Comparative physiology and transcriptome response patterns in cold-tolerant and cold-sensitive varieties of Solanum melongena.

BACKGROUND: Climate change has led to severe cold events, adversely impacting global crop production. Eggplant (Solanum melongena L.), a significant economic crop, is highly susceptible to cold damage, affecting both yield and quality. Unraveling the molecular mechanisms governing cold resistance, including the identification of key genes and comprehensive transcriptional regulatory pathways, is crucial for developing new varieties with enhanced tolerance. RESULTS: In this study, we conducted a comparative analysis of leaf physiological indices and transcriptome sequencing results. The orthogonal partial least squares discriminant analysis (OPLS-DA) highlighted peroxidase (POD) activity and soluble protein as crucial physiological indicators for both varieties. RNA-seq data analysis revealed that a total of 7024 and 6209 differentially expressed genes (DEGs) were identified from variety "A" and variety "B", respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of DEGs demonstrated that the significant roles of starch and sucrose metabolism, glutathione metabolism, terpenoid synthesis, and energy metabolism (sucrose and starch metabolism) were the key pathways in eggplant. Weighted gene co-expression network analysis (WGCNA) shown that the enrichment of numerous cold-responsive genes, pathways, and soluble proteins in the MEgrep60 modules. Core hub genes identified in the co-expression network included POD, membrane transporter-related gene MDR1, abscisic acid-related genes, growth factor enrichment gene DELLA, core components of the biological clock PRR7, and five transcription factors. Among these, the core transcription factor MYB demonstrated co-expression with signal transduction, plant hormone, biosynthesis, and metabolism-related genes, suggesting a pivotal role in the cold response network. CONCLUSION: This study integrates physiological indicators and transcriptomics to unveil the molecular mechanisms responsible for the differences in cold tolerance between the eggplant cold-tolerant variety "A" and the cold-sensitive variety "B". These mechanisms include modulation of reactive oxygen species (ROS), elevation in osmotic carbohydrate and free proline content, and the expression of terpenoid synthesis genes. This comprehensive understanding contributes valuable insights into the molecular underpinnings of cold stress tolerance, ultimately aiding in the improvement of crop cold tolerance.

Identification and Molecular Characterization of the CAMTA Gene Family in Solanaceae with a Focus on the Expression Analysis of Eggplant Genes under Cold Stress.

Calmodulin-binding transcription activator (CAMTA) is an important calmodulin-binding protein with a conserved structure in eukaryotes which is widely involved in plant stress response, growth and development, hormone signal transduction, and other biological processes. Although CAMTA genes have been identified and characterized in many plant species, a systematic and comprehensive analysis of CAMTA genes in the Solanaceae genome is performed for the first time in this study. A total of 28 CAMTA genes were identified using bioinformatics tools, and the biochemical/physicochemical properties of these proteins were investigated. CAMTA genes were categorized into three major groups according to phylogenetic analysis. Tissue-expression profiles indicated divergent spatiotemporal expression patterns of SmCAMTAs. Furthermore, transcriptome analysis of SmCAMTA genes showed that exposure to cold induced differential expression of many eggplant CAMTA genes. Yeast two-hybrid and bimolecular fluorescent complementary assays suggested an interaction between SmCAMTA2 and SmERF1, promoting the transcription of the cold key factor SmCBF2, which may be an important mechanism for plant cold resistance. In summary, our results provide essential information for further functional research on Solanaceae family genes, and possibly other plant families, in the determination of the development of plants.

Multifunctional Drugs-Loaded Carbomol Hydrogel Promotes Diabetic Wound Healing via Antimicrobial and Immunoregulation.

Diabetic wound healing poses a significant clinical dilemma. Bacterial infection and immune dysregulation are the predominant reasons. However, conventional wound dressings with a single treatment approach often limit therapeutic efficacy and continue working with difficulty. These limitations cause high treatment failure for diabetic wounds. In this study, we developed a multiple drug-loaded carbomer hydrogel containing Que/Van/Rif (QVR-CBMG) for the simultaneous treatment of infection and immune dysregulation. Honeycomb-like QVR-CBMG hydrogel exhibits excellent abilities to eliminate bacterial infection and biofilms in vitro. Moreover, QVR-CBMG hydrogel possesses an immunomodulatory capacity via affecting the Sirt3/SOD2 signaling pathway to promote M2 macrophages. Furthermore, QVR-CBMG hydrogel effectively promotes wound healing in diabetic rats through several mechanisms. The multidrug-loaded wound dressing not only eliminates bacterial infection and facilitated angiogenesis but also promotes collagen deposition and remodulates the local immune microenvironment in the areas of wounds. In summary, this synthetic strategy to eliminate infection and regulate immune disorders has potential translational value for the prevention and management of diabetic wounds.

Genome-Wide Survey and Functional Verification of the NAC Transcription Factor Family in Wild Emmer Wheat.

The NAC transcription factor (TF) family is one of the largest TF families in plants, which has been widely reported in rice, maize and common wheat. However, the significance of the NAC TF family in wild emmer wheat (Triticum turgidum ssp. dicoccoides) is not yet well understood. In this study, a genome-wide investigation of NAC genes was conducted in the wild emmer genome and 249 NAC family members (TdNACs) were identified. The results showed that all of these genes contained NAM/NAC-conserved domains and most of them were predicted to be located on the nucleus. Phylogenetic analysis showed that these 249 TdNACs can be classified into seven clades, which are likely to be involved in the regulation of grain protein content, starch synthesis and response to biotic and abiotic stresses. Expression pattern analysis revealed that TdNACs were highly expressed in different wheat tissues such as grain, root, leaves and shoots. We found that TdNAC8470 was phylogenetically close to NAC genes that regulate either grain protein or starch accumulation. Overexpression of TdNAC8470 in rice showed increased grain starch concentration but decreased grain Fe, Zn and Mn contents compared with wild-type plants. Protein interaction analysis indicated that TdNAC8470 might interact with granule-bound starch synthase 1 (TdGBSS1) to regulate grain starch accumulation. Our work provides a comprehensive understanding of the NAC TFs family in wild emmer wheat and establishes the way for future functional analysis and genetic improvement of increasing grain starch content in wheat.

Trehalose inhibits ferroptosis via NRF2/HO-1 pathway and promotes functional recovery in mice with spinal cord injury.

Spinal cord injury (SCI) is the main cause of severe damage to the central nervous system and leads to irreversible tissue loss and neurological dysfunction. Ferroptosis is a cell death pattern, newly discovered in recent years. Ferroptosis is an oxidizing cell death induced by small molecules, and is an iron-dependent process caused by the imbalance between the generation and degradation of lipid reactive oxygen species (ROS) in cells. As an antioxidant, trehalose can effectively prevent lipid peroxidation. Studies have reported that trehalose can improve the prognosis of SCI. However, it is unclear whether these benefits are related to ferroptosis. In this study, we demonstrated for the first time that trehalose reduces the degeneration and iron accumulation of neurons by inhibiting the production of ROS and ferroptosis caused by lipid peroxides after SCI, thus promoting the survival of neurons and improving the recovery of motor function. More specifically, we found that trehalose inhibited the expansion of cavities in the nerve tissue of mice with SCI, inhibited neuron loss, and improved functional recovery. In terms of mechanism, our results indicate that the neuroprotective effect of trehalose is due to the activation of the NRF2/HO-1 pathway, which in turn inhibits ferroptosis and ferroptosis-related inflammation. Our findings provide important insights into the previously unknown role of trehalose in SCI, as well as new evidence supporting the hypothesis that suppression of ferroptosis plays a key neuroprotective role in SCI.

Genome-Wide Investigation and Functional Verification of the ZIP Family Transporters in Wild Emmer Wheat.

The zinc/iron-regulated transporter-like protein (ZIP) family has a crucial role in Zn homeostasis of plants. Although the ZIP genes have been systematically studied in many plant species, the significance of this family in wild emmer wheat (Triticum turgidum ssp. dicoccoides) is not yet well understood. In this study, a genome-wide investigation of ZIPs genes based on the wild emmer reference genome was conducted, and 33 TdZIP genes were identified. Protein structure analysis revealed that TdZIP proteins had 1 to 13 transmembrane (TM) domains and most of them were predicted to be located on the plasma membrane. These TdZIPs can be classified into three clades in a phylogenetic tree. They were annotated as being involved in inorganic ion transport and metabolism. Cis-acting analysis showed that several elements were involved in hormone, stresses, grain-filling, and plant development. Expression pattern analysis indicated that TdZIP genes were highly expressed in different tissues. TdZIP genes showed different expression patterns in response to Zn deficiency and that 11 genes were significantly induced in either roots or both roots and shoots of Zn-deficient plants. Yeast complementation analysis showed that TdZIP1A-3, TdZIP6B-1, TdZIP6B-2, TdZIP7A-3, and TdZIP7B-2 have the capacity to transport Zn. Overexpression of TdZIP6B-1 in rice showed increased Zn concentration in roots compared with wild-type plants. The expression levels of TdZIP6B-1 in transgenic rice were upregulated in normal Zn concentration compared to that of no Zn. This work provides a comprehensive understanding of the ZIP gene family in wild emmer wheat and paves the way for future functional analysis and genetic improvement of Zn deficiency tolerance in wheat.

Identification and Mapping of QTL for Stripe Rust Resistance in the Chinese Wheat Cultivar Shumai126.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a damaging disease of wheat globally, and breeding resistant cultivars is the best control strategy. The Chinese winter wheat cultivar Shumai126 (SM126) exhibited strong resistance to P. striiformis f. sp. tritici in the field for more than 10 years. The objective of this study was to identify and map quantitative trait loci (QTL) for resistance to stripe rust in a population of 154 recombinant inbred lines (RILs) derived from a cross between cultivars Taichang29 (TC29) and SM126. The RILs were tested in six field environments with a mixture of the Chinese prevalent races (CYR32, CYR33, CYR34, Zhong4, and HY46) of P. striiformis f. sp. tritici and in growth chamber with race CYR34 and genotyped using the Wheat55K single nucleotide polymorphism (SNP) array. Six QTL were mapped on chromosomes 1BL, 2AS, 2AL, 6AS, 6BS, and 7BL, respectively. All QTL were contributed by SM126 except QYr.sicau-2AL. The QYr.sicau-1BL and QYr.sicau-2AS had major effects, explaining 27.00 to 39.91% and 11.89 to 17.11% of phenotypic variances, which may correspond to known resistance genes Yr29 and Yr69, respectively. The QYr.sicau-2AL, QYr.sicau-6AS, and QYr.sicau-6BS with minor effects are likely novel. QYr.sicau-7BL was only detected based on growth chamber seedling data. Additive effects were detected for the combination of QYr.sicau-1BL, QYr.sicau-2AS, and QYr.sicau-2AL. SNP markers linked to QYr.sicau-1BL (AX-111056129 and AX-108839316) and QYr.sicau-2AS (AX-111557864 and AX-110433540) were converted to breeder-friendly Kompetitive allele-specific PCR (KASP) markers that would facilitate the deployment of stripe rust resistance genes in wheat breeding.

Risk Factors for Refracture following Primary Osteoporotic Vertebral Compression Fractures.

BACKGROUND: In the aging population, osteoporosis and related complications have become a global public health problem. Osteoporotic vertebral compression fractures are among the most common type of osteoporotic fractures and patients are at risk of secondary vertebral compression fracture. OBJECTIVES: To identify risk factors for secondary vertebral compression fracture following primary osteoporotic vertebral compression fractures. STUDY DESIGN: Retrospective study. SETTING: Department of Orthopedic, an affiliated hospital of a medical university. METHODS: This retrospective cohort study evaluated the risk factors for secondary vertebral compression fracture in 317 consecutive patients with systematic osteoporotic vertebral compression fractures who received percutaneous vertebroplasty and kyphoplasty or conservative treatment. Patients were divided into secondary vertebral compression fracture (n = 43) and non- secondary vertebral compression fracture (n = 274) groups. We retrospectively analyzed clinical characteristics and radiographic parameters, including gender, age, body mass index, number of primary fractures, primary treatment (percutaneous vertebroplasty and kyphoplasty or conservative treatment), nonspinal fracture history before primary fracture, primary fracture at the thoracolumbar junction, steroid use, bisphosphonate therapy, and Hounsfield units value of L1. RESULTS: Comparison between the groups showed significant differences in age (P = 0.001), nonspinal fracture history (P < 0.001), and Hounsfield units value of L1 (P < 0.001). The receiver operating characteristic curves demonstrated that the optimal thresholds for age and Hounsfield units value of L1 were 75 (sensitivity: 55.8%; specificity: 67.5%) and 50 (sensitivity: 88.3%; specificity: 67.4%), respectively. In multivariate logistic regression analysis, nonspinal fracture history (OR = 6.639, 95% CI = 1.809 - 24.371, P = 0.004) and Hounsfield units value of L1 < 50 (OR = 15.260, 95% CI = 6.957 - 33.473, P < 0.001) were independent risk factors for secondary vertebral compression fracture. LIMITATIONS: The main limitation is the retrospective nature of this study. CONCLUSION: Patients with low Hounsfield units value of L1 or non-spinal fracture history are an important population to target for secondary fracture prevention.

Comparison of the Agronomic, Cytological, Grain Protein Characteristics, as Well as Transcriptomic Profile of Two Wheat Lines Derived From Wild Emmer.

Two advanced wheat lines BAd7-209 and BAd23-1 without the functional gene GPC-B1 were obtained from a cross between common wheat cultivar Chuannong 16 (CN16) and wild emmer wheat accession D97 (D97). BAd7-209 showed superior quality parameters than those of BAd23-1 and CN16. We found that the components of glutenins and gliadins in BAd7-209 and BAd23-1 were similar, whereas BAd7-209 had higher amount of glutenins and gliadins than those of BAd23-1. RNA sequencing analysis on developing grains of BAd7-209 and BAd23-1 as well as their parents revealed 382 differentially expressed genes (DEGs) between the high-grain protein content (GPC) (D97 + BAd7-209) and the low-GPC (CN16 + BAd23-1) groups. DEGs were mainly associated with transcriptional regulation of the storage protein genes, protein processing in endoplasmic reticulum, and protein export pathways. The upregulated gluten genes and transcription factors (e.g., NAC, MYB, and bZIP) may contribute to the high GPC in BAd7-209. Our results provide insights into the potential regulation pathways underlying wheat grain protein accumulation and contribute to make use of wild emmer for wheat quality improvement.

Comparative analysis of developing grain transcriptome reveals candidate genes and pathways improving GPC in wheat lines derived from wild emmer.

The grain protein content (GPC) in modern wheat is inherently low. Wild emmer wheat (Triticum turgidum ssp. dicoccoides, 2n = 4x = 28, AABB) gene pool harbors wide genotypic variations in GPC. However, the characterization of candidate genes associated with high GPC is a challenge due to the complex characteristic of this trait. In the current study, we performed RNA-seq analysis on developing grains of wild emmer genotype D1, common wheat CN16, and their hexaploid wide hybrid BAd107-4 with contrasting GPC. We have found a total of 39,795 expressed genes on chromosomes A and B, of which 24,152 were shared between D1, CN16, and BAd107-4. From 1744 differentially expressed genes (DEGs), 1203 were downregulated and 541 were upregulated in the high GPC (D1+BAd107-4) compared with low GPC (CN16) groups. The majority of DEGs were associated with protein processing in endoplasmic reticulum, starch and sucrose metabolism, galactose metabolism, and protein export pathways. Expression levels of nine randomly selected genes were verified by qRT-PCR, which was consistent with the transcriptome data. The present database will help us to understand the potential regulation networks underlying wheat grain protein accumulation and provide the foundation for simultaneous improvement of grain protein content and yield in wheat breeding programs.

Transcriptome analysis provides insights into the mechanisms underlying wheat cultivar Shumai126 responding to stripe rust.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a destructive fungal disease of wheat globally. Breeding resistance cultivars is one of the most cost-effective methods to control Pst. Shumai126 (SM126), a high-yielding commercial wheat cultivar, showed strong stripe rust resistance for more than ten years. However, the molecular mechanisms and the responsive genes underlying the SM126 resistance to Pst have not been explored yet. In the present study, RNA-seq was used to analyze changes in the transcriptome at different time points of Pst infection in seedling leaves of SM126. In total, 520, 148 and 1439 differentially expressed genes (DEGs) were found to be up- or down-regulated after Pst infection at 1, 3, and 7 days post inoculation, respectively. The majority of DEGs exhibited transient expression patterns during Pst infection at different time points. GO and KEGG enrichment analysis revealed that many biological processes, such as photosynthesis, flavonoid biosynthesis, oxidative phosphorylation, MAPK signaling pathway, and phenylalanine metabolism are involved in SM126 response to Pst. Expression of genes involved in the plant-pathogen interaction pathway was detected and some key genes showed differential expression. DEGs encoded R proteins and transcription factors were also identified. Our study suggests the gene resources in SM126 related to stripe rust response could be valuable for understanding the mechanisms involved in stripe rust resistance and improvement of wheat resistance to Pst.

Exosomal Transfer of LCP1 Promotes Osteosarcoma Cell Tumorigenesis and Metastasis by Activating the JAK2/STAT3 Signaling Pathway.

Increasing evidence indicates that lymphocyte cytosolic protein 1 (LCP1) overexpression contributes to tumor progression; however, its role in osteosarcoma (OS) remains unclear. We aimed to investigate the potential effect of LCP1 in OS and the underlying mechanisms. We first demonstrated that LCP1 is upregulated in OS cell lines and tissues. Then, we found that aberrant expression of LCP1 could induce the proliferation and metastasis of OS cells in vitro and in vivo by destabilizing neuregulin receptor degradation protein-1 (Nrdp1) and subsequently activating the JAK2/STAT3 signaling pathway. When coculturing OS cells with bone marrow-derived mesenchymal stem cells (BMSCs) in vitro, we validated that oncogenic LCP1 in OS was transferred from BMSCs via exosomes. Moreover, microRNA (miR)-135a-5p, a tumor suppressor, was found to interact upstream of LCP1 to counteract the pro-tumorigenesis effects of LCP1 in OS. In conclusion, BMSC-derived exosomal LCP1 promotes OS proliferation and metastasis via the JAK2/STAT3 pathway. Targeting the miR-135a-5p/LCP1 axis may have potential in treating OS.

Neuron-derived exosomes-transmitted miR-124-3p protect traumatically injured spinal cord by suppressing the activation of neurotoxic microglia and astrocytes.

BACKGROUND: Spinal cord injury (SCI) is a catastrophic injury that can cause irreversible motor dysfunction with high disability. Exosomes participate in the transport of miRNAs and play an essential role in intercellular communication via transfer of genetic material. However, the miRNAs in exosomes which derived from neurons, and the underlying mechanisms by which they contribute to SCI remain unknown. METHODS: A contusive in vivo SCI model and a series of in vitro experiments were carried out to explore the therapeutic effects of exosomes. Then, a miRNA microarray analysis and rescue experiments were performed to confirm the role of neuron-derived exosomal miRNA in SCI. Western blot, luciferase activity assay, and RNA-ChIP were used to investigate the underlying mechanisms. RESULTS: The results indicated that neuron-derived exosomes promoted functional behavioral recovery by suppressing the activation of M1 microglia and A1 astrocytes in vivo and in vitro. A miRNA array showed miR-124-3p to be the most enriched in neuron-derived exosomes. MYH9 was identified as the target downstream gene of miR-124-3p. A series of experiments were used to confirm the miR-124-3p/MYH9 axis. Finally, it was found that PI3K/AKT/NF-κB signaling cascades may be involved in the modulation of microglia by exosomal miR-124-3p. CONCLUSION: A combination of miRNAs and neuron-derived exosomes may be a promising, minimally invasive approach for the treatment of SCI.

MicroRNA-421-3p-abundant small extracellular vesicles derived from M2 bone marrow-derived macrophages attenuate apoptosis and promote motor function recovery via inhibition of mTOR in spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) has a very disabling central nervous system impact but currently lacks effective treatment. Bone marrow-derived macrophages (BMDMs) are recruited to the injured area after SCI and participate in the regulation of functional recovery with microglia. Previous studies have shown that M2 microglia-derived small extracellular vesicles (SEVs) have neuroprotective effects, but the effects of M2 BMDM-derived sEVs (M2 BMDM-sEVs) have not been reported in SCI treatment. RESULTS: In this study, we investigated the role of M2 BMDM-sEVs in vivo and in vitro for SCI treatment and its mechanism. Our results indicated that M2 BMDM-sEVs promoted functional recovery after SCI and reduced neuronal apoptosis in mice. In addition, M2 BMDM-sEVs targeted mammalian target of rapamycin (mTOR) to enhance the autophagy level of neurons and reduce apoptosis. MicroRNA-421-3P (miR-421-3p) can bind to the 3' untranslated region (3'UTR) of mTOR. MiR-421-3p mimics significantly reduced the activity of luciferase-mTOR 3'UTR constructs and increased autophagy. At the same time, tail vein injection of inhibitors of SEVs (Inh-sEVs), which were prepared by treatment with an miR-421-3p inhibitor, showed diminished protective autophagy of neuronal cells in vivo. CONCLUSIONS: In conclusion, M2 BMDM-sEVs inhibited the mTOR autophagy pathway by transmitting miR-421-3p, which reduced neuronal apoptosis and promoted functional recovery after SCI, suggesting that M2 BMDM-sEVs may be a potential therapy for SCI.

miR-210-5p promotes epithelial-mesenchymal transition by inhibiting PIK3R5 thereby activating oncogenic autophagy in osteosarcoma cells.

Osteosarcoma (OS) is a malignant bone tumor which occurs mainly in adolescents with frequent pulmonary metastasis and a high mortality rate. Accumulating evidence has indicated that microRNAs (miRNAs) play a vital role in various tumors by modulating target genes as well as signal pathways, and aberrant expression of miRNAs may contribute to OS progression. This study aimed to determine the association between miR-210-5p expression and OS progression and to investigate its potential underlying mechanism. Using reverse transcription-polymerase chain reaction (RT-PCR), miR-210-5p was found to be upregulated in clinical OS specimens and cell lines. Further functional analysis demonstrated that miR-210-5p promoted epithelial-mesenchymal transition (EMT) and induced oncogenic autophagy. Luciferase reporter assay, RNA-ChIP, and western blot analysis confirmed that PIK3R5, an essential regulator in the AKT/mTOR signaling pathway, is a target downstream gene of miR-210-5p. Overexpression or knockdown of PIK3R5 reversed the functional role of overexpression or knockdown of miR-210-5p, respectively. Silencing autophagy-related gene 5 (ATG5) abolished the functional effects of miR-210-5p upregulation or PIK3R5 knockdown in OS cells. In vivo, miR-210-5p overexpression promoted OS tumor growth and pulmonary metastasis. Taken together, our results demonstrated that miR-210-5p promoted EMT and oncogenic autophagy by suppressing the expression of PIK3R5 and regulating the AKT/mTOR signaling pathway. Therefore, inhibition of miR-210-5p may represent a promising treatment for OS.

Exosome-shuttled miR-216a-5p from hypoxic preconditioned mesenchymal stem cells repair traumatic spinal cord injury by shifting microglial M1/M2 polarization.

BACKGROUND: Spinal cord injury (SCI) can lead to severe motor and sensory dysfunction with high disability and mortality. In recent years, mesenchymal stem cell (MSC)-secreted nano-sized exosomes have shown great potential for promoting functional behavioral recovery following SCI. However, MSCs are usually exposed to normoxia in vitro, which differs greatly from the hypoxic micro-environment in vivo. Thus, the main purpose of this study was to determine whether exosomes derived from MSCs under hypoxia (HExos) exhibit greater effects on functional behavioral recovery than those under normoxia (Exos) following SCI in mice and to seek the underlying mechanism. METHODS: Electron microscope, nanoparticle tracking analysis (NTA), and western blot were applied to characterize differences between Exos and HExos group. A SCI model in vivo and a series of in vitro experiments were performed to compare the therapeutic effects between the two groups. Next, a miRNA microarray analysis was performed and a series of rescue experiments were conducted to verify the role of hypoxic exosomal miRNA in SCI. Western blot, luciferase activity, and RNA-ChIP were used to investigate the underlying mechanisms. RESULTS: Our results indicate that HExos promote functional behavioral recovery by shifting microglial polarization from M1 to M2 phenotype in vivo and in vitro. A miRNA array showed miR-216a-5p to be the most enriched in HExos and potentially involved in HExos-mediated microglial polarization. TLR4 was identified as the target downstream gene of miR-216a-5p and the miR-216a-5p/TLR4 axis was confirmed by a series of gain- and loss-of-function experiments. Finally, we found that TLR4/NF-κB/PI3K/AKT signaling cascades may be involved in the modulation of microglial polarization by hypoxic exosomal miR-216a-5p. CONCLUSION: Hypoxia preconditioning represents a promising and effective approach to optimize the therapeutic actions of MSC-derived exosomes and a combination of MSC-derived exosomes and miRNAs may present a minimally invasive method for treating SCI.

Hypoxic mesenchymal stem cell-derived exosomes promote bone fracture healing by the transfer of miR-126.

Increasing evidence has suggested that paracrine mechanisms might be involved in the underlying mechanism of mesenchymal stem cells (MSCs) transplantation, and exosomes are an important component of this paracrine role. However, MSCs are usually exposed to normoxia (21% O2) in vitro but experience large differences in oxygen concentration in the body under hypoxia. Indeed, hypoxic precondition of MSCs can enhance their paracrine effects. The main purpose of this study was to determine whether exosomes derived from MSCs under hypoxia (Hypo-Exos) exhibit greater effects on bone fracture healing than those under normoxia (Exos). Using in vivo bone fracture model and in vitro experiments including cell proliferation assay, cell migration assay and so on, we confirmed that Hypo-Exos administration promoted angiogenesis, proliferation and migration to a greater extent when compared to Exos. Furthermore, utilizing a series in vitro and in vivo gain and loss of function experiments, we confirmed a functional role for exosomal miR-126 in the process of bone fracture healing. Meanwhile, we found that knockdown of hypoxia inducible factor 1 (HIF-1α) resulted in a significant decrease of miR-126 in MSCs and exosomes, thereby abolishing the effects of Hypo-Exos. In conclusion, our results demonstrated a mechanism by which Hypo-Exos promote bone fracture healing through exosomal miR-126. Moreover, hypoxia preconditioning mediated enhanced production of exosomal miR-126 through the activation of HIF-1α. Hypoxia preconditioning represents an effective and promising method for the optimization of the therapeutic actions of MSC-derived exosomes for bone fracture healing. STATEMENT OF SIGNIFICANCE: Studies have confirmed that transplantation of exosomes exhibit similar therapeutic effects and functional properties to directly-transplanted stem cells but have less significant adverse effects. However, during in vitro culture conditions, MSCs are usually exposed to normoxia (21% O2) which is very different to the oxygen concentrations found in the body under natural physiological conditions. Our results demonstrated a mechanism by which Hypo-Exos promote bone fracture healing through exosomal miR-126 and the SPRED1/Ras/Erk signaling pathway. Moreover, hypoxia preconditioning mediated enhanced production of exosomal miR-126 through the activation of HIF-1α. Hypoxia preconditioning represents an effective and promising method for the optimization of the therapeutic actions of MSC-derived exosomes for bone fracture healing.

miR-624-5p promoted tumorigenesis and metastasis by suppressing hippo signaling through targeting PTPRB in osteosarcoma cells.

BACKGROUND: Accumulating evidence indicates that aberrant microRNA (miRNA) expression contributes to osteosarcoma progression. This study aimed to elucidate the association between miR-624-5p expression and osteosarcoma (OS) development and to investigate its underlying mechanism. METHODS: We analyzed GSE65071 from the GEO database and found miR-624-5p was the most upregulated miRNA. The expression of miR-624-5p and its specific target gene were determined in human OS specimens and cell lines by RT-PCR and western blot. The effects of miR-624-5p depletion or ectopic expression on OS proliferation, migration and invasion were evaluated in vitro using CCK-8 proliferation assay, colony formation assay, transwell assay, would-healing assay and 3D spheroid BME cell invasion assay respectively. We investigated in vivo effects of miR-624-5p using a mouse tumorigenicity model. Besides, luciferase reporter assays were employed to identify interactions between miR-624-5p and its specific target gene. RESULTS: miR-624-5p expression was upregulated in OS cells and tissues, and overexpressing miR-624-5p led to a higher malignant level of OS, including cell proliferation, migration and invasion in vitro and in vivo. Protein tyrosine phosphatase receptor type B (PTPRB) was negatively correlated with miR-624-5p expression in OS tissues. Using the luciferase reporter assay and Western blotting, PTPRB was confirmed as a downstream target of miR-624-5p. PTPRB restored the effects of miR-624-5p on OS migration and invasion. The Hippo signaling pathway was identified as being involved in the miR-624-5p/PTPRB axis. CONCLUSIONS: In conclusion, our results suggest that miR-624-5p is a negative regulator of PTPRB and a risk factor for tumor metastasis in OS progression.

Enriching LMW-GS alleles and strengthening gluten properties of common wheat through wide hybridization with wild emmer.

Two advanced lines (BAd7-209 and BAd7-213) with identical high-molecular-weight glutenin subunit composition were obtained via wide hybridization between low-gluten cultivar chuannong16 (CN16) and wild emmer D97 (D97). BAd7-209 was better than BAd7-213, and both of them were much better than CN16 in a dough quality test. We found that BAd7-209 had more abundant and higher expression levels of low-molecular-weight glutenin subunit (LMW-GS) proteins than those of BAd7-213. Twenty-nine novel LMW-GS genes at Glu-A3 locus were isolated from BAd7-209, BAd7-213 and their parents. We found that all 29 LMW-GS genes possessed the same primary structure shared by other known LMW-GSs. Twenty-seven genes encode LMW-m-type subunits, and two encode LMW-i-type subunits. BAd7-209 had a higher number of LMW-GS genes than BAd7-213, CN16, and D97. Two wild emmer genes MG574329 and MG574330 were present in the two advanced lines. Most of the LMW-m-type genes showed minor nucleotide variations between wide hybrids and their parents that could be induced through the wide hybridization process. Our results demonstrated that the wild emmer LMW-GS alleles could be feasibly transferred and integrated into common wheat background via wide hybridization and the potential value of the wild emmer LMW-GS alleles in breeding programs designed to improve wheat flour quality.

Genome-Wide Association Study Reveals Novel Genomic Regions Associated With High Grain Protein Content in Wheat Lines Derived From Wild Emmer Wheat.

Grain protein content (GPC) and yield are of two important traits in wheat, but their negative correlation has hampered their simultaneous improvement in conventional breeding. Wild emmer wheat (Triticum turgidum ssp. dicoccoides) is an important genetic resource for wheat quality improvement. In this study, we report a genome-wide association study (GWAS) using 13116 DArT-seq markers to characterize GPC in 161 wheat lines derived from wild emmer. Using a general linear model, we identified 141 markers that were significantly associated with GPC, and grouped into 48 QTL regions. Using both general linear model and mixed linear model, we identified four significant markers that were grouped into two novel QTL regions on chromosomes 2BS (QGpc.cd1-2B.1) and 7BL (QGpc.cd1-7B.2). The two QTLs have no negative effects on thousand kernel weight (TKW) and should be useful for simultaneous improvement of GPC and TKW in wheat breeding. Searches of public databases revealed 61 putative candidate/flanking genes related to GPC. The putative proteins of interest were grouped in four main categories: enzymes, kinase proteins, metal transport-related proteins, and disease resistance proteins. The linked markers and associated candidate genes provide essential information for cloning genes related to high GPC and performing marker-assisted breeding in wheat.