Hitchhiking of Cas9 with nucleus-localized proteins impairs its controllability and leads to efficient genome editing of NLS-free Cas9.

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.

Codon-Optimized and de novo-Synthesized E-Selectin/AAV2 Dose-Response Study for Vascular Regeneration Gene Therapy.

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 2×109 VG to 2×1011 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 (2×1011 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 2×1011 VG aliquots injected IM.

Establishment of orthotopic osteosarcoma animal models in immunocompetent rats through muti-rounds of in-vivo selection.

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.

Fibroblasts in Diabetic Foot Ulcers.

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.

Cancer-associated fibroblasts contribute to the immunosuppressive landscape and influence the efficacy of the combination therapy of PD-1 inhibitors and antiangiogenic agents in hepatocellular carcinoma.

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.

Premetastatic Niche Mimicking Bone-On-A-Chip: A Microfluidic Platform to Study Bone Metastasis in Cancer Patients.

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.

A Novel Oncogenic Role of FDX1 in Human Melanoma Related to PD-L1 Immune Checkpoint.

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.

Combined overexpression of four transcription factors promotes effector T cell dedifferentiation toward early phenotypes.

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.

The effect of inulin-type fructans on the intestinal immune function of antibiotic-treated mice.

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 ß1 (TGF-ß1) 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-ß1 and SIgA. • ITFs delayed the recovery of Treg and B cells in the LP.

Inhibition of melanoma by survivin-specific lymphocytes combined with CCL17 and granulocyte-macrophage colony-stimulating factor in a mouse syngeneic model.

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.

Efficient Editing of an Adenoviral Vector Genome with CRISPR/Cas9.

Immunotherapy based on genetic modification of T cells has played an important role in the treatment of tumors and viral infections. Moreover, adenoviral vectors engineered with improved safety due to their inability to integrate into the host genome have been key in the clinical application of T cell therapy. However, the commonly used adenoviral vector Ad5 exhibits low efficiency of infection of human T cells and the details of the intracellular trafficking pathway of adenoviral vectors in human primary T cells remains unclear. Resolution of these issues will depend on successful modification of the adenoviral vector. To this end, here we describe the successful establishment of a simple and efficient method for editing adenoviral vectors in vitro using the CRISPR-Cas9 gene editing system to target the adenoviral fiber gene.

Overexpression of early T cell differentiation-specific transcription factors transforms the terminally differentiated effector T cells into less differentiated state.

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 (TEFF) 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 TEFF cells, which were activated by in vitro stimulation, to promote dedifferentiation into early T cells. In the mature TEFF 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 TEFF cells were significantly downregulated (such as GZMB, PRF1, and GNLY). Additionally, the TEFF 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 TEFF cells were reprogrammed into a less differentiated state after overexpression of the eight specific TFs.

Intracellular Notch1 Signaling in Cancer-Associated Fibroblasts Dictates the Plasticity and Stemness of Melanoma Stem/Initiating Cells.

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-DFNotch1-/- prompted cocultured melanoma cells to form more spheroids and acquire the phenotype (CD271+ and Nestin+ ) of melanoma stem/initiating cells (MICs), whereas MSC-DFN1IC+/+ suppressed melanoma cell sphere formation and mitigated properties of MICs. MSC-DFNotch1-/- 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-DFNotch1-/- increased, but MSC-DFN1IC+/+ 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.

The effect of antibiotic cocktails on host immune status is dynamic and does not always correspond to changes in gut microbiota.

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 3ß (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.

Intramuscular E-selectin/adeno-associated virus gene therapy promotes wound healing in an ischemic mouse model.

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.

A Reliable Mouse Model of Hind limb Gangrene.

BACKGROUND: Lack of a reliable hind limb gangrene animal model limits preclinical studies of gangrene, a severe form of critical limb ischemia. We develop a novel mouse hind limb gangrene model to facilitate translational studies. METHODS: BALB/c, FVB, and C57BL/6 mice underwent femoral artery ligation (FAL) with or without administration of NG-nitro-L-arginine methyl ester (L-NAME), an endothelial nitric oxide synthase inhibitor. Gangrene was assessed using standardized ischemia scores ranging from 0 (no gangrene) to 12 (forefoot gangrene). Laser Doppler imaging (LDI) and DiI perfusion quantified hind limb reperfusion postoperatively. RESULTS: BALB/c develops gangrene with FAL-only (n = 11/11, 100% gangrene incidence), showing mean limb ischemia score of 12 on postoperative days (PODs) 7 and 14 with LDI ranging from 0.08 to 0.12 on respective PODs. Most FVB did not develop gangrene with FAL-only (n = 3/9, 33% gangrene incidence) but with FAL and L-NAME (n = 9/9, 100% gangrene incidence). Mean limb ischemia scores for FVB undergoing FAL with L-NAME were significantly higher than for FVB receiving FAL-only. LDI score and capillary density by POD 28 were significantly lower in FVB undergoing FAL with L-NAME. C57BL/6 did not develop gangrene with FAL-only or FAL and L-NAME. CONCLUSIONS: Reproducible murine gangrene models may elucidate molecular mechanisms for gangrene development, facilitating therapeutic intervention.

[Effect of miRNA from Glycyrrhiza uralensis decoction on gene expression of human immune cells].

MicroRNAs(miRNA) are small non-coding RNAs that regulate the expression of protein coding genes by repressing translation of protein coding mRNA or enhancing mRNA degradation. Its functions have attracted more and more attention from the public. In recent years, the cross-border regulation of miRNA has become a new research direction, and provides a new perspective for people to comprehensively understand the functions of miRNA. Plant miRNA is usually methylated and not easy to degrade. According to our previous researches, there were abundant small RNAs in the decoction of dried liquorice, which provides a new way to study the mechanism of action of licorice. In this study, small RNAs extracted from Glycyrrhiza uralensis decoction and synthesized miRNA mimics were used to treat peripheral blood mononuclear cells(PBMC) isolated from healthy volunteers. The gene expression of toll-like receptors(TLRs), some transcription factors, signal molecules and cytokines were analyzed by RT-PCR. The results showed that glycyrrhiza miRNA could significantly regulate PBMC by inhibiting the expression of genes involved in T cell differentiation, inflammation and apoptosis. The study brought new ideas to us in comprehensively studying the mechanism of licorice and developing the traditional Chinese medicine.

[A novel assay for analysis of adenovirusmediated endosome lysis of T lymphocytes].

Objective: To analyze adenovirus-mediated endosome lysis of T cells, we developed a novel approach based on pHrodo dextran (pH-sensitive fluorescent dye). Methods: After incubating Jurkat cells (T cell leukemia) with serotype 5 adenovirus (Ad5) and pHrodo dextran, we determined the optimal incubation time and concentration of pHrodo dextran. To assess viral lysis of the endosome, we monitored the ratio changes of mean fluorescence intensity in different time points by laser scanning confocal microscopy. Results: After incubating Jurkat cells with Ad5 and 80 µg/mL pHrodo dextran for 10 minutes, we observed the fluorescence intensity was significantly reduced at 30 minutes compared with that of endosomes at 0 minute. However, we found the mean fluorescence intensity was only slightly reduced by inhibiting V-ATPase with the bafilomycin A1 treatment. Conclusion: The method based on pH-sensitive dye can be used to analyze the adenovirus-mediated endosome lysis of T cells.

[Extraction of miRNA from Glycyrrhiza uralensis Decoction and Its Effect on Immune Cells].

OBJECTIVE: To extract microRNA(miRNA) from Glycyrrhiza uralensis(liquorice) decoction and to explore its effect on mmune cells. METHODS: With dried processed liquorice, the water decoction was prepared according to the conventional method and subsequently concentrated by rotary evaporation. The concentrated decoction was further freeze-dried by freeze dryer, and miRNAs were extracted with Plant MicroRNA Extraction Kit. The extracted miRNA was digested by DNase I and then analyzed through the agarose gell electrophoresis. The PBMC was isolated from healthy volunteers and treated respectively by liquorice water extract, glycyrrhizic acid, glycyrrhetic acid and liquorice miRNAs. After 24 hours, the cells numbers were counted, and the changes of cell morphology were observed. The expression of CD3, CD56 and HLA-DR were analyzed by flow cytometry to identify the change of cell subsets in PBMC. RESULTS: miRNAs could be extracted from the decoction of dried liquorice which further confirmed the stability of miRNAs. The in vitro culture experiment showed that,compared with the controls, PBMC treated with liquorice miRNAs appeared apparent cell aggregation and increased cell number and HLA-DR+ cells proportion. CONCLUSION: The miRNAs are successfully extracted from the freeze-dried decoction of dried liquorice. It is indicated that liquorice miRNAs have significant stimulative effects on the growth of PBMC and HLA-DR+ cells subset.

Generating bulk RNA-Seq gene expression data based on generative deep learning models and utilizing it for data augmentation.

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.