Synovitis-driven inflammation and oxidative stress are key drivers of osteoarthritis (OA) progression. As a master regulator of antioxidant and anti-inflammatory defenses, nuclear factor erythroid 2-related factor 2 (Nrf2) represents a promising therapeutic target. However, current strategies for Nrf2 activation remain limited in achieving durable synovial gene expression and pathology-adaptive release. Here, we developed an injectable inflammation-responsive nanocolloidal hydrogel enabling sustained and on-demand Nrf2 activation within OA joints. The hydrogel was fabricated by crosslinking polyvinyl alcohol (PVA) with phenylboronic acid (PBA)-functionalized nanoparticles encapsulating Nrf2 plasmids. Dynamic boronate ester linkages between PBA and PVA enabled rapid in situ gelation after intra-articular injection. In the ROS-enriched inflammatory microenvironment of OA, cleavage of boronate ester bonds triggered the release of Nrf2 plasmid-loaded nanoparticles. The released nanoparticles were efficiently internalized by fibroblast-like synoviocytes (FLSs) and promoted Nrf2 expression, thereby suppressing oxidative stress and inflammatory responses. In ACLT-induced OA mice, the hydrogel markedly alleviated synovial inflammation, preserved cartilage matrix, and reduced the OARSI score by approximately 70%. These findings highlight its potential as a promising strategy for inflammation-adaptive gene regulation in OA therapy.

ABO-incompatible kidney transplantation is widely used to meet the escalating need for organs. Current recipient-centric desensitization protocols involving antibody depletion through plasmapheresis increase the risk of infections, perioperative bleeding events and costs. Here we present a donor-centric desensitization protocol, converting type-A kidneys into enzyme-converted O kidneys during hypothermic perfusion to remove the A antigen from the kidneys. An ex vivo model resulted in no antibody-mediated injury. Encouraged by this, an enzyme-converted O kidney was transplanted into a type-O brain-dead recipient with a high titre of anti-A antibody, and no hyperacute rejection was observed. The graft was well tolerated with no evidence of antibody-mediated rejection for 2 days. Antibody-mediated lesions and complement deposition were found starting 3 days post-transplant, coinciding with A-antigen regeneration, and later higher Banff scores, suggesting an immune-mediated response. Single-cell sequencing confirms the elevated expression of accommodation-related genes, suggesting the potential for longer-term tolerance. This study provides a donor-centric organ engineering strategy and has the potential to broaden the reach of ABO-incompatible kidney transplantation, improving the fairness of and access to organ allocation. An ex vivo model and pre-clinical study in a brain-dead recipient provide enzyme-converted O organs to avoid hyperacute rejection in ABO-incompatible kidney transplant patients.

Although the combination of radiotherapy and immunotherapy is regarded as a promising clinical treatment strategy, numerous clinical trials have failed to demonstrate synergistic effects. One of the key reasons is that conventional radiotherapies inevitably damage intratumoral effector immune cells. Boron Neutron Capture Therapy (BNCT) is a precise radiotherapy that selectively kills tumor cells while sparing adjacent normal cells, by utilizing 10B agents and neutron irradiation. Therefore, combinational BNCT-immunotherapy holds promise for achieving more effective synergistic effects. Here it develops a 10B-containing polymer that self-assembled with PD-L1 siRNA to form 10B/siPD-L1 nanoparticles for combinational BNCT-immunotherapy. Unlike antibodies, PD-L1 siRNA can inhibit intracellular PD-L1 upregulated by BNCT, activating T-cell immunity while also suppressing DNA repair. This can enhance BNCT-induced DNA damage, promoting immunogenic cell death (ICD) and further amplifying the antitumor immune effect. The results demonstrated that BNCT using 10B/siPD-L1 nanoparticles precisely killed tumor cells while sparing adjacent T cells and induced a potent antitumor immune response, inhibiting distal and metastatic tumors.

The accurate and timely detection of disease biomarkers at the point-of-care is essential to ensuring effective treatment and epidemiological surveillance. Here, we report the selection and engineering of RNA-cleaving DNAzymes that respond to specific genetic markers and amplify detection signals. Because the target-specific activation of gene-specific DNAzymes (gDz) is like the trans-cleavage activity of clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated (Cas) machinery, we further developed a CRISPR-like assay using RNA-cleaving DNAzyme coupled with isothermal sequence and signal amplification (CLARISSA) for nucleic acid detection in clinical samples. Building on the high sequence specificity and orthogonality of gDzs, CLARISSA is highly versatile and expandable for multiplex testing. Upon integration with an isothermal recombinase polymerase amplification, CLARISSA enabled the detection of human papillomavirus (HPV) 16 in 189 cervical samples collected from cervical cancer screening participants (n = 189) with 100% sensitivity and 97.4% specificity, respectively. A multiplexed CLARISSA further allowed the simultaneous analyses of HPV16 and HPV18 in 46 cervical samples, which returned clinical sensitivity of 96.3% for HPV16 and 83.3% for HPV18, respectively. No false positives were found throughout our tests. Besides the fluorescence readout using fluorogenic reporter probes, CLARISSA is also demonstrated to be fully compatible with a visual lateral flow readout. Because of the high sensitivity, accessibility, and multiplexity, we believe CLARISSA is an ideal CRISPR-Dx alternative for clinical diagnosis in field-based and point-of-care applications.

Hemophilic articular cartilage damage presents a significant challenge for surgeons, characterized by recurrent intraarticular bleeding, a severe inflammatory microenvironment, and limited self-repair capability of cartilage tissue. Currently, there is a lack of tissue engineering-based integrated therapies that address both early hemostasis, anti-inflammation, and long-lasting chondrogenesis for hemophilic articular cartilage defects. Herein, we developed an adhesive hydrogel using oxidized chondroitin sulfate and gelatin, loaded with exosomes derived from bone marrow stem cells (BMSCs) (Hydrogel-Exos). This hydrogel demonstrated favorable injectability, self-healing, biocompatibility, biodegradability, swelling, frictional and mechanical properties, providing a comprehensive approach to treating hemophilic articular cartilage defects. The adhesive hydrogel, featuring dynamic Schiff base bonds and hydrogen bonds, exhibited excellent wet tissue adhesiveness and hemostatic properties. In a pig model, the hydrogel could be smoothly injected into the knee joint cartilage defect site and gelled in situ under fluid-irrigated arthroscopic conditions. Our in vitro and in vivo experiments confirmed that the sustained release of exosomes yielded anti-inflammatory effects by modulating macrophage M2 polarization through the NF-κB pathway. This immunoregulatory effect, coupled with the extracellular matrix components provided by the adhesive hydrogel, enhanced chondrogenesis, promoted the cartilage repair and joint function restoration after hemophilic articular cartilage defects. In conclusion, our results highlight the significant application potential of Hydrogel-Exos for early hemostasis, immunoregulation, and long-term chondrogenesis in hemophilic patients with cartilage injuries. This innovative approach is well-suited for application during arthroscopic procedures, offering a promising solution for addressing the complex challenges associated with hemophilic articular cartilage damage.

Osteomyelitis induced by Staphylococcus aureus (S. aureus) is a persistent and deep-seated infection that affects bone tissue. The main challenges in treating osteomyelitis include antibiotic resistance, systemic toxicity, and the need for multiple recurrent surgeries. An ideal therapeutic strategy involves the development of materials that combine physical, chemical, and immunomodulatory synergistic effects. In this work, we prepared injectable microspheres consisting of an interpenetrating network of ionic-cross-linked sodium alginate (SA) and genipin (Gp)-cross-linked gelatin (Gel) incorporated with tannic acid (TA) and copper ions (Cu2+). The Gp-cross-linked Gel acted as a "naturally-derived" photothermal therapy (PTT) agent. The results showed that the microspheres exhibited efficient and rapid bactericidal effects against both S. aureus and Escherichia coli (E. coli) under the irradiation of near-infrared light at 808 nm wavelength; moreover, the release of Cu2+ also induced sustained inhibitory effects against bacteria during the nonirradiation period. The in vitro cell culture results indicated that when combined with PTT, the microspheres could adaptively modulate macrophage M1 and M2 phenotypes in sequence. Additionally, these microspheres were found to enhance the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). In vivo studies conducted in a rat femur osteomyelitis model with bone defects showed that under multiple laser irradiation the microspheres effectively controlled bacterial infection, improved the pathological immune microenvironment, and significantly enhanced the repair and regeneration of bone tissues in the affected area.

Rheumatoid arthritis (RA) progression involves multiple cell types, and sequential drug action on target cells is necessary for RA treatment. Nanocarriers are widely used for RA treatment; however, the targeted delivery and on-demand release of multiple drugs remains challenging. Therefore, in this study, a dual-sensitive polymer is developed using chondroitin sulfate (CS) for the co-delivery of the cartilage repair agent, glucosamine (GlcN), and anti-inflammatory drug, tofacitinib (Tof). In the joint cavity, acidic pH facilitates the cleavage of GlcN from CS polymer to repair the cartilage damage. Subsequently, macrophage uptake via CS-CD44 binding and intracellular reactive oxygen species (ROS) mediate conversion of (methylsulfanyl)propylamine to a hydrophilic segment jointly triggered rapid Tof/GlcN release via micelle disassembly. The combined effects of Tof, GlcN, and ROS depletion promote the M1-to-M2 polarization shift to attenuate inflammation. The synergistic effects of these agents against RA are confirmed in vitro and in vivo. Overall, the dual pH/ROS-sensitive CS nanoplatform simultaneously delivers GlcN and Tof, providing a multifunctional approach for RA treatment with synergistic drug effects. An amphiphilic polymeric prodrug for the co-delivery of tofacitinib (Tof) and glucosamine (GlcN) is developed using polymer poly(aspartic acid-(methylsulfanyl)propylamine)-grafted chondroitin sulfate (-glucosamine) (CS(-GlcN)-g-PAsp(MSPA)). This dual pH- and ROS-sensitive nanocarrier can specifically control the release of GlcN and Tof in the inflamed joint to reduce pro-inflammatory factors and repair cartilage damage, respectively, with a synergistic anti-RA therapy.image

Over the past three decades, significant advancements have been made in mutation enrichment methods, driven by the increasing need for precise and efficient identification of rare genetic variants associated with diseases. Mutation-enrichment methods have emerged to boost sensitivity and enable easy detection of low-frequency mutations. These methods are crucial in genomics research and clinical diagnostics, allowing for the detection of low-frequency mutations within large genomic datasets. This review presents a summary of technological developments in rare mutation enrichment and emphasizes their mechanisms and applications in liquid biopsies.

Cationic materials currently serve as the primary materials for siRNA encapsulation; however, inherent cationic toxicity and limited shelf-life have hindered their clinical applicability. Although several approaches for encapsulating siRNA without relying on cationic materials exist, achieving a high encapsulation efficiency remains a major challenge. In this study, a feasible strategy is developed for efficiently encapsulating siRNAs in nanogels, without the use of cationic materials. The use of amphiphilic monomers facilitated a higher concentration of monomers at the interlayer, presenting a superior polymerization capability in the inverse emulsion polymerization system. This facilitates the entrapment of siRNA within the nanogel rather than its extrusion. The pH-responsiveness of the monomer and the enzyme sensitivity of the crosslinkers conferred several benefits to the siRNA-loaded nanogels, including charge reversal, endo/lysosomal escape, and controlled siRNA release. The efficiency of the nanogel for siRNA delivery and gene inhibition is confirmed through a series of experiments. Thus, a feasible platform is developed for siRNA delivery that is free of cationic toxicity. Amphiphilic monomers involved in inverse miniemulsion polymerization achieve high siRNA encapsulation efficiency independent of electrostatic interaction; MMP2-sensitive crosslinkers induce a charge reversal from nearly neutral to positive to enhance cellular uptake; cleavage of CTSB-sensitive crosslinker within the lysosome enables disassembly of the nanomedicine and subsequent release of siRNA into the cytoplasm. image

DNA hybridization probes are commonly used tools to discriminate clinically important single nucleotide variants (SNVs) but often work at elevated temperatures with very narrow temperature intervals (ΔT). Herein, we investigated the thermodynamic basis of the narrow ΔT both in silico and experimentally. Our study revealed that the high entropy penalty of classic hybridization probe designs was the key attributor for the narrow ΔT. Guided by this finding, we further introduced an entropy-compensate probe (Sprobe) design by coding intrinsic disorder into a stem-loop hybridization probe. Sprobe expanded ΔT from less than 10 °C to over 30 °C. Moreover, both ΔT and the optimal reaction temperature can be fine-tuned by simply altering the length of the loop domain. Sprobe was clinically validated by analyzing EGFR L858R mutation in 36 pairs of clinical tumor tissue samples collected from lung cancer patients, which revealed 100 % clinical sensitivity and specificity. We anticipate that our study will serve as a general guide for designing thermal robust hybridization probes for clinical diagnostics.

Healing of large calvarial bone defects remains challenge but may be improved by stimulating bone regeneration of implanted cells. The aim of this study is to specially co-activate transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF-A) genes expressions in pre-osteoblast MC3T3-E1 cells through the non-viral CRISPR activation (CRISPRa) system to promote osteogenesis. A cationic copolymer carrying nucleus localizing peptides and proton sponge groups dimethyl-histidine was synthesized to deliver CRISPRa system into MC3T3-E1 cells with high cellular uptake, lysosomal escape, and nuclear translocation, which activated VEGF-A and TGF-β1 genes expressions and thereby additively or synergistically induced several osteogenic genes expressions. A tunable dual-crosslinked hydrogel was developed to implant the above engineered cells into mice calvaria bone defect site to promote bone healing in vivo. The combination of multi-genes activation through non-viral CRISPRa system and tunable dual-crosslinked hydrogel provides a versatile strategy for promoting bone healing with synergistic effect.

Osteomyelitis is commonly developed via hematogenous spreading or direct inoculation of bacteria from orthopedics trauma. Pathogens-induced bone destruction impedes the penetration of antibiotics to the infection site, and the severe inflammation further compromises the traditional treatment outcome. In this work, vancomycin-loaded oligochitosan nanoparticles (Van-NPs) with antibacterial, antibiofilm, antioxidant as well as bone regenerative properties are prepared using sodium tripolyphosphate (TPP) as a crosslinker, and employed for the treatment of osteomyelitis. Van-NPs exhibit strong interactions with dissociative S. aureus and biofilms due to the positive zeta potential, the additional effect between vancomycin (Van) and oligochitosan (OCS) further contributes to an enhanced antibacterial and antibiofilm outcome. The in vitro osteogenic differentiation of rBMSCs is facilitated by the antioxidant ability of Van-NPs and the TPP-induced activation of ERK1/2 and p38 signaling pathways. Moreover, the combination of Van-NPs with PLGA-PEG-PLGA gel (Gel/Van-NPs) achieves successful localized treatment of osteomyelitis in terms of enhanced bacteria elimination, inflammatory modulation, and accelerated bone regeneration. Therefore, Gel/Van-NPs may serve as a promising biomaterial for the optimal treatment of osteomyelitis.

The therapeutic effect of photodynamic therapy (PDT) is highly dependent on the intracellular production of reactive oxygen species (ROS). However, the ROS generated by photosensitizers can be consumed by the highly concentrated glutathione (GSH) in tumor cells, severely impairing the therapeutic effect of PDT. Herein, we synthesized a GSH-scavenging copolymer to deliver photosensitizer chlorin e6 (Ce6). The pyridyl disulfide groups, which have faster reactivity with the thiol groups of GSH than other disulfide groups, were grafted onto a hydrophobic block to encapsulate the Ce6. Under NIR irradiation, the Ce6 generated ROS to kill tumor cells, and the pyridyl disulfide groups depleted the GSH to prevent ROS consumption, which synergistically enhanced the therapeutic effect of PDT. In vitro and in vivo experiments confirmed the combinatory antitumor effect of Ce6-induced ROS generation and the pyridyl disulfide group-induced GSH depletion. Therefore, the pyridyl disulfide group-grafted amphiphilic copolymer provides a more efficient strategy for enhancing PDT and has promising potential for clinical application.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems have revolutionized biological and biomedical sciences in many ways. The last few years have also seen tremendous interest in deploying the CRISPR-Cas toolbox for analytical and diagnostic assay development because CRISPR-Cas is one of the most powerful classes of molecular machineries for the recognition and manipulation of nucleic acids. In the short period of development, many CRISPR-enabled assays have already established critical roles in clinical diagnostics, biosensing, and bioimaging. We describe in this review the recent advances and design principles of CRISPR mediated analytical tools with an emphasis on the functional roles of CRISPR-Cas machineries as highly efficient binders and molecular scissors. We highlight the diverse engineering approaches for molecularly modifying CRISPR-Cas machineries and for devising better readout platforms. We discuss the potential roles of these new approaches and platforms in enhancing assay sensitivity, specificity, multiplexity, and clinical outcomes. By illustrating the biochemical and analytical processes, we hope this review will help guide the best use of the CRISPR-Cas toolbox in detecting, quantifying and imaging biologically and clinically important molecules and inspire new ideas, technological advances and engineering strategies for addressing real-world challenges such as the on-going COVID-19 pandemic.

Soybean provides superior and readily available protein for human and livestock. However, nutritional value of soybean is limited due to the deficiency of an essential amino acid, methionine. To improve total methionine content of soybean, a methionine-rich seed storage protein, β-zein, was introduced into soybean cultivar ’Jack’ under the control of legumin B4 promoter or CaMV 35S promoter. Totally 4 T3 transgenic lines exhibited higher expression levels of foreign genes, and legumin B4 promoter directed a stronger accumulation of β-zein protein than CaMV 35S promoter. Compared to wild type plant, total methionine content in transgenic soybean seeds significantly increased by up to approximately 15%. Although the introduction of β-zein gene improved total methionine content, the level was negligible compared to native soybean storage proteins, implying that the inadequate soluble methionine is the limiting factor. Based on these observations, a new strategy for simultaneously increasing the “source” and “sink” of methionine metabolism is proposed to further improvement of total methionine content in soybean seed.

Concepts and strategies offered by constitutional dynamic chemistry (CDC) hold great promise for designing molecular computing systems adaptive to external environments. Despite demonstrable success in storing and processing chemical information using CDC, further employment of such constitutional dynamic networks (CDNs) for processing more complex digital information has not been realized yet. Herein, we introduced a supramolecular CDN based on the aggregation of cyanineMTC(Agg-CDN), which is composed of four reversibly interconvertible constituents,i.e.monomers, dimers, J-aggregates, and H-aggregates. We demonstrated that the equilibrated Agg-CDN is reconfigurable through constituent exchange in response to well-defined chemical inputs. More importantly, the equilibrated states of the Agg-CDN are spectroscopically distinguishable because of the unique optical properties ofMTC. We further tuned the Agg-CDN to at least nine unique states for transforming the chemical inputs into digital outputs, and successfully employed it for encoding and encrypting complex digital information, such as multi-pixel images.

The CRISPR/Cas9 system, consisting of Cas9 nuclease and single guide RNA (sgRNA), is an emerging gene editing technology that can perform gene reprogramming operations such as deletion, insertion, and point mutation on DNA sequences targeted by sgRNA. In addition, CRISPR/dCas9 (a mutant that loses Cas9 nuclease activity) still retains the ability of sgRNA to target DNA. The fusion of dCas9 protein with transcriptional activator (CRISPRa) can activate the expression of the target gene, and fusion transcriptional repressors (CRISPRi) can also be used to suppress target gene expression. Efficient delivery of the CRISPR/Cas9 system is one of the main problems limiting its wide clinical application. Viral vectors are widely used to efficiently deliver CRISPR/Cas9 elements, but non-viral vector research is more attractive in terms of safety, simplicity, and flexibility. In this review, we summarize the principles and research advances of CRISPR technology, including CRISPR/ Cas9 delivery vectors, delivery methods, and obstacles to the delivery, and review the progress of CRISPR-based research in bone and cartilage tissue engineering. Finally, the challenges and future applications of CRISPR technology in bone and cartilage tissue engineering are discussed.

Due to low content of sulfur containing amino acid in soybean, understanding of cystathionine gamma-synthase coding gene GmCGS which plays a key role in synthesis of methionine, at transcriptional level is important to solving this problem. In this study, the transcriptional level of CGS in transgenic soybean lines overexpressing At D-CGS and wild type (WT) was detected after sulfate or methionine treatment designed to identify their effects on regulating the expression of GmCGS. After treated with 4. 5 mmol/L sulfate, the relative expression level of GmCGS was significantly increased in WT, whereas the transcription level of GmCGS in transgenic lines overexpressing At D-CGS was significantly decreased. No significant difference was observed after methionine treatment.Compared to WT, the transcriptional level of GmCGS was significantly decreased in transgenic lines. The results indicated that the effect of cultivation technique on expression of GmCGS was too limited to improve methionine content. All of these provided reference for increasing content of Met in soybean.

Soybean is one of the world's most important oil and protein crops. Efficient transformation is a key factor for the improvement of soybean by genetic modification. We describe an optimized protocol for the Agrobacterium rhizogenes-mediated transformation of soybean and the induction of hairy root development in vitro. Cotyledons with 0.5-cm hypocotyls were cut from 5-day-old seedlings and used as explants. After infection and co-cultivation, hairy roots were produced in induction culture medium after 10–12days. Using this method, 90%–99% of the infected explants of five different cultivars produced hairy roots within one month. Observations using reporter constructs showed that 30%–60% of the hairy roots induced were transformed. Based on high transformation efficiency and short transformation period, this method represents an efficient and rapid platform for study of soybean gene function.

As a genetically modified crop, transgenic soybean occupies the largest global scale with its food, nutritional, industrial, and pharmaceutical uses.Efficient transformation is a key factor for the improvement of genetically modified soybean. At present, the Agrobacterium-mediated method is primarily used for soybean transformation, but the efficiency of this method is still relatively low (below 5%) compared with rice (above 90%). In this study, we examined the influence of l-glutamine and/or l-asparagine on Agrobacterium-mediated transformation in soybean and explored the probable role in the process of Agrobacterium-mediated transformation. The results showed that when the amino acids l-glutamine and l-asparagine were added separately or together to the culture medium, the shoot induction frequency, elongation rate, and transformation frequency were improved. The combined effects of l-glutamine and l-asparagine were better than those of l-glutamine and l-asparagine alone. The 50 mg/L l-glutamine and 50 mg/L l-asparagine together can enhance the transformation frequency of soybean by attenuating the expression level of GmPRs (GmPR1, GmPR4, GmPR5, and GmPR10) and suppression of the plant defense response. The transgene was successfully transmitted to the T1 generation. This study will be useful in genetic engineering of soybean.