Development of an NLR-ID Toolkit and Identification of Novel Disease-Resistance Genes in Soybean.

The recognition of pathogen effectors through the nucleotide-binding leucine-rich repeat receptor (NLR) family is an important component of plant immunity. In addition to typical domains such as TIR, CC, NBS, and LRR, NLR proteins also contain some atypical integrated domains (IDs), the roles of which are rarely investigated. Here, we carefully screened the soybean (Glycine max) genome and identified the IDs that appeared in the soybean TNL-like proteins. Our results show that multiple IDs (36) are widely present in soybean TNL-like proteins. A total of 27 Gm-TNL-ID genes (soybean TNL-like gene encoding ID) were cloned and their antiviral activity towards the soybean mosaic virus (SMV)/tobacco mosaic virus (TMV) was verified. Two resistance (R) genes, SRA2 (SMV resistance gene contains AAA_22 domain) and SRZ4 (SMV resistance gene contains zf-RVT domain), were identified to possess broad-spectrum resistance characteristics towards six viruses including SMV, TMV, plum pox virus (PPV), cabbage leaf curl virus (CaLCuV), barley stripe mosaic virus (BSMV), and tobacco rattle virus (TRV). The effects of Gm-TNL-IDX (the domain of the Gm-TNL-ID gene after the TN domain) on the antiviral activity of a R protein SRC7TN (we previously reported the TN domain of the soybean broad-spectrum resistance gene SRC7) were validated, and most of Gm-TNL-IDX inhibits antiviral activity mediated by SRC7TN, possibly through intramolecular interactions. Yeast-two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that seven Gm-TNL-IDX interacted with SMV-component proteins. Truncation analysis on a broad-spectrum antiviral protein SRZ4 indicated that SRZ4TIR is sufficient to mediate antiviral activity against SMV. Soybean cDNA library screening on SRZ4 identified 48 interacting proteins. In summary, our results indicate that the integration of IDs in soybean is widespread and frequent. The NLR-ID toolkit we provide is expected to be valuable for elucidating the functions of atypical NLR proteins in the plant immune system and lay the foundation for the development of engineering NLR for plant-disease control in the future.

Observation of Zn Dendrite Growth via Operando Digital Microscopy and Time-Lapse Tomography.

The zinc-ion battery is one of the promising candidates for next-generation energy storage devices beyond lithium technology due to the earth's abundance of Zn materials and their high volumetric energy density (5855 mA h cm-3). To date, the formation of Zn dendrites during charge-discharge cycling still hinders the practical application of zinc-ion batteries. It is, therefore, crucial to understand the formation mechanism of the zinc dendritic structure before effectively suppressing its growth. Here, the application of operando digital optical microscopy and in situ lab-based X-ray computed tomography (X-ray CT) is demonstrated to probe and quantify the morphologies of zinc electrodeposition/dissolution under multiple galvanostatic plating/stripping conditions in symmetric Zn||Zn cells. With the combined microscopy approaches, we directly observed the dynamic nucleation and subsequent growth of Zn deposits, the heterogeneous transportation of charged clusters/particles, and the evolution of 'dead' Zn particles via partial dissolution. Zn electrodeposition at the early stage is mainly attributed to activation, while the subsequent dendrite growth is driven by diffusion. The high current not only facilitates the formation of sharp dendrites with a larger mean curvature at their tips but also leads to dendritic tip splitting and the creation of a hyper-branching morphology. This approach offers a direct opportunity to characterize dendrite formation in batteries with a metal anode in the laboratory.

Decoy engineering of the receptor-like cytoplasmic kinase StPBS1 to defend against virus infection in potato.

Potato virus Y (PVY) is an important pathogen of potato (Solanum tuberosum). Although the PBS1-RPS5 immune system is well documented in Arabidopsis thaliana, it has not been reported in potato. In Arabidopsis, the bacterial effector AvrPphB cleaves AtPBS1 to trigger an immune response. Here, we show that the AvrPphB-triggered immune response is mediated by StPBS1, a close homologue of AtPBS1 in potato. However, downstream signalling of StPBS1 was mediated by unknown resistance (R) proteins other than potato orthologues of AtRPS5 and HvPBR1, which is important for HvPBS1 signalling in barley. Immune signalling of StPBS1 is mediated by the AvrPphB C-terminal cleavage domain and an STKPQ motif, in contrast to AtPBS1-mediated immunity in which both AvrPphB cleavage fragments and an SEMPH motif are essential. The cleavage sequence of AvrPphB in StPBS1 was replaced with that of the PVY NIa-Pro protease to obtain StPBS1NIa . StPBS1NIa overexpression potato displayed stronger immunity to PVY infection than did the StPBS1 transgenic lines. StPBS1NIa was cleaved at the expected target site by NIa-Pro protease from PVY. Thus, we characterized the function of StPBS1 in potato immunity and provide a biotechnology control method for PVY via transformation of decoy-engineered StPBS1NIa .

In-Situ Li-Ion Pouch Cell Diagnostics Utilising Plasmonic Based Optical Fibre Sensors.

As the drive to improve the cost, performance characteristics and safety of lithium-ion batteries increases with adoption, one area where significant value could be added is that of battery diagnostics. This paper documents an investigation into the use of plasmonic-based optical fibre sensors, inserted internally into 1.4 Ah lithium-ion pouch cells, as a real time and in-situ diagnostic technique. The successful implementation of the fibres inside pouch cells is detailed and promising correlation with battery state is reported, while having negligible impact on cell performance in terms of capacity and columbic efficiency. The testing carried out includes standard cycling and galvanostatic intermittent titration technique (GITT) tests, and the use of a reference electrode to correlate with the anode and cathode readings separately. Further observations are made around the sensor and analyte interaction mechanisms, robustness of sensors and suggested further developments. These finding show that a plasmonic-based optical fibre sensor may have potential as an opto-electrochemical diagnostic technique for lithium-ion batteries, offering an unprecedented view into internal cell phenomena.

A Multiscale X-Ray Tomography Study of the Cycled-Induced Degradation in Magnesium-Sulfur Batteries.

Rechargeable Mg/S batteries have the potential to provide a compelling battery for a range of applications owing to their high capacity and gravimetric energy density, safety, and low-cost construction. However, the Mg/S energy storage is not widely developed and deployed due to technical challenges, which include short cycle lifespan and lack of suitable electrolyte. To study the microstructure degradation of Mg/S batteries, multiscale X-ray tomography, an inherently nondestructive method, is performed on dismantled Swagelok Mg/S cells comprising a graphene-sulfur cathode and a super-P separator. For the first time, 3D microstructure visualization and quantification reveal the dissolution (volume fraction decreases from 13.5% to 0.7%, surface area reduces from 2.91 to 1.74 µm2 µm-3 ) and agglomeration of sulfur particles, and the carbon binder densification after 10 cycles. Using tomography data, the image-based simulations are then performed. The results show that the insoluble polysulfides can inevitably block the Mg2+ transportation via shuttle effect. The representative volume should exceed 8200 µm3 to represent bulk cathode. This work elucidates that the Mg/S cell performance is significantly affected by microstructural degradation, and moreover demonstrates how multiscale and multimodal characterization can play an indispensable role in developing and optimizing the Mg/S electrode design.

The Identified Hub Gene GlcN in Osteoarthritis Progression and Treatment.

BACKGROUND: As a chronic disease, osteoarthritis has caused great trouble to the health of middle-aged and elderly people. Studies have shown that glucosamine (GlcN) can be used to abate the progression and improve this disease. Based on this point of view, we try to verify the connection between GlcN and osteoarthritis and find more effective biomarkers. METHODS: We downloaded the GSE72575 data set from the GEO database, and used the R language to perform DEG analysis on the gene expression profile of the samples. Next, the GO function and the KEGG signaling pathways were analyzed through the DAVID database, and then, the KEGG pathways enriched in the gene set were analyzed based on GSEA. Then, the PPI network of DEGs was constructed based on the STRING online database, and finally, the hub genes were selected by Cytoscape. RESULTS: Three GlcN-treated MH7A cell treatment groups and 3 control groups in the GSE72575 data set were studied. Through analysis, there were 52 DEGs in these samples. Then, through GO, KEGG, and GSEA, regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway, FoxO signaling pathway, JAK-STAT signaling pathway, PI3K-Akt signaling pathway, TGF-beta signaling pathway, and ECM receptor interaction were involved in the regulatory mechanisms of the osteoarthritis pathogenesis. After that, the hub genes IL6 and DDIT3 were identified through PPI network construction and analysis. And it was found that IL6 was lowly expressed in the group with GlcN-treated MH7A cells, while DDIT3 was highly expressed. CONCLUSION: The above results provide a basis for GlcN to participate in the treatment of osteoarthritis and a possibility for finding effective therapeutic targets.

miR-101-loaded exosomes secreted by bone marrow mesenchymal stem cells requires the FBXW7/HIF1α/FOXP3 axis, facilitating osteogenic differentiation.

Exosomes derived from mesenchymal stem cells (MSCs) have emerged as significant mediators of intercellular communication, with studies highlighting their role in the transmission of biological signals between cells. Dominant microRNA (miRNA)-mediated translational repression of messenger RNAs has been extensively investigated in regard to its influence in orchestrating osteogenic differentiation. In the current study, we sought to ascertain the contributory role of miRNA-101 (miR-101) encapsulated in the process of bone marrow mesenchymal stem cell (BMSC)-derived exosomes in osteogenic differentiation. Exosomes were initially extracted from BMSCs at Days 0, 3, 12, and 21 of osteogenic differentiation by ultracentrifugation. Artificial modulation of miR-101 and FBXW7 (silencing and overexpression) were performed in the BMSCs to identify its effects on osteogenic factors, alkaline phosphatase activity, and osteogenic differentiation. Mechanistic exploration was performed to evaluate the binding affinity between miR-101 and FBXW7, the FBXW7-mediated HIF1α ubiquitination, and the HIF1α enrichment in the FOXP3 promoter region. Exosomes from MSCs in the late stage of osteogenic differentiation exhibited enhanced osteogenic differentiation. Upregulated miR-101 in MSC-derived exosomes was detected during osteogenic differentiation, while diminished levels of FBXW7 expression was noted. Importantly, miR-101 was found to specifically bind to the 3'-untranslated region of FBXW7. Meanwhile, data was obtained indicating that FBXW7 could ubiquitinate and degrade HIF1α to repress its upregulation during osteogenic differentiation. HIF1α bound to the promoter region of FOXP3 to facilitate osteogenic differentiation. Ultimately, the findings of the current study demonstrate that BMSC-derived exosomal miR-101 augments osteogenic differentiation in MSCs by inhibiting FBXW7 to regulate the HIF1α/FOXP3 axis.

3D Phase Field Modeling of Multi-Dendrites Evolution in Solidification and Validation by Synchrotron X-ray Tomography.

In this paper, the dynamics of multi-dendrite concurrent growth and coarsening of an Al-15 wt.% Cu alloy was studied using a highly computationally efficient 3D phase field model and real-time synchrotron X-ray micro-tomography. High fidelity multi-dendrite simulations were achieved and the results were compared directly with the time-evolved tomography datasets to quantify the relative importance of multi-dendritic growth and coarsening. Coarsening mechanisms under different solidification conditions were further elucidated. The dominant coarsening mechanisms change from small arm melting and interdendritic groove advancement to coalescence when the solid volume fraction approaches ~0.70. Both tomography experiments and phase field simulations indicated that multi-dendrite coarsening obeys the classical Lifshitz-Slyozov-Wagner theory Rn-R0n = kc(t-t0), but with a higher constant of n = 4.3.

MicroRNA-15b shuttled by bone marrow mesenchymal stem cell-derived extracellular vesicles binds to WWP1 and promotes osteogenic differentiation.

BACKGROUND: Osteogenic differentiation is an essential process for bone regeneration involving bone marrow mesenchymal stem cells (BMSCs). BMSC-secreted extracellular vesicles (EVs) enriched with microRNAs (miRs) have vital roles to play in mediating osteogenic differentiation. Therefore, this study aimed to explore the effect of BMSC-derived EVs loaded with miR-15b on osteogenic differentiation. METHODS: Human BMSCs (hBMSCs) were cultured and treated with plasmids overexpressing or knocking down KLF2, WWP1, and miR-15b to define the role of derived EVs in osteogenic differentiation in vitro. The expression of osteogenic differentiation-related marker was measured by Western blot analysis. The interaction among miR-15b, WWP1, and ubiquitination of KLF2 was investigated by dual-luciferase reporter, immunoprecipitation, and GST pull-down assays. Moreover, EVs from hBMSCs transfected with miR-15b inhibitor (EV-miR-15b inhibitor) were injected into ovariectomized rats to verify the effect of miR-15b on bone loss in vivo. RESULTS: WWP1 was downregulated, and KLF2 was upregulated during osteogenic differentiation. After co-culture with EVs, miR-15b expression was elevated and WWP1 expression was reduced in hBMSCs. Upregulation of miR-15b or KLF2 or downregulation of WWP1 or NF-κB increased ALP activity and cell mineralization, as well as osteogenic differentiation-related marker expression in hBMSCs. Mechanistically, miR-15b targeted and inhibited WWP1, thus attenuating KLF2 degradation and inhibiting NF-κB activity. Co-culture of EVs increased the bone volume and trabecular number, but decreased bone loss in ovariectomized rats, which could be reversed after treatment with EV-miR-15b inhibitor. CONCLUSION: Collectively, BMSC-derived EVs loaded with miR-15b promoted osteogenic differentiation by impairing WWP1-mediated KLF2 ubiquitination and inactivating the NF-κB signaling pathway.

Studies on the lipid-regulating mechanism of alisol-based compounds on lipoprotein lipase.

Studies on the lipid-regulating effects of alisol compounds are reported that include alisol B, alisol A 24-acetate (24A), alisol A and an alisol B - 24A - alisol A mixture (content ratio = 1:1:1). The effects on the activity of lipoprotein lipase (LPL), a key lipid-modulating enzyme, were studied to investigate the molecular mechanism of lipid-regulating activity of alisols. The effects of alisols on regulating blood lipids and the activities of LPL were determined using a reagent kit method. The structure of LPL was obtained by homology modeling and the interactive mechanism of alisol monomers and the mixture with LPL was investigated by molecular simulation. The alisol monomer and mixture were shown to regulate blood lipids, suggesting that alisols may decrease the level of triglyceride (TG) by improving the activity of LPL. The order of intensity was: mixture > alisol A > alisol B > 24A, indicating that alisols of alismatis rhizoma feature a synergistic effect on LPL. The N- and C-terminus of LPL both represented the catalytic active domains of this lipid-regulating effect. Cys306, Gln129 and Ser166 were the key amino acid residues resulting in the lipid-regulating effect of the alisol monomer while Ser166 and Arg18 were found to be responsible for the lipid-regulating effect of the mixture. The C-terminus of LPL was indirectly involved in the enzymatic process. A folded side chain of alisols or the parent ring was found to bind somewhat weaker to LPL than an open side chain or parent ring. The hydroxyl groups on the C14-, C22-, C28-, C30- and C31-terminus in the side chain, the ring ether structure in C23-position, and the acetyl group in C29-position represented the key sites for the lipid-regulating action of alisols. Meanwhile, the C30-site hydroxyl group played an important role in the synergistic effect of the alisol mixture.

Study on antitumor molecular mechanism of Alisols based on p53DNA.

Methyl thiazolyl tetrazolium (MTT) assay, UV-vis absorption spectroscopy, fluorescence spectroscopy and molecular simulation were used to investigate the antitumor activity of alisol A, alisol B and an 1:1 mixture of both compounds, the mechanism of its interaction with anti-cancer target p53DNA and explored the antitumor mechanism of alisols. MTT assay showed that the order of antitumor activity was:alisol B > alisol A > alisol A-alisol B(1:1). Spectroscopic experiments and molecular simulation suggested that alisol A, alisol B and their mixture interact with p53DNA in by partial insertion and the strength of binding affinity was consistent with the MTT assay. The Ksv of alisol A was 9.35 × 104 L·mol-1, Kq was 9.35 × 1012 L·mol-1·s-1 and the Ksv and Kq of alisol B were 11.61 × 104 L·mol-1 and 11.61 × 1012 L·mol-1·s-1. The molecular simulation revealed that competitive antagonism was observed in the interaction between the alisol mixture and p53DNA. The critical groups and significant binding sites for the interaction between alisol monomers and p53DNA include C19-OH and C22-OH of the alisols; N2 and H21 of the guanine deoxynucleotide (DG8), N2-H21 of the DG7, O4' of the DG9 in the f-chain of p53DNA; and C2-O2 of the cytosine deoxynucleotide (DC16) in the e-chain of p53DNA. Also, the C-22 and C23- of the alisols and the DA18-DT5 base pairs of p53DNA were key factors in the interaction of the mixture with p53DNA.

Transplantation of copper-doped calcium polyphosphate scaffolds combined with copper (II) preconditioned bone marrow mesenchymal stem cells for bone defect repair.

Calcium polyphosphate is a bioactive ceramic that possesses similar mineral components to bone and possess good physicochemical properties. However, pure calcium polyphosphate scaffold is brittle, and it is insufficient in promoting vascularization and osteoinductivity. This study was conducted to assess whether copper (Cu) incorporated into calcium polyphosphate could improve the scaffolds' inherent shortcomings. In the experiments, Cu-calcium polyphosphate scaffolds' mechanical strength, biocompatibility, and biodegradability were researched primarily. And then, hypoxia-inducible factor 1-alpha expression along with angiogenesis and osteogenesis potential when the scaffolds treated with the bone marrow mesenchymal stem cells (BMMSCs) were analyzed in vitro. In in vivo studies, the Cu-calcium polyphosphate scaffolds combined with the liquid extract preconditioned BMMSCs were implanted into animal model to repair the bone defects. Meanwhile, we also evaluate the expression of angiogenic and osteogenic factors. For comparison, Cu-calcium polyphosphate, calcium polyphosphate, and blank control groups were designed. According to the results, proper content of Cu incorporated with calcium polyphosphate (0.1% Cu-calcium polyphosphate) did not significantly change the scaffold's degradation velocity, but it obtained higher compress mechanical strength and Cu-doped scaffolds were less brittle. Besides, these scaffolds incorporated with Cu showed better cytocompatibility and cell proliferation activity. Moreover, after Cu was doped, the hypoxia-inducible factor 1-alpha expression was up-regulated with the angiogenic and osteogenic factors levels increased both in in vitro and in vivo study. The bone defect healing capacity was accessed, using Cu-calcium polyphosphate combined with preconditioned BMMSCs further enhanced new bone formation and improved hypoxia-inducible factor 1-alpha, alkaline phosphatase, osteocalcin, and vascular endothelial growth factor expression. In conclusion, doped Cu into calcium polyphosphate was an alternative strategy for improving calcium polyphosphate's mechanical property and promoting the osteogenesis and angiogenesis potential. Using Cu-calcium polyphosphate scaffolds combined with Cu preconditioned BMMSCs to treat bone defect could enhance bone defect healing.

Periarticular multimodal drug injection in total knee arthroplasty.

PURPOSE: A systematic review and meta-analysis based on randomized controlled trials (RCTs) were conducted to evaluate the efficiency and safety of periarticular multimodal drug injection in total knee arthroplasty (TKA). METHODS: Periarticular injection with the use of multimodal drugs is an efficient alternative for postoperative analgesia in TKA. A systematical electronic search was performed to identify the eligible RCTs in the databases of PubMed, Embase, Cochrane Central Register of Controlled Trials, Web of Science and the Chinese Biomedical Literature Database. Two independent reviewers completed data collection and assessment of methodological quality. The quality of evidence of outcomes was judged using GRADE criteria. Meta-analysis was performed for the outcomes of pain, straight leg raise, operating time, hospital stay and complications. RESULTS: Ten RCTs including eight studies with 1,216 TKAs in 835 patients met the inclusion criteria. Periarticular injection with multimodal drugs in TKA was associated with short-term benefits in terms of pain relief, straight leg raise, narcotic consumption, and the rates of nausea, vomiting, rash and pruritus. There were no statistically significant differences in operating time, hospital stay, wound complications and deep vein thrombosis between both groups. CONCLUSIONS: The current evidence suggests that periarticular multimodal drug injection in TKA provides short-term advantages in pain relief, straight leg raise and postoperative complications.

Marrow stromal stem cell autologous transplantation in denervated fracture healing: an experimental study in rats.

OBJECTIVE: To investigate the influence of bone marrow stromal stem cell (BMSCs) transplantation on healing of fractures combined with central nerve injuries in rats. METHODS: Forty-eight healthy adult SD male rats were randomly divided into the following three groups (16 rats in each group): group A, simple (left) tibial fracture; group B, tibial fracture combined with T10 spinal cord transection (SCT); group C, tibial fracture combined with T10 SCT and BMSCs transplantation. The tibial fractures were stabilized with modular intramedullary nails and all operated hind limbs were further immobilized in plaster casts to prevent unequal load bearing. BMSCs were labeled with bromodeoxyuridine and implanted into the fractures of C group rats 2 days after creation of the model. The animals in B and C groups were evaluated by postoperative Tarlov scores. The fractured tibiae were evaluated separately radiographically (X-ray and CT) and immunohistochemically 1, 2, 3 and 4 weeks after injury to assess fracture healing. In addition, the wet weights of the left tibias were measured. RESULTS: All Tarlov score of the B and C group animals reached the requirements of the experiment. One, 2 and 3 weeks after surgery, the tibial callus widths in B and C group animals were significantly greater than those of group A rats (P < 0.05). At 4 weeks the tibial callus width in group C animals had decreased, but still differed significantly from that in group A rats (P < 0.05). One, 2, 3 and 4 weeks after surgery, the wet weights of B and C group tibias were significantly greater than those of group A (P < 0.05). Hematoxylin-eosin-stained sections showed bony union and increased bone trabecula in B and C groups and areas with particles positive for alkaline phosphatase staining were more abundant in groups B and C, especially in group C. CONCLUSION: Neural regulation plays an important role in fracture healing. Treatment with BMSCs has a positive effect on defective callus in rats that have been subjected to SCT.

Subvastus versus medial parapatellar approach in total knee arthroplasty: meta-analysis.

The subvastus and medial parapatellar approaches are 2 commonly performed techniques in total knee arthroplasty, but the optimal approach for total knee arthroplasty remains controversial. The purpose of this study was to compare the effectiveness and safety of the subvastus vs medial parapatellar approach.The PubMed, Embase, Cochrane Library, Inter-Services Intelligence Web of Knowledge, and Chinese Biomedical Literature databases were searched for eligible quasi-randomized, controlled and randomized, controlled trials. Two authors independently extracted data and assessed the methodological quality of the included studies according to the Cochrane handbook version 5.1.0. Statistical analysis was performed using Review Manager version 5.1 software. Eight randomized, controlled trials and 1 quasi-randomized, controlled trial involving 940 primary total knee arthroplasties were included for meta-analysis. Meta-analysis revealed significant differences favoring the subvastus group in Knee Society Score in terms of function at 4 to 6 weeks (weighted mean difference [WMD]=5.09; 95% confidence interval [CI], 3.08 to 7.09; P<.01) and knee score at 12 months (WMD=2.17; 95% CI, 0.01 to 4.34; P=.05) and lateral retinacular release (odds ratio=0.34; 95% CI, 0.14 to 0.79; P=.01) when compared with the medial parapatellar approach. However, both groups showed similar results in range of motion (P>.05), operative time (WMD=2.15; 95% CI, -3.61 to 7.35; P=.42), blood loss (WMD= -31.07; 95% CI, -91.89 to 29.75; P=.32), hospital stay (WMD= -0.18; 95% CI, -0.67 to 0.31; P=.47), and postoperative complications (P>.05).