Application of a Diabetic Foot Smart APP in the measurement of diabetic foot ulcers.

AIMS: In the early stage, we developed an intelligent measurement APP for diabetic foot ulcers, named Diabetic Foot Smart APP. This study aimed to validate the APP in the measurement of ulcer area for diabetic foot ulcer (DFU). METHODS: We selected 150 DFU images to measure the ulcer areas using three assessment tools: the Smart APP software package, the ruler method, and the gold standard Image J software, and compared the measurement results and measurement time of the three tools. The intra-rater and inter-rater reliability were described by Pearson correlation coefficient, intra-group correlation coefficient, and coefficient of variation. RESULTS: The Image J software showed a median ulcer area of 4.02 cm2, with a mean measurement time of 66.37 ± 7.95 s. The ruler method showed a median ulcer area of 5.14 cm2, with a mean measurement time of 171.47 ± 46.43 s. The APP software showed a median ulcer area of 3.70 cm2, with a mean measurement time of 38.25 ± 6.81 s. There were significant differences between the ruler method and the golden standard Image J software (Z = -4.123, p < 0.05), but no significant difference between the APP software and the Image J software (Z = 1.103, p > 0.05). The APP software also showed good inter-rater reliability and intra-rater reliability, with both reaching 0.99. CONCLUSION: The Diabetic Foot Smart APP is a fast and reliable measurement tool with high measurement accuracy that can be easily used in clinical practice for the measurement of ulcer areas of DFU. TRIAL REGISTRATION: Chinese clinical trial registration number: ChiCTR2100047210.

Intranasal delivery of small extracellular vesicles from specific subpopulation of mesenchymal stem cells mitigates traumatic spinal cord injury.

Vascular injury following spinal cord injury (SCI) can significantly exacerbate secondary SCI and result in neurological dysfunction. Strategies targeting angiogenesis have demonstrated potential in enhancing functional recovery post-SCI. In the context of angiogenesis, the CD146+ and CD271+ subpopulations of mesenchymal stem cells (MSCs) have been recognized for their angiogenic capabilities in tissue repair. Small extracellular vesicles (sEVs) derived from MSCs are nanoscale vesicles containing rich bioactive components that play a crucial role in tissue regeneration. However, the precise role of sEVs derived from CD146+CD271+ UCMSCs (CD146+CD271+ UCMSC-sEVs) in SCI remain unclear. In this study, CD146+CD271+ UCMSC-sEVs were non-invasively administered via intranasal delivery, demonstrating a significant capacity to stimulate angiogenesis and improve functional recovery in mice following SCI. Furthermore, in vitro assessments revealed the effective enhancement of migration and tube formation capabilities of the murine brain microvascular endothelial cell line (bEnd.3) by CD146+CD271+UCMSC-sEVs. MicroRNA array analysis confirmed significant enrichment of multiple microRNAs within CD146+CD271+ UCMSC-sEVs. Subsequent in vivo and in vitro experiments demonstrated that CD146+CD271+ UCMSC-sEVs promote enhanced angiogenesis and improved functional recovery mediated by miR-27a-3p. Further mechanistic studies revealed that miR-27a-3p sourced from CD146+CD271+ UCMSC-sEVs enhances migration and tube formation of bEnd.3 cells in vitro by suppressing the expression of Delta Like Canonical Notch Ligand 4 (DLL4), thereby promoting angiogenesis in vivo. Collectively, our results demonstrate that a crucial role of CD146+CD271+ UCMSC-sEVs in inhibiting DLL4 through the transfer of miR-27a-3p, which leads to the promotion of angiogenesis and improved functional recovery after SCI.

Kdm6a-CNN1 axis orchestrates epigenetic control of trauma-induced spinal cord microvascular endothelial cell senescence to balance neuroinflammation for improved neurological repair.

Cellular senescence assumes pivotal roles in various diseases through the secretion of proinflammatory factors. Despite extensive investigations into vascular senescence associated with aging and degenerative diseases, the molecular mechanisms governing microvascular endothelial cell senescence induced by traumatic stress, particularly its involvement in senescence-induced inflammation, remain insufficiently elucidated. In this study, we present a comprehensive demonstration and characterization of microvascular endothelial cell senescence induced by spinal cord injury (SCI). Lysine demethylase 6A (Kdm6a), commonly known as UTX, emerges as a crucial regulator of cell senescence in injured spinal cord microvascular endothelial cells (SCMECs). Upregulation of UTX induces senescence in SCMECs, leading to an amplified release of proinflammatory factors, specifically the senescence-associated secretory phenotype (SASP) components, thereby modulating the inflammatory microenvironment. Conversely, the deletion of UTX in endothelial cells shields SCMECs against senescence, mitigates the release of proinflammatory SASP factors, and promotes neurological functional recovery after SCI. UTX forms an epigenetic regulatory axis by binding to calponin 1 (CNN1), orchestrating trauma-induced SCMECs senescence and SASP secretion, thereby influencing neuroinflammation and neurological functional repair. Furthermore, local delivery of a senolytic drug reduces senescent SCMECs and suppresses proinflammatory SASP secretion, reinstating a local regenerative microenvironment and enhancing functional repair after SCI. In conclusion, targeting the UTX-CNN1 epigenetic axis to prevent trauma-induced SCMECs senescence holds the potential to inhibit SASP secretion, alleviate neuroinflammation, and provide a novel treatment strategy for SCI repair.

A Combined Treatment of BMP2 and Soluble VEGFR1 for the Enhancement of Tendon-Bone Healing by Regulating Injury-Activated Skeletal Stem Cell Lineage.

BACKGROUND: Bone morphogenetic protein 2 (BMP2) is an appealing osteogenic and chondrogenic growth factor for promoting tendon-bone healing. Recently, it has been reported that soluble vascular endothelial growth factor (VEGF) receptor 1 (sVEGFR1) (a VEGF receptor antagonist) could enhance BMP2-induced bone repair and cartilage regeneration; thus, their combined application may represent a promising treatment to improve tendon-bone healing. Moreover, BMP2 could stimulate skeletal stem cell (SSC) expansion and formation, which is responsible for wounded tendon-bone interface repair. However, whether the codelivery of BMP2 and sVEGFR1 increases tendon enthesis injury-activated SSCs better than does BMP2 alone needs further research. PURPOSE: To study the effect of BMP2 combined with sVEGFR1 on tendon-bone healing and injury-activated SSC lineage. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 128 C57BL/6 mice that underwent unilateral supraspinatus tendon detachment and repair were randomly assigned to 4 groups: (1) untreated control group; (2) hydrogel group, which received a local injection of the blank hydrogel at the injured site; (3) BMP2 group, which received an injection of hydrogel with BMP2; and (4) BMP2 with sVEGFR1 group, which received an injection of hydrogel with BMP2 and sVEGFR1. Histology, micro-computed tomography, and biomechanical tests were conducted to evaluate tendon-bone healing at 4 and 8 weeks after surgery. In addition, flow cytometry was performed to detect the proportion of SSCs and their downstream differentiated subtypes, including bone, cartilage, and stromal progenitors; osteoprogenitors; and pro-chondrogenic progenitors within supraspinatus tendon enthesis at 1 week postoperatively. RESULTS: The repaired interface in BMP2 with sVEGFR1 group showed a significantly improved collagen fiber continuity, increased fibrocartilage, greater newly formed bone, and elevated mechanical properties compared with the other 3 groups. There were more SSCs; bone, cartilage, and stromal progenitors; osteoprogenitors; and pro-chondrogenic progenitors in the BMP2 with sVEGFR1 group than that in the other groups. CONCLUSION: Our study suggests that the combined delivery of BMP2 and sVEGFR1 could promote tendon-bone healing and stimulate the expansion of SSCs and their downstream progeny within the injured tendon-bone interface. CLINICAL RELEVANCE: Combining BMP2 with sVEGFR1 may be a good clinical treatment for wounded tendon enthesis healing.

Monitoring drug metabolic pathways through extracellular vesicles in mouse plasma.

The ability to monitor the response of metabolic enzymes to drug exposure in individuals is highly appealing and critical to personalized medicine. Although pharmacogenomics assesses genotypic differences, it does not report changes in metabolic enzyme activities due to environmental factors such as drug interactions. Here, we report a quantitative proteomics strategy to monitor drug metabolic pathways by profiling metabolic enzymes in circulating extracellular vesicles (EVs) upon drug exposure. Mass spectrometry (MS)-based measurement revealed that changes in metabolic enzyme abundance in EVs paralleled those in hepatic cells isolated from liver tissue. Coupling with multiplexed isotopic labeling, we temporally quantified 34 proteins involved in drug absorption, distribution, metabolism, and excretion (ADME) pathways. Out of 44 known ADME proteins in plasma EVs, previously annotated mouse cytochrome P450 3A11 (Cyp3a11), homolog to human CYP3A4, and uridine 5'-diphospho (UDP) glucuronosyltransferase 2A3 (Ugt2a3), increased upon daily rifampicin dosage. Dasatinib, a tyrosine kinase inhibitor to treat leukemia, also elevated Cyp3a11 levels in plasma EVs, but to a lesser extent. Altogether, this study demonstrates that measuring drug enzymes in circulating EVs as an effective surrogate is highly feasible and may transform today's drug discovery and development for personalized medicine.

Extracellular vesicles from UTX-knockout endothelial cells boost neural stem cell differentiation in spinal cord injury.

BACKGROUND: Vascular endothelial cells are pivotal in the pathophysiological progression following spinal cord injury (SCI). The UTX (Ubiquitously Transcribed Tetratripeptide Repeat on Chromosome X) serves as a significant regulator of endothelial cell phenotype. The manipulation of endogenous neural stem cells (NSCs) offers a compelling strategy for the amelioration of SCI. METHODS: Two mouse models were used to investigate SCI: NSCs lineage-traced mice and mice with conditional UTX knockout (UTX KO) in endothelial cells. To study the effects of UTX KO on neural differentiation, we harvested extracellular vesicles (EVs) from both UTX KO spinal cord microvascular endothelial cells (SCMECs) and negative control SCMECs. These EVs were then employed to modulate the differentiation trajectory of endogenous NSCs in the SCI model. RESULTS: In our NSCs lineage-traced mice model of SCI, a marked decrease in neurogenesis was observed post-injury. Notably, NSCs in UTX KO SCMECs mice showed enhanced neuronal differentiation compared to controls. RNA sequencing and western blot analyses revealed an upregulation of L1 cell adhesion molecule (L1CAM), a gene associated with neurogenesis, in UTX KO SCMECs and their secreted EVs. This aligns with the observed promotion of neurogenesis in UTX KO conditions. In vivo administration of L1CAM-rich EVs from UTX KO SCMECs (KO EVs) to the mice significantly enhanced neural differentiation. Similarly, in vitro exposure of NSCs to KO EVs resulted in increased activation of the Akt signaling pathway, further promoting neural differentiation. Conversely, inhibiting Akt phosphorylation or knocking down L1CAM negated the beneficial effects of KO EVs on NSC neuronal differentiation. CONCLUSIONS: In conclusion, our findings substantiate that EVs derived from UTX KO SCMECs can act as facilitators of neural differentiation following SCI. This study not only elucidates a novel mechanism but also opens new horizons for therapeutic interventions in the treatment of SCI. Video Abstract.

Biosynthesis of Bt-Ag2O nanoparticles using Bacillus thuringiensis and their pesticidal and antimicrobial activities.

Nanosilver oxide exhibits strong antibacterial and photocatalytic properties and has shown great application potential in food packaging, biochemical fields, and other fields involving diseases and pest control. In this study, Ag2O nanoparticles were synthesized using Bacillus thuringiensis (Bt-Ag2O NPs). The physicochemical characteristics of the Bt-Ag2O NPs were analyzed by UV‒vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM), inductively coupled plasma emission spectrometry (ICP), high-resolution transmission electron microscopy (HR-TEM), and zeta potential. The phis-chemical characterization revealed that the Bt-Ag2O NPs are in spherical shape with the small particle size (18.24 nm), high crystallinity, well dispersity, and stability. The biopesticidal and antifungal effects of Bt-Ag2O NPs were tested against Tribolium castaneum, Aspergillus flavus, and Penicillium chrysogenum. The survival, growth, and reproduction of tested pests and molds were significantly inhibited by Bt-Ag2O NPs in a dose-dependent manner. Bt-Ag2O NPs showed higher pesticidal activities against T. castaneum than Bt and commercial Ag2O NPs. The LC50 values of Bt, Ag2O NPs, and Bt-Ag2O NPs were 0.139%, 0.072%, and 0.06% on day 14, respectively. The Bt-Ag2O NPs also showed well antifungal activities against A. flavus and P. chrysogenum, while it resulted a small inhibition zone than commercial Ag2O NPs did. In addition, A. flavus showed much more sensitive to Bt-Ag2O NP treatments, compared to P. chrysogenum. Our results revealed that Bt-Ag2O NPs synthesized using B. thuringiensis could act as pesticides and antifungal agents in stored-product fields. KEY POINTS: • Bt-Ag2O NPs could be synthesized using Bacillus thuringiensis (Bt). • The NPs showed a high degree of crystallinity, spherical shape, and small particle size. • The NPs also showed excellent insecticidal and antifungal activity.

Exosomes derived from CD271+CD56+ bone marrow mesenchymal stem cell subpopoulation identified by single-cell RNA sequencing promote axon regeneration after spinal cord injury.

Rationale: Spinal cord injury (SCI) results in neural tissue damage. However, the limited regenerative capacity of adult mammals' axons upon SCI leads to persistent neurological dysfunction. Thus, exploring the pathways that can enhance axon regeneration in injured spinal cord is of great significance. Methods: Through the utilization of single-cell RNA sequencing in this research, a distinct subpopulation of bone marrow mesenchymal stem cells (BMSCs) that exhibits the capacity to facilitate axon regeneration has been discovered. Subsequently, the CD271+CD56+ BMSCs subpopulation was isolated using flow cytometry, and the exosomes derived from this subpopulation (CD271+CD56+ BMSC-Exos) were extracted and incorporated into a hydrogel to create a sustained release system. The aim was to investigate the therapeutic effects of CD271+CD56+ BMSC-Exos and elucidate the underlying mechanisms involved in promoting axon regeneration and neural function recovery. Results: The findings indicate that CD271+CD56+ BMSC-Exos share similar physical and chemical properties with conventional exosomes. Importantly, in an SCI model, in situ implantation of CD271+CD56+ BMSC-Exos hydrogel resulted in increased expression of NF and synaptophysin, markers associated with axon regeneration and synapse formation, respectively. This intervention also contributed to improved neural function recovery. In vitro experiments demonstrated that CD271+CD56+ BMSC-Exos treatment significantly enhanced axon extension distance and increased the number of branches in dorsal root ganglion axons. Moreover, further investigation into the molecular mechanisms underlying CD271+CD56+ BMSC-Exos-mediated axon regeneration revealed the crucial involvement of the miR-431-3p/RGMA axis. Conclusion: In summary, the implantation of CD271+CD56+ BMSC-Exos hydrogel presents a promising and effective therapeutic approach for SCI.

Optimized Allogenic Decellularized Meniscal Scaffold Modified by Collagen Affinity Stromal Cell-Derived Factor SDF1α for Meniscal Regeneration: A 6- and 12-Week Animal Study in a Rabbit Model.

BACKGROUND: Total meniscectomy for treating massive meniscal tears may lead to joint instability, cartilage degeneration, and even progressive osteoarthritis. The meniscal substitution strategies for advancing reconstruction of the meniscus deserve further investigation. HYPOTHESIS: A decellularized meniscal scaffold (DMS) modified with collagen affinity stromal cell-derived factor (C-SDF1α) may facilitate meniscal regeneration and protect cartilage from abrasion. STUDY DESIGN: Controlled laboratory study. METHODS: The authors first modified DMS with C-SDF1α to fabricate a new meniscal graft (DMS-CBD [collagen-binding domain]). Second, they performed in vitro studies to evaluate the release dynamics, biocompatibility, and differentiation inducibility (osteogenic, chondrogenic, and tenogenic differentiation) on human bone marrow mesenchymal stem cells. Using in vivo studies, they subjected rabbits that received medial meniscectomy to a transplantation procedure to implement their meniscal graft. At postoperative weeks 6 and 12, the meniscal regeneration outcomes and chondroprotective efficacy of the new meniscal graft were evaluated by macroscopic observation, histology, micromechanics, and immunohistochemistry tests. RESULTS: In in vitro studies, the optimized DMS-CBD graft showed notable biocompatibility, releasing efficiency, and chondrogenic inducibility. In in vivo studies, the implanted DMS-CBD graft after total meniscectomy promoted the migration of cells and extracellular matrix deposition in transplantation and further facilitated meniscal regeneration and protected articular cartilage from degeneration. CONCLUSION: The new meniscal graft (DMS-CBD) accelerated extracellular matrix deposition and meniscal regeneration and protected articular cartilage from degeneration. CLINICAL RELEVANCE: The results demonstrate that the DMS-CBD graft can serve as a potential meniscal substitution after meniscectomy.

FIONA1-mediated mRNA m6 A methylation regulates the response of Arabidopsis to salt stress.

N6 -methyladenosine (m6 A) is an mRNA modification widely found in eukaryotes and plays a crucial role in plant development and stress responses. FIONA1 (FIO1) is a recently identified m6 A methyltransferase that regulates Arabidopsis (Arabidopsis thaliana) floral transition; however, its role in stress response remains unknown. In this study, we demonstrate that FIO1-mediated m6 A methylation plays a vital role in salt stress response in Arabidopsis. The loss-of-function fio1 mutant was sensitive to salt stress. Importantly, the complementation lines expressing the wild-type FIO1 exhibited the wild-type phenotype, whereas the complementation lines expressing the mutant FIO1m , in which two critical amino acid residues essential for methyltransferase activity were mutated, did not recover the wild-type phenotype under salt stress, indicating that the salt sensitivity is associated with FIO1 methyltransferase activity. Furthermore, FIO1-mediated m6 A methylation regulated ROS production and affected the transcript level of several salt stress-responsive genes via modulating their mRNA stability in an m6 A-dependent manner in response to salt stress. Importantly, FIO1 is associated with salt stress response by specifically targeting and differentially modulating several salt stress-responsive genes compared with other m6 A writer. Collectively, our findings highlight the molecular mechanism of FIO1-mediated m6 A methylation in the salt stress adaptation.

ECT12, an YTH-domain protein, is a potential mRNA m6A reader that affects abiotic stress responses by modulating mRNA stability in Arabidopsis.

N6-methyladenosine (m6A), the most abundant modification found in eukaryotic mRNAs, is interpreted by m6A "readers," thus playing a crucial role in regulating RNA metabolism. The YT521-B homology-domain (YTHD) proteins, also known as EVOLUTIONARILY CONSERVED C-TERMINAL REGION (ECT), are recognized as m6A reader proteins in plants and animals. Among the 13 potential YTHD family proteins in Arabidopsis thaliana, the functions of only a few members are known. In this study, we determined the function of ECT12 (YTH11) as a potential m6A reader that plays a crucial role in response to abiotic stresses. The loss-of-function ect12 mutants showed no noticeable developmental defects under normal conditions but displayed hypersensitivity to salt or dehydration stress. The salt- or dehydration-hypersensitive phenotypes were correlated with altered levels of several m6A-modified stress-responsive transcripts. Notably, the increased or decreased transcript levels were associated with each transcript's reduced or enhanced decay, respectively. Electrophoretic mobility shift and RNA-immunoprecipitation assays showed that ECT12 binds to m6A-modified RNAs both in vitro and in planta, suggesting its role as an m6A reader. Collectively, these results indicate that the potential m6A reader ECT12 regulates the stability of m6A-modified RNA transcripts, thereby facilitating the response of Arabidopsis to abiotic stresses.

Phylogenetic and functional analyses of N6-methyladenosine RNA methylation factors in the wheat scab fungus Fusarium graminearum.

In eukaryotes, N6-methyladenosine (m6A) RNA modification plays a crucial role in governing the fate of RNA molecules and has been linked to various developmental processes. However, the phyletic distribution and functions of genetic factors responsible for m6A modification remain largely unexplored in fungi. To get insights into the evolution of m6A machineries, we reconstructed global phylogenies of potential m6A writers, readers, and erasers in fungi. Substantial copy number variations were observed, ranging from up to five m6A writers in early-diverging fungi to a single copy in the subphylum Pezizomycotina, which primarily comprises filamentous fungi. To characterize m6A factors in a phytopathogenic fungus Fusarium graminearum, we generated knockout mutants lacking potential m6A factors including the sole m6A writer MTA1. However, the resulting knockouts did not exhibit any noticeable phenotypic changes during vegetative and sexual growth stages. As obtaining a homozygous knockout lacking MTA1 was likely hindered by its essential role, we generated MTA1-overexpressing strains (MTA1-OE). The MTA1-OE5 strain showed delayed conidial germination and reduced hyphal branching, suggesting its involvement during vegetative growth. Consistent with these findings, the expression levels of MTA1 and a potential m6A reader YTH1 were dramatically induced in germinating conidia, followed by the expression of potential m6A erasers at later vegetative stages. Several genes including transcription factors, transporters, and various enzymes were found to be significantly upregulated and downregulated in the MTA1-OE5 strain. Overall, our study highlights the functional importance of the m6A methylation during conidial germination in F. graminearum and provides a foundation for future investigations into m6A modification sites in filamentous fungi.IMPORTANCEN6-methyladenosine (m6A) RNA methylation is a reversible posttranscriptional modification that regulates RNA function and plays a crucial role in diverse developmental processes. This study addresses the knowledge gap regarding phyletic distribution and functions of m6A factors in fungi. The identification of copy number variations among fungal groups enriches our knowledge regarding the evolution of m6A machinery in fungi. Functional characterization of m6A factors in a phytopathogenic filamentous fungus Fusarium graminearum provides insights into the essential role of the m6A writer MTA1 in conidial germination and hyphal branching. The observed effects of overexpressing MTA1 on fungal growth and gene expression patterns of m6A factors throughout the life cycle of F. graminearum further underscore the importance of m6A modification in conidial germination. Overall, this study significantly advances our understanding of m6A modification in fungi, paving the way for future research into its roles in filamentous growth and potential applications in disease control.

Local delivery of EGFR+NSCs-derived exosomes promotes neural regeneration post spinal cord injury via miR-34a-5p/HDAC6 pathway.

Spinal cord injury (SCI) causes severe axon damage, usually leading to permanent paraparesis, which still lacks effective regenerative therapy. Recent studies have suggested that exosomes derived from neural stem cells (NSCs) may hold promise as attractive candidates for SCI treatment. Epidermal Growth Factor Receptor positive NSC (EGFR+NSC) is a subpopulation of endogenous NSCs, showing strong regenerative capability in central nervous system disease. In the current study, we isolated exosomes from the EGFR+NSCs (EGFR+NSCs-Exos) and discovered that local delivery of EGFR+NSCs-Exos can effectively promote neurite regrowth in the injury site of spinal cord-injured mice and improve their neurological function recovery. Using the miRNA-seq, we firstly characterized the microRNAs (miRNAs) cargo of EGFR+NSCs-Exos and identified miR-34a-5p which was highly enriched in EGFR+NSCs derived exosomes. We further interpreted that exosomal miR-34a-5p could be transferred to neurons and inhibit the HDAC6 expression by directly binding to its mRNA, contributing to microtubule stabilization and autophagy induction for aiding SCI repair. Overall, our research demonstrated a novel therapeutic approach to improving neurological functional recovery by using exosomes secreted from a subpopulation of endogenous NSCs and providing a precise cell-free treatment strategy for SCI repair.

Inhibition of UTX/KDM6A improves recovery of spinal cord injury by attenuating BSCB permeability and macrophage infiltration through the MLCK/p-MLC pathway.

Spinal cord injury (SCI) can prompt an immediate disruption to the blood-spinal cord barrier (BSCB). Restoring the integrity of this barrier is vital for the recovery of neurological function post-SCI. The UTX protein, a histone demethylase, has been shown in previous research to promote vascular regeneration and neurological recovery in mice with SCI. However, it is unclear whether UTX knockout could facilitate the recovery of the BSCB by reducing its permeability. In this study, we systematically studied BSCB disruption and permeability at different time points after SCI and found that conditional UTX deletion in endothelial cells (ECs) can reduce BSCB permeability, decrease inflammatory cell infiltration and ROS production, and improve neurological function recovery after SCI. Subsequently, we used RNA sequencing and ChIP-qPCR to confirm that conditional UTX knockout in ECs can down-regulate expression of myosin light chain kinase (MLCK), which specifically mediates myosin light chain (MLC) phosphorylation and is involved in actin contraction, cell retraction, and tight junctions (TJs) protein integrity. Moreover, we found that MLCK overexpression can increase the ratio of p-MLC/MLC, further break TJs, and exacerbate BSCB deterioration. Overall, our findings indicate that UTX knockout could inhibit the MLCK/p-MLC pathway, resulting in decreased BSCB permeability, and ultimately promoting neurological recovery in mice. These results suggest that UTX is a promising new target for treating SCI.

Identification and targeting of treatment resistant progenitor populations in T-cell Acute Lymphoblastic Leukemia.

Refractoriness to initial chemotherapy and relapse after remission are the main obstacles to cure in T-cell Acute Lymphoblastic Leukemia (T-ALL). Biomarker guided risk stratification and targeted therapy have the potential to improve outcomes in high-risk T-ALL; however, cellular and genetic factors contributing to treatment resistance remain unknown. Previous bulk genomic studies in T-ALL have implicated tumor heterogeneity as an unexplored mechanism for treatment failure. To link tumor subpopulations with clinical outcome, we created an atlas of healthy pediatric hematopoiesis and applied single-cell multiomic (CITE-seq/snATAC-seq) analysis to a cohort of 40 cases of T-ALL treated on the Children's Oncology Group AALL0434 clinical trial. The cohort was carefully selected to capture the immunophenotypic diversity of T-ALL, with early T-cell precursor (ETP) and Near/Non-ETP subtypes represented, as well as enriched with both relapsed and treatment refractory cases. Integrated analyses of T-ALL blasts and normal T-cell precursors identified a bone-marrow progenitor-like (BMP-like) leukemia sub-population associated with treatment failure and poor overall survival. The single-cell-derived molecular signature of BMP-like blasts predicted poor outcome across multiple subtypes of T-ALL within two independent patient cohorts using bulk RNA-sequencing data from over 1300 patients. We defined the mutational landscape of BMP-like T-ALL, finding that NOTCH1 mutations additively drive T-ALL blasts away from the BMP-like state. We transcriptionally matched BMP-like blasts to early thymic seeding progenitors that have low NR3C1 expression and high stem cell gene expression, corresponding to a corticosteroid and conventional cytotoxic resistant phenotype we observed in ex vivo drug screening. To identify novel targets for BMP-like blasts, we performed in silico and in vitro drug screening against the BMP-like signature and prioritized BMP-like overexpressed cell-surface (CD44, ITGA4, LGALS1) and intracellular proteins (BCL-2, MCL-1, BTK, NF-κB) as candidates for precision targeted therapy. We established patient derived xenograft models of BMP-high and BMP-low leukemias, which revealed vulnerability of BMP-like blasts to apoptosis-inducing agents, TEC-kinase inhibitors, and proteasome inhibitors. Our study establishes the first multi-omic signatures for rapid risk-stratification and targeted treatment of high-risk T-ALL.

Targeted Delivery of RGD-CD146+CD271+ Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Promotes Blood-Spinal Cord Barrier Repair after Spinal Cord Injury.

Spinal cord injury (SCI) disrupts the blood-spinal cord barrier (BSCB), potentially exacerbating nerve damage and emphasizing the criticality of preserving the BSCB integrity during SCI treatment. This study explores an alternative therapeutic approach for SCI by identifying a subpopulation of exosomes with stable BSCB function and achieving a specific targeted delivery. Specific subpopulations of CD146+CD271+ umbilical cord mesenchymal stem cells (UCMSCs) were isolated, from which engineered exosomes (RGD-CD146+CD271+ UCMSC-Exos) with targeted neovascularization function were obtained through gene transfection. In vivo and in vitro experiments were performed to explore the targeting and therapeutic effects of RGD-CD146+CD271+ UCMSC-Exos and the potential mechanisms underlying BSCB stabilization and neural function recovery. The results demonstrated that RGD-CD146+CD271+ UCMSC-Exos exhibited physical and chemical properties similar to those of regular exosomes. Notably, following intranasal administration, RGD-CD146+CD271+ UCMSC-Exos exhibited enhanced aggregation at the SCI center and demonstrated the specific targeting of neovascular endothelial cells. In the SCI model, intranasal administration of RGD-CD146+CD271+ UCMSC-Exos reduced Evans blue dye leakage, increased tight junction protein expression, and improved neurological function recovery. In vitro testing revealed that RGD-CD146+CD271+ UCMSC-Exos treatment significantly reduced the permeability of bEnd.3 cells subjected to oxygen-glucose deprivation, thereby restoring the integrity of tight junctions. Moreover, further exploration of the molecular mechanism underlying BSCB stabilization by CD146+CD271+ UCMSC-Exos identified the crucial role of the miR-501-5p/MLCK axis in this process. In conclusion, targeted delivery of RGD-CD146+CD271+ UCMSC-Exos presents a promising and effective treatment option for SCI.

Hypoxia-treated adipose mesenchymal stem cell-derived exosomes attenuate lumbar facet joint osteoarthritis.

BACKGROUND: Lumbar facet joint osteoarthritis (LFJ OA) is a common disease, and there is still a lack of effective disease-modifying therapies. Our aim was to determine the therapeutic effect of hypoxia-treated adipose mesenchymal stem cell (ADSC)-derived exosomes (Hypo-ADSC-Exos) on the protective effect against LFJ OA. METHODS: The protective effect of Hypo-ADSC-Exos against LFJ OA was examined in lumbar spinal instability (LSI)-induced LFJ OA models. Spinal pain behavioural assessments and CGRP (Calcitonin Gene-Related Peptide positive) immunofluorescence were evaluated. Cartilage degradation and subchondral bone remodelling were assessed by histological methods, immunohistochemistry, synchrotron radiation-Fourier transform infrared spectroscopy (SR-FTIR), and 3D X-ray microscope scanning. RESULTS: Hypoxia enhanced the protective effect of ADSC-Exos on LFJ OA. Specifically, tail vein injection of Hypo-ADSC-Exos protected articular cartilage from degradation, as demonstrated by lower FJ OA scores of articular cartilage and less proteoglycan loss in lumbar facet joint (LFJ) cartilage than in the ADSC-Exo group, and these parameters were significantly improved compared to those in the PBS group. In addition, the levels and distribution of collagen and proteoglycan in LFJ cartilage were increased in the Hypo-ADSC-Exo group compared to the ADSC-Exo or PBS group by SR-FTIR. Furthermore, Hypo-ADSC-Exos normalized uncoupled bone remodelling and aberrant H-type vessel formation in subchondral bone and effectively reduced symptomatic spinal pain caused by LFJ OA in mice compared with those in the ADSC-Exo or PBS group. CONCLUSIONS: Our results show that hypoxia is an effective method to improve the therapeutic effect of ADSC-Exos on ameliorating spinal pain and LFJ OA progression.

Single-cell RNA sequencing reveals cellular and molecular heterogeneity in fibrocartilaginous enthesis formation.

The attachment site of the rotator cuff (RC) is a classic fibrocartilaginous enthesis, which is the junction between bone and tendon with typical characteristics of a fibrocartilage transition zone. Enthesis development has historically been studied with lineage tracing of individual genes selected a priori, which does not allow for the determination of single-cell landscapes yielding mature cell types and tissues. Here, in together with open-source GSE182997 datasets (three samples) provided by Fang et al., we applied Single-cell RNA sequencing (scRNA-seq) to delineate the comprehensive postnatal RC enthesis growth and the temporal atlas from as early as postnatal day 1 up to postnatal week 8. And, we furtherly performed single-cell spatial transcriptomic sequencing on postnatal day 1 mouse enthesis, in order to deconvolute bone-tendon junction (BTJ) chondrocytes onto spatial spots. In summary, we deciphered the cellular heterogeneity and the molecular dynamics during fibrocartilage differentiation. Combined with current spatial transcriptomic data, our results provide a transcriptional resource that will support future investigations of enthesis development at the mechanistic level and may shed light on the strategies for enhanced RC healing outcomes.

Targeted delivery of CD163+ macrophage-derived small extracellular vesicles via RGD peptides promote vascular regeneration and stabilization after spinal cord injury.

Targeted delivery of small extracellular vesicles (sEVs) with low immunogenicity and fewer undesirable side effects are needed for spinal cord injury (SCI) therapy. Here, we show that RGD (Arg-Gly-Asp) peptide-decorated CD163+ macrophage-derived sEVs can deliver TGF-ß to the neovascular endothelial cells of the injured site and improve neurological function after SCI. CD163+ macrophages are M2 macrophages that express TGF-ß and are reported to promote angiogenesis and vascular stabilization in various diseases. Enriched TGF-ß EVs were crucial in angiogenesis and tissue repair. However, TGF-ß also boosts the formation of fibrous or glial scars, detrimental to neurological recovery. Our results found RGD-modified CD163+ sEVs accumulated in the injured region and were taken up by neovascular endothelial cells. Furthermore, RGD-CD163+ sEVs promoted vascular regeneration and stabilization in vitro and in vivo, resulting in substantial functional recovery post-SCI. These data suggest that RGD-CD163+ sEVs may be a potential strategy for treating SCI.

UTX deletion promotes M2 macrophage polarization by epigenetically regulating endothelial cell-macrophage crosstalk after spinal cord injury.

Macrophages polarized to the M2 subtype after spinal cord injury (SCI) are beneficial for promoting neurological recovery. The crosstalk between endothelial cells (ECs) and macrophages is crucial for the imbalance between proinflammatory and pro-resolving responses caused by macrophage heterogeneity; however, this crosstalk is strengthened post-SCI, leading to inflammatory cascades and second damage. As a powerful means to regulate gene expression, epigenetic regulation of the interaction between immune cells and ECs in SCI is still largely unknown. Our previous research demonstrated that the histone demethylase UTX deletion in ECs (UTX-/- ECs) promotes neurological recovery, while the precise mechanism is unrevealed. Here, we discovered that UTX-/- ECs polarize macrophages toward the M2 subtype post-SCI. Macrophage deficiency could block the neurological recovery caused by the knockdown of UTX. The exosomes from UTX-/- ECs mediate this crosstalk. In addition, we found UTX, H3K27, and miR-467b-3p/Sfmbt2 promoters forming a regulatory complex that upregulates the miR-467b-3p in UTX-/- ECs. And then, miR-467b-3p transfers to macrophages by exosomes and activates the PI3K/AKT/mTOR signaling by decreasing PTEN expression, finally polarizing macrophage to the M2 subtype. This study reveals a mechanism by epigenetic regulation of ECs-macrophages crosstalk and identifies potential targets, which may provide opportunities for treating SCI.