Diabetic chronic wounds represent a major clinical challenge characterized by sustained oxidative stress, excessive inflammation, poor angiogenesis, and high susceptibility to bacterial infection. Single-component biomaterials are unable to address the multiple pathological disorders of diabetic wound microenvironments, resulting in unsatisfactory therapeutic efficacy. Although materials inspired by mussel foot proteins (MFPs) have shown potential for wound repair, the direct application of these proteins to diabetic wound therapy remains largely unexplored. In this study, three functional mussel foot proteins (Mcfp3, Mcfp5, Mcfp6) were recombinantly expressed and assembled into a ternary protein complex Mcfps(3,5,6). An arginine/serine-modified chitosan (ASCS) hydrogel carrying Mcfps(3,5,6) was further fabricated for diabetic chronic wound therapy. In vitro results revealed that Mcfps(3,5,6) significantly promoted cell adhesion, migration, and angiogenesis, efficiently eliminated ROS, increased SOD and GSH-Px activities, and drove macrophage polarization from pro-inflammatory M1 toward anti-inflammatory M2 phenotype. The hydroxylation rate of the ternary complex reached 43.84%, much higher than that of individual proteins. The ASCS hydrogel gelled rapidly at 37°C, exhibited good biocompatibility and effective antibacterial activity. In full-thickness diabetic mouse wounds, ASCS/Mcfps(3,5,6) hydrogel markedly accelerated wound closure, with a healing rate of 98.91% at day 14. It also reduced inflammatory infiltration and ROS levels, enhanced collagen deposition, and promoted angiogenesis. Mechanistically, the composite hydrogel restored redox homeostasis, and facilitated extracellular matrix remodeling. This multifunctional recombinant protein-hydrogel system possesses antioxidant, anti-inflammatory, pro-angiogenic capacities and acts as a promising biomacromolecular platform to treat chronic diabetic wound.

Single gene encoded phage lysis proteins offer a promising strategy for bacterial ghost production, yet their host-dependent regulatory mechanisms remain poorly understood. Here, we investigated the lysis protein E from phage ID52 (ID52-E), which exhibits stronger lytic activity than φX174 E. By screening ID52-E-resistant mutants, we identified a four-base insertion in secB as the genetic alteration associated with lysis resistance in BL21, and CRISPR-Cas9-mediated secB disruption confirmed that SecB is required for ID52-E-mediated lysis. Proteomic analysis revealed altered protein expression in resistant mutants despite preserved bacterial morphology. Co-immunoprecipitation and biolayer interferometry supported an apparent interaction between SecB and ID52-E, with an apparent KD of 3.541 × 10- 8 M under the tested 1:1 fitting model. Molecular docking, molecular dynamics simulations, mutagenesis, lysis assays, and binding measurements further implicated SecB Ala145 as a key interface residue. Together, these findings identify SecB as a host factor that facilitates ID52-E-mediated bacterial lysis and provide mechanistic insight for improving bacterial ghost production.

Several HSP90 inhibitors are in use or late-stage trials: pimitespib is approved in Japan for intestinal tumors, hypericin sodium under US regulatory review, and WP-1303 in Phase III development. However, their efficacy depends on tumor HSP90 expression levels, necessitating probes for subtype-specific detection in vivo. Translation is limited by inadequate validation in specific subtypes and off-target accumulation in kidneys and liver. We developed and optimized an HSP90-targeted radiotracer addressing these limitations. Chemical modifications enhanced tumor uptake in colorectal and gastric cancer models (10.02 ± 2.05% and 5.02 ± 0.08% ID/g), while reducing liver and kidney retention (∼2% and ∼5% ID/g), yielding tumor/muscle ratios of 23.28 ± 9.70 and 16.73 ± 2.80. Clinical evaluation confirmed translational potential, enabling tumor delineation and high-contrast imaging (SUVmax ∼ 5). This probe supports comprehensive cancer management and may guide clinical application of emerging HSP90 inhibitors.

BACKGROUND:Epidemiological studies have shown an association between obstructive sleep apnea (OSA) and metabolic dysfunction-associated steatotic liver disease (MASLD), yet the precise role of OSA in MASLD severity remains unclear. We aimed to evaluate the association of OSA with liver histological severity in Chinese bariatric surgery patients, especially metabolic dysfunction-associated steatohepatitis (MASH) and significant fibrosis. METHODS:This prospective cross-sectional study enrolled 582 consecutive bariatric surgery patients. Key exclusions were other liver diseases, significant alcohol intake, and prior OSA treatment. All participants underwent preoperative polysomnography and intraoperative liver biopsy. Patients were stratified by biopsy-proven MASLD severity and OSA severity (apnea-hypopnea index, AHI). The primary outcomes were the associations of OSA with histologically defined MASH and significant fibrosis (stage ≥2). RESULTS:More severe MASLD subtypes exhibited higher AHI and greater hypoxia (p < 0.05). OSA severity was positively associated with histological features, including steatosis, ballooning, lobular inflammation, and fibrosis (all p < 0.05), as well as with higher NAFLD activity score (NAS) (p < 0.001) and MASH activity grade (p < 0.001). Multivariable analysis identified OSA as an independent predictor associated with histologically confirmed MASLD (odds ratio [OR] 1.86, 95% confidence interval [CI], 1.08-3.22), lobular inflammation (OR 95%CI, 1.18-2.79), MASH (OR 95%CI, 1.09-2.71), and significant fibrosis (OR 95%CI, 1.49-7.08). A linear dose-response relationship existed between AHI and both MASH and significant fibrosis (p < 0.05). CONCLUSIONS:OSA is associated with more severe histological features of MASLD and contributes to the severity of MASH and significant fibrosis in patients with obesity.

In vitro oocyte maturation (IVM) is a pivotal process influencing the success of embryo production in laboratory and clinical settings. However, oxidative stress (OS) often compromises oocyte quality during IVM. Antioxidants such as melatonin and epigallocatechin-3-gallate (EGCG) are known to mitigate OS by neutralizing reactive oxygen species (ROS) and bolstering antioxidant defenses. Despite extensive studies on their individual effects, the synergistic impact of melatonin and EGCG remains underexplored. Utilizing a mouse model, this study evaluated their combined effect on oocyte maturation, focusing on nuclear and cytoplasmic development, intracellular ROS, glutathione (GSH) levels, and subsequent embryonic competence. The results demonstrated that melatonin and EGCG significantly enhanced the polar body extrusion rate (p < 0.05), with the combination group achieving the highest rate of 91.96%. Cumulus expansion was observed to improve across all treated groups, with the combination treatment showing the highest cumulus expansion index (CEI) of 3.06. Furthermore, the combination treatment significantly reduced ROS levels and increased GSH content, indicating enhanced antioxidant capacity (p < 0.01). Embryonic development outcomes, including cleavage and blastocyst rates, were markedly higher in the combination group at 75.23% and 53.97%, respectively, demonstrating superior developmental potential (p < 0.01). These findings suggest that the melatonin-EGCG combination offers a novel and effective strategy to combat oxidative damage during IVM, thereby improving oocyte quality and embryonic development potential in mice.

An advanced and highly sensitive analytical platform for SARS-CoV-2 is of crucial for public health. In this study, a machine learning-assisted platform that utilizes ratiometric fluorescent paper sensors based on the metal- -organic framework Al3+/Au NCs@ZIF-90 was developed for precise and sensitive point-of-care testing (POCT) of SARS-CoV-2. This platform employs RdRp gene-induced hyperbranched rolling circle amplification (HRCA) to produce pyrophosphate (PPi) as a by-product, which triggers fluorescence quenching in ratiometric fluorescent paper sensors. Under ultraviolet (UV) excitation, the blue fluorescence emitted by ZIF-90 within Al3+/Au NCs@ZIF-90 serves as a reference signal, whereas the red fluorescence from Al3+/Au NCs acts as the analytical signal, with the fluorescence intensity being proportional to the PPi concentration. This approach not only ensures achieves high sensitivity but also exhibits a visible change of fluorescence color, achieving a limit of detection (LOD) of 0.3 pM specifically for SARS-CoV-2. By leveraging these distinctive fluorescence signals, the machine learning-assisted platform, which employs the Residual Neural Network (ResNet) algorithm, analyzes fluorescence images to discern SARS-CoV-2 RNA concentrations with an accuracy rate exceeding 99 %. The innovative platform integrates ratiometric fluorescent paper sensors with machine learning, offering a promising solution for point-of-care testing (POCT) of COVID-19 and potentially facilitating the early diagnosis of various diseases.

Purpose:Insulin resistance, a hallmark feature of type 2 diabetes and cardiovascular diseases, is critically influenced by liver-adipose tissue crosstalk, offering a novel therapeutic strategy for its management. Emerging evidence indicates that extracellular vesicles (EVs) secreted from adipose tissue serve as essential carriers of miRNA-mediated interorgan communication. This study aimed to investigate the regulatory effects of adipose tissue-derived EVs on obesity-induced hepatic insulin resistance and to elucidate the underlying molecular mechanisms by which EV-mediated signaling contributes to metabolic dysfunction. Methods:EVs with miR-141-3p knockout or overexpression were constructed and administered to both in vitro cell models and in vivo mouse models to investigate the regulatory role and underlying mechanisms of miR-141-3p-mediated adipose tissue-derived EVs in obesity-induced hepatic insulin resistance. Results:miR-141-3p is significantly upregulated in adipose tissue-derived EVs from high-fat diet (HFD)-fed mice, as well as in other obesity-related conditions. Furthermore, the knockdown of miR-141-3p in EVs from chow diet (CD-EVs) counteracted the effect in improving obesity-induced hepatic insulin resistance, whereas the overexpression of miR-141-3p in HFD-EVs improved hepatic insulin resistance. Mechanistically, EVs-derived miR-141-3p directly targets PTEN to promote PI3K/AKT signaling, thereby mediating hepatic glucose homeostasis through the regulation of hepatic gluconeogenesis and glycogen synthesis. Conclusion:In summary, our results highlight the emerging role of miR-141-3p in mediating adipose tissue-derived EVs to alleviate obesity-induced hepatic insulin resistance, providing potential therapeutic targets for type 2 diabetes.

A simple, rapid, low-cost, and multiplex detection platform is crucial for the diagnosis of infectious diseases, especially for on-site pathogen screening. However, current methods are difficult to satisfy the requirements for minimal instrument and multiplexed point-of-care testing (POCT). Herein, we propose a versatile and easy-to-use platform (FARPA-chip) by combining multiplex FARPA with an autosampling microfluidic chip. A pair of universal recombinase polymerase amplification (RPA) primers introduced during double-stranded cDNA (ds-cDNA) preparation are employed to amplify multiple targets, followed by amplicon-decoding with the chip, indicating no bias in amplifying different targets due to the universal RPA primers. FARPA-chip exhibits that as low as 10 copies of each target RNA in the starting sample can be sensitively detected by 12-plex detection of vector-borne viruses within 45 min and no cross-talk is observed between different targets. The feasibility of this platform is confirmed by designing a 9-plex FARPA-chip to detect 6 kinds of clinically common respiratory viruses from 16 clinical samples of nasopharyngeal swabs, and the results are completely consistent with RT-qPCR. Furthermore, by integrating quick extraction reagent, the turnaround time can be significantly decreased to <50 min, highlighting that our FARPA-chip enables a cost-effective on-site pathogen screening with a relatively high level of multiplexing. Depending on the number of chambers in the chip, the current design is theoretically capable of detecting up to 24 different pathogens, which should fulfill most clinical purposes. We believe that the proposed platform could provide an effective way for a series of healthcare-related applications in resource-limited settings.

Antimicrobial susceptibility testing (AST) is crucial for guiding timely antibiotic therapy. However, the conventional AST requires several days to issue a report. Although digital polymerase chain reaction (dPCR) can reduce AST turnaround time by determining bacterial susceptibility after short antibiotic exposure, the process remains time-consuming. Recombinase polymerase amplification (RPA) offers greater efficiency since it runs at room temperature and avoids amplicon denaturing or primer annealing. Nevertheless, adapting RPA to a digital format has been challenging because amplification begins prematurely before compartmentalization, inevitably leading to overestimation. Here, we proposed a photo-initiated droplet digital RPA (ddRPA) method that eliminates pre-amplification. We modified three bases within RPA primers with photocleavable groups to efficiently block RPA, and ddRPA starts only after irradiation. Due to the high efficacy of RPA in droplets, quantification is completed within only 8 min. Based on the photo-controlled strategy, we developed an ultra-fast AST platform (PIRATE) for bloodstream infections (BSI) pathogens. Using Klebsiella pneumoniae and Enterococcus faecalis against quinolones as model systems, PIRATE demonstrates 100 % concordance with standard AST results across 33 clinical isolates and 8 blood samples while reducing the turnaround time from 3 days to under 6 h. This approach shows significant potential for guiding precise antibiotic prescription.

The early rapid and ultrasensitive detection of pathogenic bacteria is crucial for preventing foodborne illnesses and addressing bacterial infections effectively. Herein, we have developed an ultrasensitive and rapid method for detecting Salmonella typhimurium based on specific phage tail spike protein-magnetic separation, dual signal amplification by four-in-one AuPt nanoparticles coated enzyme-antibiotic-inorganic nanoflowers and smartphone-assisted paper sensor. The generation of nanoflowers involved the encapsulation of horseradish peroxidase (HRP) and polymyxin B (PMB) within inorganic nanocrystal composites in Ca2 + solution, imitating the natural biomineralization process through nanoarchitectonics. Subsequently, the nanoenzyme AuPt nanoparticles were coupled to the nanoflower surface by electrostatic adsorption for enhanced catalytic activity. The prepared HRP-PMB-CaHPO4@AuPt nanoflowers could both target gram-negative bacteria using the biorecognition unit (PMB) and alleviate enzymatic activity by dual signal amplification elements (HRP and AuPt). The phage tail spike protein-magnetic beads were utilized for specific separation of S. typhimurium. Following this, a paper-based sensor containing 3,3 ',5,5 '-tetramethyl benzidine (TMB) catalyzed by HRP and AuPt was introduced to induce a color change. Finally, a smartphone application (APP) was used to analyze the RGB (redgreen-blue) values of the paper sensor, enabling the direct detection of S. typhimurium. The limit of detection (LOD) was 3.28 x 101 CFU/mL and the entire detection took 30 min, showing exceptional performance. This proposed assay, serving as a rapid and highly sensitive platform, successfully addresses the requirement for an efficient detection method for S. typhimurium, particularly in limited-resources settings.

Loop-mediated isothermal amplification (LAMP) is a detection method widely used in pathogen detection and clinical diagnosis. Nevertheless, it is highly constrained by thermal stability, catalytic activity, and resistance to inhibitors of Bst DNA polymerase. In this study, a novel DNA polymerase was characterized from Clostridium thermocellum, exhibiting potential in LAMP detection. Through bioinformatics analysis, the enzyme and the DNA-binding domain (DBD) from Pyrococcus abyssi were mutated for enhanced interaction between proteins and DNA. A chimeric mutant DBDE146K-S738R reaches the detection threshold 13 min earlier than wild-type Cth DNA polymerase in real-time LAMP detection with a template concentration of 1.58 × 105 fg/µL. It also showed the highest enzymatic activity at pH 9.0 and 65°C. The chimeric enzyme DBDE146K-S738R exhibits good thermal stability, capable of performing LAMP reactions after treatment at 73°C or 70°C for 8 h. Moreover, it maintains high activity even under the inhibitory conditions of 50 U/mL heparin, 1.6 mM EDTA, 200 mM NaCl, 10% ethanol, 1.2 M urea, or 0.8% phenol. Notably, it was able to detect 1.58 × 102 ag/µL of the genome and 1.03 CFU/mL of the colony in Salmonella typhimurium detection. The enzyme's performance is superior to commercial Bst 2.0 and comparable to commercial Bst 3.0. The results suggest that DBDE146K-S738R in LAMP exhibits great potential for molecular biological studies and clinical diagnostic analysis.

High-fat diet (HFD)-induced obesity leads endothelial dysfunction and contributes to cardiovascular diseases. NLRP3-mediated pyroptosis plays a key role in endothelial injury induced by HFD. Pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide belonging to the secretin family, has demonstrated diverse beneficial effects. However, its impact on a high-fat-induced pyroptosis remains unexplored. The purpose of this study is to evaluate the effect of PACAP in alleviating high-fat-induced pyroptosis of human umbilical vein endothelial cells (HUVECs) and to elucidate its potential mechanisms. The results show that palmitic acid (PA) induces HUVECs injury and pyroptosis, while PACAP alleviates PA-induced HUVECs injury and pyroptosis. In addition, PACAP also has a protective effect on vascular damage in the thoracic aorta of obese mice. We further found that PACAP reduced PA-induced intracellular Reactive Oxygen Species (ROS) in HUVECs, while also mitigating PA-induced HUVECs pyroptosis. Moreover, PACAP can inhibit PA-induced ROS and pyroptosis through activation of SIRT1, and the effects of PACAP are reversed by a SIRT1 inhibitor. In conclusion, our study demonstrates that PACAP can inhibit PA-induced oxidative stress and pyroptosis in HUVECs, and its action is closely related to the SIRT1 pathway.

Recombinant protein technology, a pivotal subset of biotechnology, facilitates the generation of specific proteins via recombinant DNA techniques, predominantly within heterologous expression systems [...]

Hyperpigmentation is a skin disorder characterized by the abnormal accumulation of melanin, in which the melanocortin-1 receptor (MC1R) was recognized as a key target for melanin production. Antisense oligonucleotide (ASO), by targeting mRNA to inhibit protein synthesis, holds a promising gene therapy for hyperpigmentation. However, challenges such as nonspecific activation, off-target toxicity, and limited cellular permeability hinder their broader clinical application. Inspired by the Chinese proverb "Turn an enemy into a friend", we introduce a dual-gated, ultraviolet (UV)-tyrosinase (TYR) cascade-responsive modified ASO (m-ASO) that enables spatiotemporally controlled silencing of MC1R. The engineered m-ASO is caged via thymidine modification, rendering it inactive until sequentially exposed to exogenous UV and intracellular TYR. UV irradiation, conventionally hyperpigmentation trigger, is repurposed to initiate decaging of m-ASO into an intermediate form, ph-ASO. This intermediate is then enzymatically activated by TYR overexpressed in melanocytes within pigmented tissues, ultimately restoring the native conformation and therapeutic function of the ASO. This "enemy-to-ally" strategy allows for disease-specific, precision activation of ASO therapeutics. Notably, to enhance transdermal delivery, the m-ASO nanocomplex is integrated into a biocompatible, dissolvable microneedle system for painless and localized epidermal administration. The resulting platform demonstrates precise MC1R silencing and effective melanin suppression, while sparing normal melanocytes. Together, this cascade-caged, cell-selective, and minimally invasive strategy presents a novel framework for programmable nucleic acid therapeutics in dermatological and enzyme-overexpressing diseases.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme involved in DNA repair and cellular redox regulation, and is frequently overexpressed in tumor cells. This highlights the urgent need for a rapid and high-sensitive point-of-care testing (POCT) strategy for APE1 to facilitate early cancer diagnosis. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification method; however, its application in APE1 detection remains rare. Here, we introduce a versatile RCA-Lock (Rlock) conversion platform that enables the transformation of RCA-based nucleic acid detection technologies into APE1-responsive assays. Building upon this platform, we further developed a novel POCT method for APE1 detection─Rlock-mediated, CRISPR/Cas12a-driven RCA cycle (RCRE)─which, for the first time, integrates CRISPR/Cas12a with RCA into a one-pot APE1 detection system. The RCRE assay achieves a limit of detection of 8.86 × 10-4 U/mL within 30 min, while requiring minimal equipment, low cost, and no complex handling procedures. This Rlock-based conversion strategy represents a transformative advance in the field of APE1 diagnostics and offers conceptual inspiration for the design of programmable nucleic acid-based biosensors. The resulting RCRE assay significantly broadens the technological landscape for early cancer detection and paves the way for future clinical translation.

Framework nucleic acids effectively meet the demands for precise size control and accurate targeting in the design of drug delivery systems, while developing a controllable drug delivery system with low immunogenicity and high efficiency for delivering nucleic acid drugs to the tumor immune microenvironment (TIME) remains significant challenge. Inspired by ancient Chinese mortise and tenon joint structures, this study develops an intelligent self-assembling DNA tetrahedron (TDN@siCSF-1R), which consists of a gapped DNA tetrahedron (TDN) and a therapeutic siRNA against Colony-Stimulating Factor-1 Receptor (siCSF-1R) that non-covalently bind with TDN via its gap, aiming to target tumor-associated macrophages (TAMs) and inhibit the CSF-1R pathway. Additionally, a CD206 mRNA-responsive sequence is introduced into the gapped TDN, triggering the site-specific release of siCSF-1R in M2-like TAMs, thereby achieving the precise targeting of CSF-1R in M2-like TAMs and reducing off-target effect. The mortise-and-tenon-like TDN@siCSF-1R synchronously combines the self-assembly flexibility and structural stability, significantly inhibiting 4T1 tumor growth, lung metastasis, and tumor recurrence after resection in vivo. Furthermore, it repolarizes M2-like TAMs and activates infiltrating T cells in TIME, thereby reshaping the immunosuppressive microenvironment, and offering a promising strategy for the clinical application of cancer immunotherapy.