for ischemic tissue conditions, for wound healing, and for limb salvage. Co-authors, Z.J.L. and O.C.V., serve as consultants and chief scientific and medical advisory officers to Ambulero Inc. ; are co-Inventors of the technologies; and are minority shareholders in Ambulero Inc. Co-authors, Z.J.L. and O.C.V. are also funded for this work by the NIH/NHLBI and Philanthropy (Eloise & David Kimmelman Foundation) in ongoing preclinical investigations of these technologies., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

T cells utilized in adoptive T cell immunotherapy are typically activated in vitro. Although these cells demonstrate proliferation and anti-tumor activity following activation, they often face difficulties in sustaining long-term survival post-reinfusion. This issue is attributed to the induction of T cells into a terminal differentiation state upon activation, whereas early-stage differentiated T cells exhibit enhanced proliferation potential and survival capabilities. In previous study, we delineated four T cell subsets at varying stages of differentiation: T N , T SCM , T CM , and T EM , and acquired their miRNA expression profiles via high-throughput sequencing. In the current study, we performed a differential analysis of miRNA across these subsets, identifying a distinct miRNA, hsa-miR-744-5p, characterized by progressively increasing expression levels upon T cell activation. This miRNA is not expressed in T SCM but is notably present in T EM . Target genes of miR-744-5p were predicted, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, revealing that these genes predominantly associate with pathways related to the 'Wnt signaling pathway'. We established that miR-744-5p directly targets STK11, influencing its expression. Further, we investigated the implications of miR-744-5p on T cell differentiation and functionality. Overexpression of miR-744-5p in T cells resulted in heightened apoptosis, reduced proliferation, an increased proportion of late-stage differentiated T cells, and elevated secretion of the cytokine TNF-α. Moreover, post-overexpression of miR-744-5p led to a marked decline in the expression of early-stage differentiation-associated genes in T cells (CCR7, CD62L, LEF1, BCL2) and a significant rise in late-stage differentiation-associated genes (KLRG1, PDCD1, GZMB). In conclusion, our findings affirm that miR-744-5p contributes to the progressive differentiation of T cells by downregulating the STK11 gene expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Immunodeficient murine models are usually used as the preclinical models of osteosarcoma. Such models do not effectively simulate the process of tumorigenesis and metastasis. Establishing a suitable animal model for understanding the mechanism of osteosarcoma and the clinical translation is indispensable. The UMR-106 cell suspension was injected into the marrow cavity of Balb/C nude mice. Tumor masses were harvested from nude mice and sectioned. The tumor fragments were transplanted into the marrow cavities of SD rats immunosuppressed with cyclosporine A. Through muti-rounds selection in SD rats, we constructed orthotopic osteosarcoma animal models using rats with intact immune systems. The primary tumor cells were cultured in-vitro to obtain the immune-tolerant cell line. VX2 tumor fragments were transplanted into the distal femur and parosteal radius of New Zealand white rabbit to construct orthotopic osteosarcoma animal models in rabbits. The rate of tumor formation in SD rats (P1 generation) was 30%. After four rounds of selection and six rounds of acclimatization in SD rats with intact immune systems, we obtained immune-tolerant cell lines and established the orthotopic osteosarcoma model of the distal femur in SD rats. Micro-CT images confirmed tumor-driven osteolysis and the bone destruction process. Moreover, the orthotopic model was also established in New Zealand white rabbits by implanting VX2 tumor fragments into rabbit radii and femurs. We constructed orthotopic osteosarcoma animal models in rats with intact immune systems through muti-rounds in-vivo selection and the rabbit osteosarcoma model., (© 2024. The Author(s).)

Osteochondral defect (OCD) is a common but challenging condition in orthopaedics that imposes huge socioeconomic burdens in our aging society. It is imperative to accelerate the R&D of regenerative scaffolds using osteochondral tissue engineering concepts. Yet, all innovative implant-based treatments require animal testing models to verify their feasibility, biosafety, and efficacy before proceeding to human trials. Rabbit models offer a more clinically relevant platform for studying OCD repair than smaller rodents, while being more cost-effective than large animal models. The core-decompression drilling technique to produce full-thickness distal medial femoral condyle defects in rabbits can mimic one of the trauma-relevant OCD models. This model is commonly used to evaluate the implant's biosafety and efficacy of osteochondral dual-lineage regeneration. In this article, we initially indicate the methodology and describe a minimally-invasive surgical protocol in a step-wise manner to generate a standard and reproducible rabbit OCD for scaffold implantation. Besides, we provide a detailed procedure for sample collection, processing, and evaluation by a series of subsequent standardized biochemical, radiological, biomechanical, and histological assessments. In conclusion, the well-established, easy-handling, reproducible, and reliable rabbit OCD model will play a pivotal role in translational research of osteochondral tissue engineering., Competing Interests: The 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Zhou, Ho, Zheng, Xu, Chen, Ye, Zou, Li, Chang, Shao, Li, Long, Nie, Stoddart, Lai and Qin.)

Ligilactobacillus murinus, a member of the Ligilactobacillus genus, holds significant potential as a probiotic. While research on Ligilactobacillus murinus has been relatively limited compared to well-studied probiotic lactic acid bacteria such as Limosilactobacillus reuteri and Lactobacillus gasseri, a mounting body of evidence highlights its extensive involvement in host intestinal metabolism and immune activities. Moreover, its abundance exhibits a close correlation with intestinal health. Notably, beyond the intestinal context, Ligilactobacillus murinus is gaining recognition for its contributions to metabolism and regulation in the oral cavity, lungs, and vagina. As such, Ligilactobacillus murinus emerges as a potential probiotic candidate with a pivotal role in supporting host well-being. This review delves into studies elucidating the multifaceted roles of Ligilactobacillus murinus. It also examines its medicinal potential and associated challenges, underscoring the imperative to delve deeper into unraveling the mechanisms of its actions and exploring its health applications., Competing Interests: Conflict of Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

CRISPR-Cas9 is the most commonly used genome-editing tool in eukaryotic cells. To modulate Cas9 entry into the nucleus to enable control of genome editing, we constructed a light-controlled CRISPR-Cas9 system to control exposure of the Cas9 protein nuclear localization signal (NLS). Although blue-light irradiation was found to effectively control the entry of Cas9 protein into the nucleus with confocal microscopy observation, effective gene editing occurred in controls with next-generation sequencing analysis. To further clarify this phenomenon, a CRISPR-Cas9 editing system without the NLS and a CRISPR-Cas9 editing system containing a nuclear export signal were also constructed. Interestingly, both Cas9 proteins could achieve effective editing of target sites with significantly reduced off-target effects. Thus, we speculated that other factors might mediate Cas9 entry into the nucleus. However, NLS-free Cas9 was found to produce effective target gene editing even following inhibition of cell mitosis to prevent nuclear import caused by nuclear membrane disassembly. Furthermore, multiple nucleus-localized proteins were found to interact with Cas9, which could mediate the "hitchhiking" of NLS-free Cas9 into the nucleus. These findings will inform future attempts to construct controllable gene-editing systems and provide new insights into the evolution of the nucleus and compatible protein functions., Competing Interests: Declaration of interests W.Z. and H.S. are employees of Guangdong Pharmaceutical University. C.G., Y.J., Haozheng Wang, Z.L., Hui Wang, X.L., M.L., and Y.W. are graduate students of W.Z., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Fibroblasts are stromal cells ubiquitously distributed in the body of nearly every organ tissue. These cells were previously considered to be "passive cells", solely responsible for ensuring the turnover of the extracellular matrix (ECM). However, their versatility, including their ability to switch phenotypes in response to tissue injury and dynamic activity in the maintenance of tissue specific homeostasis and integrity have been recently revealed by the innovation of technological tools such as genetically modified mouse models and single cell analysis. These highly plastic and heterogeneous cells equipped with multifaceted functions including the regulation of angiogenesis, inflammation as well as their innate stemness characteristics, play a central role in the delicately regulated process of wound healing. Fibroblast dysregulation underlies many chronic conditions, including cardiovascular diseases, cancer, inflammatory diseases, and diabetes mellitus (DM), which represent the current major causes of morbidity and mortality worldwide. Diabetic foot ulcer (DFU), one of the most severe complications of DM affects 40 to 60 million people. Chronic non-healing DFU wounds expose patients to substantial sequelae including infections, gangrene, amputation, and death. A complete understanding of the pathophysiology of DFU and targeting pathways involved in the dysregulation of fibroblasts are required for the development of innovative new therapeutic treatments, critically needed for these patients.

Large-scale high-throughput transcriptome sequencing data holds significant value in biomedical research. However, practical challenges such as difficulty in sample acquisition often limit the availability of large sample sizes, leading to decreased reliability of the analysis results. In practice, generative deep learning models, such as Generative Adversarial Networks (GANs) and Diffusion Models (DMs), have been proven to generate realistic data and may be used to solve this promblem. In this study, we utilized bulk RNA-Seq gene expression data to construct different generative models with two data preprocessing methods: Min-Max-GAN, Z-Score-GAN, Min-Max-DM, and Z-Score-DM. We demonstrated that the generated data from the Min-Max-GAN model exhibited high similarity to real data, surpassing the performance of the other models significantly. Furthermore, we trained the models on the largest dataset available to date, achieving MMD (Maximum Mean Discrepancy) of 0.030 and 0.033 on the training and independent datasets, respectively. Through SHAP (SHapley Additive exPlanations) explanations of our generative model, we also enhanced our model's credibility. Finally, we applied the generated data to data augmentation and observed a significant improvement in the performance of classification models. In summary, this study establishes a GAN-based approach for generating bulk RNA-Seq gene expression data, which contributes to enhancing the performance and reliability of downstream tasks in high-throughput transcriptome analysis., Competing Interests: Declaration of competing interest The authors have declared that no competing interest exists., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system is a powerful genomic DNA editing tool. The increased applications of gene editing tools, including the CRISPR-Cas system, have contributed to recent advances in biological fields, such as genetic disease therapy, disease-associated gene screening and detection, and cancer therapy. However, the major limiting factor for the wide application of gene editing tools is gene editing efficiency. This review summarizes the recent advances in factors affecting the gene editing efficiency of the CRISPR-Cas9 system and the CRISPR-Cas9 system optimization strategies. The homology-directed repair efficiency-related signal pathways and the form and delivery method of the CRISPR-Cas9 system are the major factors that influence the repair efficiency of gene editing tools. Based on these influencing factors, several strategies have been developed to improve the repair efficiency of gene editing tools. This review provides novel insights for improving the repair efficiency of the CRISPR-Cas9 gene editing system, which may enable the development and improvement of gene editing tools.

Primary cancer modulates the bone microenvironment to sow the seeds of dormancy and metastasis in tumor cells, leading to multiple organ metastasis and death. In this study, 3D printing and bone-on-a-chip (BOC) are combined to develop a BOC platform that mimics the pre-metastatic niches (PMNs) and facilitates elucidation of the interactions between bone-resident cells and metastatic tumor cells under the influence of primary cancer. Photocrosslinkable gelatin methacrylate (GelMA) is used as a 3D culturing hydrogel to encapsulate cells, and circulate tumor culture medium (CM) adjacent to the hydrogel to verify the critical role of mesenchymal stem cells (MSCs) and osteoclasts (RAW264.7s). Three niches: the dormancy niche, the perivascular niche, and the "vicious cycle" niche, are devised to recapitulate bone metastasis in one chip with high cell viability and excellent nutrient exchange. With respect to tumor dormancy and reactivation, the invadopodia formation of A549 lung cancer cells in communication with MSCs and RAW264.7 via the cortactin pathway is researched. As a proof of concept, the functionality and practicality of the platform are demonstrated by analyzing the invadopodia formation and the influence of various cells, and the establishment of the dynamic niches paves the way to understanding PMN formation and related drug discovery., (© 2023 Wiley-VCH GmbH.)

Background: Chronic inflammation is considered the most critical predisposing factor of hepatocellular carcinoma (HCC), with inflammatory cell heterogeneity, hepatic fibrosis accumulation, and abnormal vascular proliferation as prominent features of the HCC tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) play a key role in HCC TME remodeling. Therefore, the level of abundance of CAFs may significantly affect the prognosis and outcome in HCC patients., Methods: Unsupervised clustering was performed based on 39 genes related to CAFs in HCC identified by single-cell RNA sequencing data. Patients of bulk RNA were grouped into CAF low abundance cluster and high abundance clusters. Subsequently, prognosis, immune infiltration landscape, metabolism, and treatment response between the two clusters were investigated and validated by immunohistochemistry., Results: Patients in the CAF high cluster had a higher level of inflammatory cell infiltration, a more significant immunosuppressive microenvironment, and a significantly worse prognosis than those in the low cluster. At the metabolic level, the CAF high cluster had lower levels of aerobic oxidation and higher angiogenic scores. Drug treatment response prediction indicated that the CAF high cluster could have a better response to PD-1 inhibitors and conventional chemotherapeutic agents for HCC such as anti-angiogenic drugs, whereas CAF low cluster may be more sensitive to transarterial chemoembolization treatment., Conclusions: This study not only revealed the TME characteristics of HCC with the difference in CAF abundance but also further confirmed that the combination therapy of PD-1 inhibitors and anti-angiogenic drugs may be more valuable for patients with high CAF abundance., (© 2023 American Cancer Society.)

In this paper, a novel bifunctional cellulase gene cel1 was cloned from Thermoascus aurantiacus by PCR and heterologously expressed in Pichia pastoris GS115. Bioinformatics and other related tools were used to compare the nucleotide homology of target genes, and analyze the signal peptide, transmembrane domain, hydrophilicity, secondary and tertiary structure of proteins. It was concluded that cel1 has similar endoglucanase nucleotide sequences and falls under the GH5 family. It was also found that cel1 has nucleotide sequences similar to glucosidase, which can infer that cel1 may have the properties of glucosidase, indicating that cel1 is multifunctional. At the same time, a part of the nucleotide sequence of the gene was removed to obtain a new gene cel2, and after highly efficient heterologous expression, its specific activity was found to be 2.1 times higher. Its enhancement is related to the exposure of the protein's hollow three-dimensional structure. This paper provides good material for exploring the relationship between the structure of bifunctional enzymes and their functions, which lays a solid foundation for further research and applications, and provides useful insight for gene mining of other novel enzymes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Hongwei Shao. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Osteosarcoma (OS) is a malignant bone tumor that threatens human health. Surgical removal of the tumor and followed by implantation with a graft is the golden standard for its clinical treatment. However, avoiding recurrence by enhancing the antitumor properties of the implants and improving osteogenesis around the implants remain a challenge. Here, we developed a layered double hydroxide (LDH)-coated magnesium (Mg) alloy and loaded it with celastrol. The celastrol-loaded Mg alloy exhibited enhanced corrosion resistance and sustained release of celastrol. In vitro cell culture suggested that the modified Mg alloy loaded with an appropriate amount of celastrol significantly inhibited the proliferation and migration of bone tumor cells while having little influence on normal cells. A mechanistic study revealed that the celastrol-loaded Mg alloy upregulated reactive oxygen species (ROS) generation in bone tumor cells, resulting in mitochondrial dysfunction due to reduced membrane potential, thereby inducing bone tumor cell apoptosis. Furthermore, it was found that celastrol-induced autophagy in tumor cells inhibited cell apoptosis in the initial 6 h. After ≥12 h of culture, inhibition of the PI3K-Akt-mTOR signaling pathway was noted, resulting in excessive autophagy in tumor cells, finally causing cell apoptosis. The celatsrol-loaded Mg alloy also exhibited effective antitumor properties in a subcutaneous tumor model. In vitro tartrate-resistant acid phosphatase (TRAP) staining and gene expression results revealed that the modified Mg alloy reduced the viability of osteoclasts, inducing a potential pathway for the increased bone regeneration around the modified Mg alloy seen in vivo . Together, the results of our study show that the celatsrol-loaded Mg alloy might be a promising implant for treating OS.

Organelle-targeted therapy guided by fluorescence imaging is promising for precise cancer treatment. However, most current organelle-targeted therapeutics can only destruct single organelles, which suffer from limited therapeutic efficacy. To address this challenge, a photoactivatable probe was developed for sequential photodynamic destruction of multiorganelles in cancer cells, including lysosomes, lipid droplets, and mitochondria. This photoactivatable probe not only exhibits efficient cancer cell eradication in vitro but also can suppress tumor growth in vivo . Simultaneously, the photoactivatable probe enables sequential destruction of multiple organelles in cancer cells, which can be observed in situ through the conversion of green-to-red fluorescence facilitated by a photooxidative dehydrogenation reaction. We believe this photoactivatable probe for sequential destruction of multiple organelles associated with fluorescence color conversion provides a new strategy for cancer treatment with greatly improved efficacy.

The aim of this study was to evaluate the association between Ferredoxin 1 (FDX1) expression and the prognostic survival of tumor patients and predict the efficacy of immunotherapy response to antitumor drug sensitivity. FDX1 plays an oncogenic role in thirty-three types of tumors, based on TCGA and GEO databases, and further experimental validation in vitro was provided through multiple cell lines. FDX1 was expressed highly in multiple types of cancer and differently linked to the survival prognosis of tumorous patients. A high phosphorylation level was correlated with the FDX1 site of S177 in lung cancer. FDX1 exhibited a significant association with infiltrated cancer-associated fibroblasts and CD8 + T cells. Moreover, FDX1 demonstrated correlations with immune and molecular subtypes, as well as functional enrichments in GO/KEGG pathways. Additionally, FDX1 displayed relationships with the tumor mutational burden (TMB), microsatellite instability (MSI), DNA methylation, and RNA and DNA synthesis (RNAss/DNAss) within the tumor microenvironment. Notably, FDX1 exhibited a strong connection with immune checkpoint genes in the co-expression network. The validity of these findings was further confirmed through Western blotting, RT-qPCR, and flow cytometry experiments conducted on WM115 and A375 tumor cells. Elevated FDX1 expression has been linked to the enhanced effectiveness of PD-L1 blockade immunotherapy in melanoma, as observed in the GSE22155 and GSE172320 cohorts. Autodocking simulations have suggested that FDX1 may influence drug resistance by affecting the binding sites of antitumor drugs. Collectively, these findings propose that FDX1 could serve as a novel and valuable biomarker and represent an immunotherapeutic target for augmenting immune responses in various human cancers when used in combination with immune checkpoint inhibitors.

The response to ischemia in peripheral artery disease (PAD) depends on compensatory neovascularization and coordination of tissue regeneration. Identifying novel mechanisms regulating these processes is critical to the development of nonsurgical treatments for PAD. E-selectin is an adhesion molecule that mediates cell recruitment during neovascularization. Therapeutic priming of ischemic limb tissues with intramuscular E-selectin gene therapy promotes angiogenesis and reduces tissue loss in a murine hindlimb gangrene model. In this study, we evaluated the effects of E-selectin gene therapy on skeletal muscle recovery, specifically focusing on exercise performance and myofiber regeneration. C57BL/6J mice were treated with intramuscular E-selectin/adeno-associated virus serotype 2/2 gene therapy (E-sel/AAV) or LacZ/AAV2/2 (LacZ/AAV) as control and then subjected to femoral artery coagulation. Recovery of hindlimb perfusion was assessed by laser Doppler perfusion imaging and muscle function by treadmill exhaustion and grip strength testing. After three postoperative weeks, hindlimb muscle was harvested for immunofluorescence analysis. At all postoperative time points, mice treated with E-sel/AAV had improved hindlimb perfusion and exercise capacity. E-sel/AAV gene therapy also increased the coexpression of MyoD and Ki-67 in skeletal muscle progenitors and the proportion of Myh7 + myofibers. Altogether, our findings demonstrate that in addition to improving reperfusion, intramuscular E-sel/AAV gene therapy enhances the regeneration of ischemic skeletal muscle with a corresponding benefit on exercise performance. These results suggest a potential role for E-sel/AAV gene therapy as a nonsurgical adjunct in patients with life-limiting PAD., Competing Interests: Z-JL and OCV along with the University of Miami hold intellectual property of the E-selectin/AAV gene therapy-based cell modification technologies and have been licensed to Ambulero, Inc., a new start-up company out of the University of Miami that focuses on developing new vascular treatments for ischemic conditions., (Copyright © 2023 Antoine J. Ribieras et al.)

Osteosarcoma is one of the most common malignant tumors in children and tends to occur around the knee. Problems such as recurrence and metastasis are the outcomes of traditional treatment methods. One of the reasons for these issues is the infiltration of tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). Photothermal immunotherapy has emerged as one of the most potent approaches for cancer treatment. In this study, we designed a biodegradable, injectable, and photothermal hydrogel that functions to reprogram TAMs into classically activated macrophages (M1) based on hydroxypropyl chitin (HPCH), tannic acid and ferric ions (HTA). We found that HTA had better photothermal efficiency than a pure hydrogel; its photothermal repeatability is good and it can be NIR (808 nm) irradiated as needed. In addition, the precooled hydrogel solution can be injected into the tumor and it can rapidly gel in situ . In vitro , HTA with NIR irradiation (HTA + NIR) induced the apoptosis of K7M2 cancer cells. In vivo , the local administration of HTA + NIR exerted photothermal killing of primary tumors and reprogramming of TAMs into M1-type macrophages in the TME. Therefore, the injectable photothermally active antitumor hydrogel has great potential for modulating the TME to treat bone tumors.

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based genome editing system exhibits marked potential for both gene editing and gene therapy, and its continuous improvement contributes to its great clinical potential. However, the largest hindrance to its application in clinical practice is the presence of off-target effects (OTEs). Thus, in addition to continuous optimization of the CRISPR system to reduce and eventually eliminate OTEs, further development of unbiased genome-wide detection of OTEs is key for its successful clinical application. This article summarizes detection strategies for OTEs of different CRISPR systems, to provide detailed guidance for the detection of OTEs in CRISPR-based genome editing.

Cancer therapies usually suffer from poor targeting ability and serious side effects. Photoactivatable cancer therapy has the significant advantage of a high spatiotemporal resolution, but most photoactivatable prodrugs require decoration with stoichiometric photocleavable groups, which are only responsive to ultraviolet irradiation and suffer from low reaction efficiency. To tackle these challenges, we herein propose a photoactivation strategy with biogenic riboflavin as the photosensitizer to promote the in situ transformation of noncytotoxic dihydroalkaloid prodrugs dihydrochelerythrine (DHCHE), dihydrosanguinarine (DHSAN), and dihydronitidine (DHNIT) into anticancer alkaloid drugs chelerythrine (CHE), sanguinarine (SAN), and nitidine (NIT), respectively, which can efficiently kill cancer cells and inhibit in vivo tumor growth. Meanwhile, the photoactivatable transformation can be in situ monitored by green-to-red fluorescence conversion, which will contribute to easy controlling of the therapeutic dose. The proposed photoactivatable transformation mechanism was also explored by density functional theory (DFT) calculations. We believe this riboflavin-promoted and imaging-guided photoactivation strategy is promising for precise cancer therapy.

T cells used in immune cell therapy, represented by T cell receptor therapy (TCR-T), are usually activated and proliferated in vitro and are induced to a terminally differentiated phenotype, with limited viability after transfusion back into the body. T cells exhibited a robust proliferative potential and in vivo viability in the early stages of progressive differentiation. In this study, we identified microRNAs that regulate T cell differentiation. After microRNA sequencing of the four subsets: Naïve T cells (T N ), stem cell-like memory T cells (T SCM ), central memory T cells (T CM ）, and effector memory T cells (T EM ), miR-150 was identified as the most highly expressed miRNA among the four subsets and was lowly expressed in the T SCM cells. We predicted the target genes of miR-150 miRNA and performed Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes analyses. We observed that the target genes of miR-150 were enriched in pathways associated with T-cell differentiation. FOXP1 and RC3H1 were identified as key target genes of miR-150 in the regulation of T-cell function. We examined the effects of miR-150 on the differentiation and function of healthy donor T-cells. We observed that miR-150 overexpression promoted T-cell differentiation to effector T-cells and effector memory T-cells, enhanced apoptosis, inhibited cell proliferation and increased secretion of pro-inflammatory cytokines such as IFN-γ and TNF-α. In addition, the expressions of early differentiation-related genes (ACTN1, CERS6, BCL2, and EOMES), advanced differentiation-related genes (KLRG1), and effector-function-related genes (PRF1 and GZMB) were significantly decreased after overexpression of miR-150. Collectively, our results suggested that miR-150 can promote progressive differentiation of T cells and the downmodulation of miR-150 expression while performing adoptive immunotherapy may inhibit T-cell differentiation and increase the proliferative potential of T cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Bone implants with the photothermal effect are promising for the treatment of bone tumor defects. Noble metal-based photothermal nanoagents are widely studied for their stable photothermal effect, but they are expensive and difficult to directly grow on implant surfaces. In contrast, non-noble metal photothermal nanoagents are economical but unstable. Herein, to develop a stable and economical photothermal film on bone implants, a Ni nanoparticle-doped oxide semiconductor film was grown in situ on Nitinol via the reduction of Ni-Ti-layered double hydroxides. Ni nanoparticles remained stable in the NiTiO 3 structure even when immersed in fluid for 1 month, and thus, the film presented a reliable photothermal effect under near-infrared light irradiation. The film also showed excellent in vitro and in vivo antitumor performance. Moreover, the nanostructure on the film allowed bone differentiation of mouse embryo cells (C3H10T1/2), and the released Ni ions supported the angiogenesis behavior of human vein endothelial cells. Bone implantation experiments further showed the enhancement of osteointegration of the modified Nitinol implant in vivo. This novel multifunctional Nitinol bone implant design offers a promising strategy for the therapy of bone tumor-related defects.

Objective: To assess the correlation between HMGB1 expression and the patient prognosis in a multicancer context., Methods: The potential oncogenic role of HMGB1 was explored in forty tumors through the TCGA, GEO, and Oncomine datasets. We analyzed the clinical prognostic value and antitumor immunotherapy of HMGB1 in a multicancer context using GEO (GSE111636)., Results: High expression of HMGB1 is present in multicancer cases, and its low expression is closely associated with the prognostic survival of patients, in terms of both overall and disease-free survival in ACC and LUAD. Further investigation revealed that the high expression of gastric and lung cancer is closely associated with low risk and better prognosis of patients based on COX and Kaplan-Meier analysis of OS, FP and PPS. HMGB1 expression was found to be significantly correlated with cancer-associated fibroblast and CD8 + T cell infiltration in the TME. The analysis of GO functional annotation/KEGG pathways indicates that HMGB1 may regulate tumor immunity-related pathways, such as the tumor immunotherapy response in colorectal cancer. The function of four genes as hubs are confirmed by in vitro HMGB1 knockdown which led to inhibition of cell proliferation and metastasis in SW620 and SW480 cells., Conclusion: HMGB1 is a potential novel biomarker for improving clinical prognosis and antitumor immunotherapy efficacy. CDK1, HMGB2, SSRP1, and H2AFV may serve as key nodes for HMGB1 in colorectal cancer.

Cytokine-induced killer (CIK) cells are a group of highly heterogeneous T cells expressing diverse T-cell antigen receptors (TCRs), but possessing wide major histocompatibility complex (MHC)-unrestricted antitumour activity, which have been used in the treatment of many tumours. However, the clonal diversity of different T-cell subsets and whether there is a certain clonal preference during CIK culture remains to be clarified. We found that the proportion of oligoclones seen in some subfamilies of freshly isolated T cells decreased in varying degrees after culturing. This indicates that the diversity of T-cell clones had been restored to a certain extent. The diversity differed among different T-cell subsets, as some TCR subfamily oligoclones mainly appeared in CD3 + CD56 + cells, which also indicates the heterogeneity of the CIK cell composition., (© 2022 Federation of European Biochemical Societies.)

Licorice has previously been shown to affect gene expression in cells; however, the underlying mechanisms remain to be clarified. We analyzed the microRNA expression profile of serum from mice treated by gavage with licorice decoction, and obtained 11 differentially expressed microRNAs (DEmiRNAs). We also screened differentially expressed genes (DEgenes) based on RNA-Seq data, and 271 common genes were identified by intersection analysis of the predicted target genes of 11 DEmiRNAs and the DEgenes. The miRNA-gene network showed that most of the hub genes were immune-related. KEGG enrichment analysis of the 271 genes identified three significant pathways, and the 21 genes involved in these three pathways, and the 11 DEmiRNAs, were constructed into a miRNA pathway-target gene network, in which mmu-miR-27a-3p stood out. Compared to ImmPort, there were 13 immune genes within the above group of 21 genes, and three intersected with the mmu-miR-27a-3p predicted target genes, Cd28 , Grap2 and Cxcl12 , of which the expression of Cd28 changed most significantly. We confirmed the regulation of Cd28 by mmu-miR-27a-3p using a dual-luciferase assay, and further confirmed that overexpression of mmu-miR-27a-3p could significantly downregulate the expression of Cd28 in lymphocytes. These results indicate that mmu-miR-27a-3p could be involved in the licorice-mediated regulation of the expression of Cd28 in mice.

For patients with chronic limb-threatening ischemia and limited revascularization options, alternate means for therapeutic angiogenesis and limb salvage are needed. E-selectin is a cell adhesion molecule that is critical for inflammation and neovascularization in areas of wound healing and ischemia. Here, we tested the efficacy of modifying ischemic limb tissue by intramuscular administration of E-selectin/AAV2/2 (adeno-associated virus serotype 2/2) to modulate angiogenic and inflammatory responses in a murine hindlimb gangrene model. Limb appearance, reperfusion, and functional recovery were assessed for 3 weeks after induction of ischemia. Mice receiving E-selectin/AAV2/2 gene therapy had reduced gangrene severity, increased limb and footpad perfusion, enhanced recruitment of endothelial progenitor cells, and improved performance on treadmill testing compared to control group. Histologically, E-selectin/AAV2/2 gene therapy was associated with increased vascularity and preserved myofiber integrity. E-selectin/AAV2/2 gene therapy also upregulated a panel of pro-angiogenic genes yet downregulated another group of genes associated with the inflammatory response. This novel gene therapy did not induce adverse effects on coagulability, or hematologic, hepatic, and renal function. Our findings highlight the potential of E-selectin/AAV2/2 gene therapy for improving limb perfusion and function in patients with chronic limb-threatening ischemia., Competing Interests: Z-JL and OV along with the University of Miami hold intellectual property of the AAV engineered E-selectin vector and have been licensed to Ambulero, Inc. 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 Ribieras, Ortiz, Li, Huerta, Le, Shao, Vazquez-Padron, Liu and Velazquez.)

The inappropriate regenerated fibrous cartilage and subchondral bone of the injured chondral defect ultimately cause degeneration of the regenerated cartilage, which eventually leads to the failure of cartilage repair. In this study, we developed a macrophage-modulated and injectable 'building block' drug delivery system comprised of porous chitosan (CS) microspheres and hydroxypropyl chitin (HPCH) hydrogel, where the dimethyloxallyl glycine (DMOG) was encapsulated in the thermosensitive HPCH hydrogel (HD) while kartogenin (KGN) was conjugated on the porous CS microspheres (CSK-PMS). The developed HD/CSK-PMS composite scaffold effectively modulated the microenvironment at the defect site, achieved local macrophage M2 polarization and promoted cartilage regeneration. The fast-degradable HD favored hyaline cartilage regeneration, while the highly stable CSK-PMS supported the endochondral ossification and regenerated the subchondral bone. In vitro and in vivo evaluations revealed that the newly developed HD/CSK-PMS as a controlled drug delivery system could effectively create M2 macrophage microenvironment and orchestrate osteochondral (OC) regeneration. These findings indicate the importance of the immune microenvironment and subchondral bone for high-quality cartilage repair, and thus the immunomodulation-based hydrogel/PMS composite system could be a promising candidate for OC regeneration., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Effector T cells, which are abundant but are short-lived after reinfusion into the body, are generally used for T-cell therapy, and antitumor immunity is typically not maintained over the long term. Genetic modification by early differentiated T cells and reinfusion has been shown to enhance antitumor immunity in vivo. This study overexpressed the characteristic transcription factors of differentiated early T cells by transfecting effector T cells with transcription factor recombinant lentivirus (S6 group: BCL6, EOMES, FOXP1, LEF1, TCF7, KLF7; S1 group: BCL6, EOMES, FOXP1, KLF7; S3 group: BCL6, EOMES, FOXP1, LEF1) to induce a sufficient number of effector T cells to dedifferentiate and optimize the transcription factor system. The results revealed that overexpression of early characteristic transcription factors in effector T cells upregulated the expression of early T cell differentiation markers (CCR7 and CD62L), with the S1 group having the highest expression level, while the rising trend of late differentiation marker (CD45RO) expression was suppressed. Moreover, the expression of early differentiation-related genes (ACTN1, CERS6, BCL2) was significantly increased, while the expression of late differentiation-related genes (KLRG-1) and effector function-related genes (GNLY, GZMB, PRF1) was significantly decreased; this difference in expression was more significant in the S1 group than in the other two experimental groups. The antiapoptotic ability of each experimental group was significantly enhanced, while the secretion ability of TNF-α and IFN-γ was weakened, with the effector cytokine secretion ability of the S1 group being the weakest. Transcriptomic analysis showed that the gene expression profile of each experimental group was significantly different from that of the control group, with differences in the gene expression pattern and number of differentially expressed genes in the S1 group compared with the other two experimental groups. The differentially expressed gene enrichment pathways were basically related to the cell cycle, cell division, and immune function. In conclusion, overexpression of early characteristic transcription factors in effector T cells induces their dedifferentiation, and induction of dedifferentiation by the S1 group may be more effective., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study aimed to evaluate the effect of supplementation with inulin-type fructans (ITFs) on the intestinal immune function in the context of dysbiosis resulting from antibiotic cocktail (ABx) treatment. BALB/c mice (8-9 weeks of age) were treated with an ABx for 3 weeks and then allowed to recover spontaneously or with ITF supplementation (5%) for 4 weeks. Our results showed that ABx treatment can induce gut microbiota dysbiosis and intestinal inflammation in mice. After 4 weeks of recovery, ITF supplementation restored the composition of the intestinal microbial community. However, compared with spontaneous recovery, ITF supplementation delayed inflammation recovery in the intestine and upregulated diamine oxidase (DAO) activity and increased lipopolysaccharide (LPS) content in serum. In addition, ITF supplementation delayed the regulatory T (Treg) cell and B cell recovery in the lamina propria (LP). Furthermore, compared with spontaneous recovery, ITF supplementation inhibited the relative expression of certain proinflammatory genes, such as for inducible nitric oxide synthase (iNOS) and tumour necrosis factor α (Tnf-α), in the colon, but it reduced the secretion of the anti-inflammatory mediator transforming growth factor β1 (TGF-β1) in serum, reduced the secretion of secretory immunoglobulin A (SIgA) in the colon and promoted the secretion of the proinflammatory cytokine interleukin (IL)-17A. In conclusion, these data supported the hypothesis that the influence of ITFs on the host's intestinal status is not always beneficial in the context of ABx-induced biological disorder. However, the significance of these findings needs to be determined by advanced studies KEY POINTS: • ITFs did not promote the recovery of microbial community composition. • ITFs delayed the recovery of ABx-induced colonic inflammation. • ITFs reduced the secretion of TGF-β1 and SIgA. • ITFs delayed the recovery of Treg and B cells in the LP., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

One of the major challenges for Ti-based implants is insufficient osteointegration, which might result in the loosening of the implant. In this study, we fabricated strontium (Sr)-containing barium titanate (BST) on the surface of Ti to improve the bioactivity for osteointegration enhancement. The introduction of Sr significantly reduced the crystallization time and improved crystallinity, which was proved by X-ray diffraction, X-ray photoelectron spectroscopy, and transmission electron microscopy. Compared with Ti, the BST film showed greater wettability surface and lower elastic modulus and hardness. Furthermore, in synergy with the release of Sr ions, the BST film improved early adhesion and followed osteogenic differentiation of rat bone mesenchymal stem cells. Furthermore, the bone implantation experiment suggested that the BST film could significantly improve the in vivo osteogenesis and osteointegration capabilities of Ti implants. In summary, this study revealed that BST-modified Ti has potential application in bone repair.

CCL17 is an important chemokine that plays a vital immunomodulatory role in the tumor microenvironment (TME). Analysis of lung adenocarcinoma (LUAD) data in Kaplan-Meier plotter databases found that the overall survival of patients in the CCL17 high-expression group was higher than that of the low-expression group, especially for patients with early (stages I and II) LUAD, which has a more positive prognostic value. Expression of CCL17 in LUAD was positively correlated with the proportion of tumor-infiltrating lymphocytes, immunostimulators, and major histocompatibility complexes using the TISIDB databases. Based on the RNA-seq and clinical data of 491 LUAD patients obtained from the TCGA database, 1,455 differential genes were found between the CCL17 high- and low-expression groups. Using WGCNA analysis confirmed that the expression of differential genes in the blue module is negatively correlated with poor survival and clinical stages of LUAD patients, and CCL17 and CCR4 genes belong to the hub genes in the blue module. Further analysis by the ESTIMATE and CIBERSORT algorithm found that the naive B cells and CD8 + T cells in the CCL17 high-expression group have a higher distribution ratio in the early LUAD patients, and the high immune score has a positive relationship with the overall survival rate. Using somatic mutation data of TCGA-LUAD, we found that 1) the tumor mutation burden values of the CCL17 high-expression group were significantly lower than those of the CCL17 low-expression group and 2) the expression levels of CCL17 and the tumor mutation burden values were negatively correlated. Transwell chemotaxis and cytotoxicity assays confirmed that CCL17 contributes to the migration of CCR4-positive lymphocytes into the H1993 LUAD TME and enhances the specific lysis of LUAD cells. In summary, high expression of CCL17 in the LUAD TME promotes local immune cell infiltration and antitumor immune response, which may contribute to the better survival and prognosis of patients with early LUAD., Competing Interests: The 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 Ye, Zhang, Dong, Liu, Zhang, Wu, Bo, Shao, Zhang and Shen.)

Aims: Novel cell-based therapeutic angiogenic treatments for patients with critical limb ischemia may afford limb salvage. Mesenchymal stem cells (MSCs) do not overexpress E-selectin; however, we have previously demonstrated the cell-adhesion molecule's vital role in angiogenesis and wound healing. Thus, we created a viral vector to overexpress E-selectin on MSCs to increase their therapeutic profile., Methods and Results: Femoral artery ligation induced hind limb ischemia in mice and intramuscular injections were administered of vehicle or syngeneic donor MSCs, transduced ex vivo with an adeno-associated viral vector to express either GFP + (MSC GFP ) or E-selectin-GFP + (MSC E-selectin-GFP ). Laser Doppler Imaging demonstrated significantly restored reperfusion in MSC E-selectin-GFP -treated mice vs. controls. After 3 weeks, the ischemic limbs in mice treated with MSC E-selectin-GFP had increased footpad blood vessel density, hematoxylin and eosin stain (H&E) ischemic calf muscle sections revealed mitigated muscular atrophy with restored muscle fiber size, and mice were able to run further before exhaustion. PCR array-based gene profiling analysis identified nine upregulated pro-angiogenic/pro-repair genes and downregulated Tumor necrosis factor (TNF) gene in MSC E-selectin-GFP -treated limb tissues, indicating that the therapeutic effect is likely achieved via upregulation of pro-angiogenic cytokines and downregulation of inflammation., Conclusion: This innovative cell therapy confers increased limb reperfusion, neovascularization, improved functional recovery, decreased muscle atrophy, and thus offers a potential therapeutic method for future clinical studies., Competing Interests: Z-JL and OV along with the University of Miami hold intellectual property of the AAV engineered E-selectin vector and have been licensed to Ambulero, Inc. 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 Quiroz, Valencia, Shao, Li, Ortiz, Parikh, Lassance-Soares, Vazquez-Padron, Liu and Velazquez.)

Most of the malignant melanomas are already in the middle and advanced stages when they are diagnosed, which is often accompanied by the metastasis and spread of other organs. Besides, the prognosis of patients is bleak. The characteristics of the local immune microenvironment in metastatic melanoma have important implications for both tumor progression and tumor treatment. In this study, data on patients with metastatic melanoma from the TCGA and GEO datasets were selected for immune, stromal, and estimate scores, and overlapping differentially expressed genes were screened. A nine-IRGs prognostic model (ALOX5AP, ARHGAP15, CCL8, FCER1G, GBP4, HCK, MMP9, RARRES2 and TRIM22) was established by univariate COX regression, LASSO and multivariate COX regression. Receiver operating characteristic curves were used to test the predictive accuracy of the model. Immune infiltration was analyzed by using CIBERSORT and Xcell in high-risk and low-risk groups. The immune infiltration of the high-risk group was significantly lower than that of the low-risk group. Immune checkpoint analysis revealed that the expression of PDCD1, CTLA4, TIGIT, CD274, HAVR2 and LAG3 demonstrated the visible difference in groups with different levels of risk scores. WGCNA analysis found that the yellow-green module contained seven genes from the nine-IRG prognostic model, and the yellow-green module had the highest correlation with risk scores. The results of GO and KEGG suggested that the genes in the yellow-green module were mainly enriched in immune-related biological processes. Finally, the expression characteristics of ALOX5AP, ARHGAP15, CCL8, FCER1G, GBP4, HCK, MMP9, RARRES2 and TRIM22 were analyzed between metastatic melanoma and normal samples. Overall, a prognostic model for metastatic melanoma based on the tumor immune microenvironment characteristics was established, which left plenty of space for further studies. It could function well in helping people to understand characteristics of the immune microenvironment in metastatic melanoma.

Primary malignant bone tumors can be life-threatening. Surgical resection of tumor plus chemotherapy is the standard clinical treatment. However, postoperative recovery is hindered due to tumor recurrence caused by residual tumor cells and bone defect caused by resection of tumor tissue. Herein, a multifunctional mussel-inspired film was fabricated on Mg alloy, that is, an inner hydrothermal-treated layer, a middle layer of polydopamine, and an outer layer of doxorubicin. The modified Mg alloy showed excellent photothermal effect and thermal/pH-controlled release of doxorubicin. The synergistic effect of chemotherapy and photothermal therapy enabled the modified Mg alloy to kill bone tumor in vitro and inhibit tumor growth in nude mice. Moreover, because of the controlled release of Mg ions and biocompatibility of polydopamine, the modified Mg alloy supported extracellular matrix mineralization, alkaline phosphatase activity, and bone-related gene expression in C3H10T1/2. Bone implantation model in rats verified that the modified Mg showed excellent osteointegration. These findings prove that the use of mussel-inspired multifunction film on Mg alloy offers a promising strategy for the therapy of primary malignant bone tumor., (© The Author(s) 2021. Published by Oxford University Press.)

Cancer-associated fibroblasts (CAFs) play a crucial role in cancer progression, drug resistance and tumor recurrence. We have recently shown that the Notch pathway determines the tumor-regulatory role of experimentally created 'CAFs'. Here, we examined the status of Notch signaling in human melanoma-associated fibroblasts (MAFs) versus their normal counterparts and tested whether manipulation of the Notch pathway activity in MAFs alters their tumor-regulatory function. Using tissue microarrays, we found that MAFs exhibit decreased Notch pathway activity compared with normal fibroblasts in adjacent and non-adjacent skin. Consistently, MAFs isolated from human metastatic melanoma exhibited lower Notch activity than did normal human fibroblasts, demonstrating that Notch pathway activity is low in MAFs. We then investigated the effect of increasing Notch pathway activity in MAF on melanoma growth in co-cultures and in a mouse co-graft model. We found that activation of the Notch pathway in MAFs significantly restricted melanoma cell growth in vitro and suppressed melanoma skin growth and tumor angiogenesis in vivo. Our study demonstrates that the Notch signaling is inhibited in MAFs. Increase of Notch pathway activity can confer tumor-suppressive function on MAFs. Thus, targeting melanoma by activating Notch signaling in MAF may represent a novel therapeutic approach., Competing Interests: The authors declare no conflict of interest.

Titanium (Ti) has been the most widely used orthopedic implant in the past decades. However, their inert surface often leads to insufficient osteointegration of Ti implant. To solve this issue, two bioactive Mg(OH) 2 films were developed on Ti surfaces using hydrothermal treatment (Ti-M1# and Ti-M2#). The Mg(OH) 2 films showed nano-flake structures: sheets on Ti-M1# with a thickness of 14.7 ± 0.7 nm and a length of 131.5 ± 2.9 nm, and on Ti-M2# with a thickness of 13.4 ± 2.2 nm and a length of 56.9 ± 5.6 nm. Both films worked as Mg ions releasing platforms. With the gradual degradation of Mg(OH) 2 films, weakly alkaline microenvironments will be established surrounding the modified implants. Benefiting from the sustained release of Mg ions, nanostructures, and weakly alkaline microenvironments, the as-prepared nano-Mg(OH) 2 coated Ti showed better in vitro and in vivo osteogenesis. Notably, Ti-M2# showed better osteogenesis than Ti-M1#, which can be ascribed to its smaller nanostructure. Moreover, whole genome expression analysis was applied to study the osteogenic mechanism of nano-Mg(OH) 2 films. For both coated samples, most of the genes related to ECM-receptor interaction, focal adhesion, and TGF-β pathways were upregulated, indicating that these signaling pathways were activated, leading to better osteogenesis. Furthermore, cells cultured on Ti-M2# showed markedly upregulated BMP-4 gene expression, suggesting that the nanostructure with Mg ion release ability can better activate BMP-4 related signaling pathways, resulting in better osteogenesis. Nano-Mg(OH) 2 films demonstrated a superior osteogenesis and are promising surface modification strategy for orthopedic applications., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.)

As a new generation of treatment, tumor immunotherapy targeting tumor-associated antigens (TAA) has attracted widespread attention. The survivin antigen belongs to TAA. It is a key inhibitor of apoptosis and a key regulator of cell cycle progression; furthermore, it may be a candidate target for tumor therapy. In addition, studies have confirmed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and CCL17 significantly affect local anti-tumor immunity in the tumor microenvironment. The mouse survivin gene was screened by BIMAS and SYFPEITHI to obtain the highest scored mouse survivin epitope peptide, which was synthesized into a peptide vaccine to immunize normal mice. Subsequently, spleen lymphocytes were isolated to induce survivin-specific cytotoxic T lymphocytes (CTL). Next, genetic engineering was used to construct the B16F10 cell line that stably expressed CCL17 and GM-CSF genes. A mouse melanoma model was used to observe the effects of the combination of the three on tumor volume and tumor weight. In-vitro survivin-specific CTL combined with CCL17 gene had a stronger inhibitory effect on B16F10 cells, while combined GM-CSF gene did not enhance the inhibitory effect of CTL on B16F10 cells. In-vivo experiments demonstrated that survivin-specific CTL combined with GM-CSF and CCL17 genes can inhibit the growth of mouse melanoma. HE staining and immunohistochemistry showed that the tumor had more necrotic cells and more infiltrating lymphocytes. The results showed that survivin-specific CTL combined with CCL17 and GM-CSF genes could inhibit tumor growth better., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: Novel therapeutic angiogenic concepts for critical limb ischemia are still needed for limb salvage. E-selectin, a cell-adhesion molecule, is vital for recruitment of the stem/progenitor cells necessary for neovascularization in ischemic tissues. We hypothesized that priming ischemic limb tissue with E-selectin/adeno-associated virus (AAV) gene therapy, in a murine hindlimb ischemia and gangrene model, would increase therapeutic angiogenesis and improve gangrene., Methods: FVB/NJ mice were given intramuscular hindlimb injections of either E-selectin/AAV or LacZ/AAV and then underwent induction of gangrene via femoral artery ligation and concomitant systemic injections of the nitric oxide synthesis inhibitor L-NAME (L-N G -Nitro arginine methyl ester; 40 mg/kg). Gangrene was evaluated via the Faber hindlimb appearance score. The rate of ischemic limb reperfusion and ischemic tissue angiogenesis were evaluated using laser Doppler perfusion imaging and DiI perfusion with confocal laser scanning microscopy of the ischemic footpads, respectively. The treadmill exhaustion test was performed on postoperative day (POD) 8 to determine hindlimb functionality., Results: The E-selectin/AAV-treated mice (n = 10) had decreased Faber ischemia scores compared with those of the LacZ/AAV-treated mice (n = 7) at both PODs 7 and 14 ( P  < .05 and P  < .01, respectively), improved laser Doppler perfusion imaging reperfusion indexes by POD 14 ( P  < .01), and greater gangrene footpad capillary density ( P  < .001). E-selectin/AAV-treated mice also had improved exercise tolerance ( P  < .05) and lower relative muscular atrophy ( P  < .01)., Conclusion: We surmised that E-selectin/AAV gene therapy would significantly promote hindlimb angiogenesis, reperfusion, and limb functionality in mice with hindlimb ischemia and gangrene. Our findings highlight the reported novel gene therapy approach to critical limb ischemia as a potential therapeutic option for future clinical studies., (© 2020 Published by Elsevier Inc. on behalf of the Society for Vascular Surgery.)

Fibroblasts play a pivotal role in wound healing. However, the molecular mechanisms determining the reparative response of fibroblasts remain unknown. Here, we identify Notch1 signaling as a molecular determinant controlling the plasticity and function of fibroblasts in modulating wound healing and angiogenesis. The Notch pathway is activated in fibroblasts of diabetic wounds but not in normal skin and non-diabetic wounds. Consistently, wound healing in the FSP-1 +/- ;ROSA LSL-N1IC+/+ mouse, in which Notch1 is activated in fibroblasts, is delayed. Increased Notch1 activity in fibroblasts suppressed their growth, migration, and differentiation into myofibroblasts. Accordingly, significantly fewer myofibroblasts and less collagen were present in granulation tissues of the FSP-1 +/- ;ROSA LSL-N1IC +/+ mice, demonstrating that high Notch1 activity inhibits fibroblast differentiation. High Notch1 activity in fibroblasts diminished their role in modulating the angiogenic response. We also identified that IL-6 is a functional Notch1 target and involved in regulating angiogenesis. These findings suggest that Notch1 signaling determines the plasticity and function of fibroblasts in wound healing and angiogenesis, unveiling intracellular Notch1 signaling in fibroblasts as potential target for therapeutic intervention in diabetic wound healing., (© 2020 Shao et al.)

Mussel inspired polydopamine (PDA) coatings have attracted a great deal of attention for their superior osteogenic property. Furthermore, recent investigations have demonstrated that vessel formation is crucial to bone regeneration. Hence, in the present study, the potential ability of polydopamine coatings with different oxidation degrees were systematically investigated in vitro to improve the angiogenic behavior of human umbilical vein endothelial cells (HUVECs). PDA was first coated on titanium (PDA-1#), and then oxidized by thermal treatment at 150 (PDA-2#) and 300 °C (PDA-3#), respectively. X-ray photoelectron spectroscopy (XPS) results revealed that phenolic hydroxyl (C-OH) and primary amino group (-NH 2 ) on PDA coatings deceased after oxidation, while quinone (C=O) increased. In vitro cell culture experiments suggested that PDA-2# sample was most beneficial for the adhesion, migration, and proliferation of HUVECs. Furthermore, HUVECs cultured on PDA-2# sample also exhibited best tube formation, CD31 expression, vascular endothelial growth factor (VEGF) secretion, as well as angiogenic-associated gene expression abilities. Our study suggests that moderate oxidation of PDA coating with balanced quinone and amino group has excellent potential to enhance bone vascularization, and is thus promising for clinical application in orthopedic implants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The in vivo proliferation and viability of transfused engineered T cells markedly limits the long-term effect of adoptive cell therapy on tumors. The therapeutic efficacy and proliferative potential of T cells are reported to be dependent on the differentiation status of T cells. The T cells at the early stage of progressive differentiation have a long lifespan, strong proliferative potential, and the ability to reconstruct intact T cell subsets. Thus, they are more suitable for adoptive immunotherapy. Previously, it was difficult to obtain a sufficient number of early differentiated T cells by inhibiting the progressive differentiation of T cells or by two-step programming. A more effective strategy is to directly reprogram and dedifferentiate the easily available terminal effector T (T EFF ) cells, which are generated in large numbers, into early T cells. This study attempted to overexpress eight (candidate) early differentiation-specific transcription factors (TFs) (LEF1, KLF7, ID3, EOMES, BCL6, TCF7, FOXP1, and FOXO1) in the T EFF cells, which were activated by in vitro stimulation, to promote dedifferentiation into early T cells. In the mature T EFF cells simultaneously overexpressing these specific TFs, the expression pattern of T cell differentiation markers (CCR7 and CD45RO) exhibited a tendency to change to the pattern observed during early differentiation. The transcriptome analysis revealed that the function of differentially expressed genes was mainly concentrated in the cell cycle, growth and development, and effector function. Moreover, many genes related to early differentiated T cells (such as BCL2 and PIM1) were significantly upregulated, while those related to the effector function of T EFF cells were significantly downregulated (such as GZMB, PRF1, and GNLY). Additionally, the T EFF cells overexpressing characteristic TFs exhibited enhanced anti-apoptotic capabilities and decreased secretion of cytokines (IFN-γ and TNF-α). Based on these results, we believe that the T EFF cells were reprogrammed into a less differentiated state after overexpression of the eight specific TFs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The disruption of the gut microbiota by treatment with an antibiotic cocktail (ABx) can trigger an imbalance in immune homeostasis. However, whether the changes in the intestinal microbiota always correspond to the changes in the physiology and immune homeostasis of the host remains unclear. Here, we analyzed the effects of ABx on immune homeostasis by analyzing the colonic transcriptome with 16S rRNA analysis of the gut microbiota on the 7th and 21st days of continuous treatment with ABx. Our results showed that the composition profile of the gut microbiota was similar after 7 and 21 days of ABx treatment. However, after 21 days of ABx treatment, the intestinal inflammation did not deteriorate further. Instead, the inflammation of the host was relieved, and half of the differentially expressed genes in the colon were restored compared with the 7 days of ABx treatment. Furthermore, the enrichment and network analysis of these restored genes indicated that expression of regenerating islet-derived protein 3β (Reg3b) and expression of regenerating islet-derived protein 3γ (Reg3g), especially Reg3b, may participate in the regulation of the inflammatory response and affect the changes in host immune homeostasis during continuous ABx treatment. Finally, Spearman's correlation analysis showed that the expression of Reg3b is correlated with the growth of Escherichia-Shigella. Our data demonstrated that even though the disruption of the gut microbiota profile induced by ABx treatment is similar, the host response and immune status will be different at different times.Key Points• Host immune status can change in different ABx treatment times.• Gut microbiota showed same exhaustion state in different ABx treatment times.• Host tried to revert to a certain extent after long-term ABx treatment.• Reg3b may affect the changes in host immune homeostasis during continuous ABx treatment.• The expression of Reg3b correlated with the growth of Escherichia-Shigella.

Alternative splicing produces multiple mRNA variants of TP53 which have diverse biologic functions. In this study, we identified a novel splice variant of TP53 lacking a 200 nt portion of exon 4 (p53ΔE4p) from a human leukemia T cell line. No protein product of p53ΔE4p was identifiable by western blot; however, forced expression of the variant in HEK-293T cells expressing wild-type p53 could inhibit cell proliferation and promote cell death. Interestingly, this novel variant also significantly enhances the expression of reporter genes. Moreover, transcriptome analysis showed that genes related to DNA binding and regulation of transcription by RNA polymerase II function were significantly upregulated following p53ΔE4p transfection, suggesting a role for this variant in the regulation of gene expression., Competing Interests: None., (IJCEP Copyright © 2020.)

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

Cancer stem cells (CSCs) play critical roles in cancer initiation, metastasis, recurrence, and drug resistance. Recent studies have revealed involvement of cancer-associated fibroblasts (CAFs) in regulating CSCs. However, the intracellular molecular mechanisms that determine the regulatory role of CAFs in modulating the plasticity of CSCs remain unknown. Here, we uncovered that intracellular Notch1 signaling in CAFs serves as a molecular switch, which modulates tumor heterogeneity and aggressiveness by inversely controlling stromal regulation of the plasticity and stemness of CSCs. Using mesenchymal stem cell-derived fibroblasts (MSC-DF) harboring reciprocal loss-of-function and gain-of-function Notch1 signaling, we found that MSC-DF Notch1-/- prompted cocultured melanoma cells to form more spheroids and acquire the phenotype (CD271 + and Nestin + ) of melanoma stem/initiating cells (MICs), whereas MSC-DF N1IC+/+ suppressed melanoma cell sphere formation and mitigated properties of MICs. MSC-DF Notch1-/- increased stemness of CD271 + MIC, which resultantly exhibited stronger aggressiveness in vitro and in vivo, by upregulating Sox2/Oct4/Nanog expression. Consistently, when cografted with melanoma cells into NOD scid gamma (NSG) mice, MSC-DF Notch1-/- increased, but MSC-DF N1IC+/+ decreased, the amounts of CD271 + MIC in melanoma tissue. The amounts of CD271 + MIC regulated by MSC-DF carrying high or low Notch1 pathway activity is well correlated with capability of melanoma metastasis, supporting that melanoma metastasis is MIC-mediated. Our data demonstrate that intracellular Notch1 signaling in CAFs is a molecular switch dictating the plasticity and stemness of MICs, thereby regulating melanoma aggressiveness, and therefore that targeting the intracellular Notch1 signaling pathway in CAFs may present a new therapeutic strategy for melanoma. Stem Cells 2019;37:865-875., (©AlphaMed Press 2019.)

Various pathogenic species are capable of penetrating plant leaves through stomata on the leaf surface for propagation by absorbing nutrients in plant interiors. Plants have evolved abilities to close stomata to restrict pathogen infections. The model plant Arabidopsis ( Arabidopsis thaliana ) closes stomata when FLAGELLIN SENSING2 (FLS2), a receptor protein localized in the plasma membrane (PM) of stomatal guard cells, detects flagellin, a pathogen-associated molecular pattern (PAMP) derived from the bacterial pathogen Pseudomonas syringae . It currently remains largely unknown how flagellin-FLS2 signaling initiates stomatal closure. Our previous studies showed that PAMP-INDUCED PEPTIDE1 (PIP1), an Arabidopsis endogenous peptide, activates immune responses through a PM-localized receptor, RECEPTOR-LIKE KINASE7 (RLK7). Here, we demonstrate that PIP1-RLK7 act downstream of FLS2 to activate stomatal immunity against the bacterial strain Pseudomonas syringe pv. tomato ( Pst ) DC3118. PIP1 promotes the expression of genes involved in salicylic acid (SA) biosynthesis. SA contributes to the expression of PIP1 preligand prePIP1 and the PIP1-induced stomatal closure. In contrast, methl jasmonate (MJ) and a pathogen-derived jasmonate mimic coronatine (COR) performs an opposite function of SA. SA also promotes the PIP1-induced production of reactive oxygen species (ROS) which is required for PIP1-induced stomatal closure. Overall, PIP1 and SA may form a positive feedback loop to regulate ROS-mediated stomatal immunity in Arabidopsis .

Background: Poor wound healing in critical limb ischemia (CLI) is attributed to impaired neovascularization and reperfusion. Optimizing the ischemic wound with adhesion molecules that enhance stem cell homing may revolutionize treatment. The purpose of this study is to test the efficacy of adhesion molecule E-selectin on wound healing in an ischemic mouse wound., Methods: Adult FVB/NJ mice underwent unilateral femoral artery and vein ligation to induce CLI. A 4-mm punch biopsy wound was created on the anterior thigh to simulate ischemic wounds. Intramuscular injection of adeno-associated virus (AAV) carrying either E-selectin (E-selectin/AAV, n = 11) or LacZ as control (LacZ/AAV, n = 10) was performed. Gross wound size was measured for 10 d postoperatively. Ischemic hindlimb reperfusion was quantified using laser Doppler imaging. Wound tissue neovascularization was visualized using DiI perfusion and confocal microscopy. E-selectin expression in wounds was verified by immunofluorescence., Results: Immunofluorescence confirmed E-selectin/AAV delivery in treatment versus control limbs. Wounds from E-selectin/AAV mice versus controls revealed surface area healing of 54% versus 20% (P < 0.01) on postoperative day (POD) 1, 78% versus 51% on POD 4 (P < 0.01), and 97% versus 84% on POD 10 (P < 0.01). Laser Doppler imaging revealed greater reperfusion in E-selectin/AAV mice versus controls by POD 10 (0.49 versus 0.27, P < 0.05). DiI perfused ligated hindlimb in E-selectin/AAV versus control mice revealed mean neovascularization intensity score of 30 versus 18 (P < 0.05) on POD 10., Conclusions: Intramuscularly injected E-selectin/AAV gene therapy in mice with CLI significantly increases wound angiogenesis and limb reperfusion, expediting overall wound healing., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cluster of differentiation (CD)147 is highly expressed in drug-resistant tumor cell lines and is involved in the formation of tumor drug resistance. Therefore, immunotherapy utilizing CD147 epitope peptides is a promising approach for the elimination of drug-resistant tumor cells. However, like most tumor-associated antigens (TAAs), CD147 belongs to the autoantigen category, and T cells that recognize high affinity, immunodominant epitopes from autoantigens are deleted though thymic negative selection. Furthermore, wild-type autoantigen peptides cannot effectively activate and expand T lymphocytes with lower affinity T cell receptors in vivo . However, mutations of TAA peptides have been demonstrated to increase the affinity of major histocompatibility complex molecules and their binding to T cell receptor molecules, leading to activation of T lymphocytes in vitro . In the present study, a high-affinity point mutation peptide, CD147 126-134 L2, was predicted by the human leukocyte antigen (HLA) binding prediction algorithm and its affinity was testified using a T2 binding assay. In addition, when peptide-specific cytotoxic T lymphocytes (CTLs) were stimulated with dendritic cells loaded with the CD147 126-134 L2 peptide under HLA-A*02:01 restriction, interferon-γ release and cytotoxicity assays showed that peptide-specific CTLs effectively cross-recognized and lysed T2 target cells loaded either with the wild-type (CD147 126-134 ) or mutated peptide (CD147 126-134 L2). Moreover, the CD147 126-134 L2 peptide-specific CTLs exerted strong cytotoxic activity against drug-resistant MCF-7/Adr cells, which express a high level of CD147 and are HLA-A*02:01-positive, but not against normal MCF-7 cells. Thus, this suggests that the wild-type peptide (CD147 126-134 ) is naturally presented on HLA-A*02:01 of CD147-expressing MCF-7/Adr cells and is cross-recognized by CTLs. In conclusion, an HLA-A*02:01-restricted CD147-point mutant epitope peptide was identified that induces CTLs to efficiently lyse drug-resistant MCF-7 cells that highly express CD147. Therefore, this immunotherapeutic approach should be explored as a potential treatment for drug-resistant tumors.