ObjectiveThe widespread adoption of portable fundus cameras for primary care and community screening is hindered by limitations in current autofocus(AF) technologies. Image-based methods relying on sharpness evaluation require iterative searches, resulting in slow convergence, while projection-based techniques are susceptible to optical artifacts and calibration errors. To address these challenges, this study introduces a novel AF system based on direct wavefront sensing, designed to deliver simultaneous high speed, high precision, and operational robustness within the compact form factor essential for portable ophthalmic devices. MethodsOur approach fundamentally reimagines the AF process by directly measuring the ocular wavefront aberration. We developed a custom portable fundus camera integrating a miniaturized Shack-Hartmann wavefront sensor (SHWS) into the optical path. An 850 nm laser diode projects a point source onto the retina via oblique illumination to minimize corneal reflections. Light scattered from this spot carries the eye’s refractive error through the imaging optics and is directed to the SHWS, positioned at a plane optically conjugate to the primary color CMOS imaging sensor. A microlens array within the SHWS samples the incident wavefront, generating a pattern of focal spots on a CCD. Real-time centroid analysis of these spots provides a map of local wavefront slopes. These measurements are processed through a singular value decomposition (SVD) algorithm to fit a Zernike polynomial basis set, enabling real-time reconstruction of the wavefront phase. The defocus component (S) is extracted from the second-order Zernike coefficients, providing a direct, quantitative measure of the refractive error in diopters. This value serves as a precise error signal in a closed-loop control system, which commands a voice-coil actuated focusing lens to its null position in a single, deterministic step, eliminating the need for iterative search algorithms. ResultsComprehensive evaluation demonstrated the system’s high performance. Testing on a calibrated model eye (OEMI-7) established a highly linear relationship between the computed defocus S and the focusing lens position across a ±20 Diopter (D) compensation range, achievable within a 5 mm mechanical travel. The system achieved a focusing precision of 0.08 D, corresponding to an 18-fold improvement over a conventional projection spot-size method tested under identical conditions. The total focus acquisition time, encompassing wavefront measurement, computation, and lens actuation, averaged under 0.5 s. Clinical validation with 25 human volunteers (50 eyes, refractive range -15 D to +10 D) confirmed practical efficacy. The wavefront-sensing AF succeeded in 92% of attempts with a mean time of 0.5 s, substantially outperforming a projection-based benchmark which achieved only a 32% success rate with an average time of 4.25 s. The system provided instantaneous directional guidance and maintained stability during minor ocular movements. Objective assessment of image quality, via amplitude contrast of retinal vasculature, showed consistent and significant enhancement following AF correction across the entire tested diopter range. ConclusionThis work successfully implements and validates a direct wavefront-sensing autofocus paradigm for portable fundus cameras. By directly quantifying and compensating for the optical defocus aberration, this method bypasses the fundamental limitations of image-processing and projection-based techniques, enabling rapid, precise, and deterministic diopter compensation. The developed system delivers an exceptional combination of a wide operational range (±20 D), high accuracy (0.08 D), fast convergence (0.5 s), and a compact physical footprint. This technology provides a practical and high-performance focusing solution capable of enhancing the reliability, throughput, and diagnostic utility of portable retinal imaging in large-scale screening applications. Future efforts will be directed towards system cost optimization and performance adaptation for diverse ocular conditions.

Inteins are unique protein insertion sequences capable of self-excision, enabling the covalent ligation of flanking extein peptides via amide bond formation. This process proceeds spontaneously without requiring external enzymes, cofactors, or chemical reagents, granting inteins exceptional biocompatibility and traceless performance in protein engineering applications. Split inteins represent a specialized and versatile subclass whose splicing domains are encoded by two separate gene fragments rather than a single continuous open reading frame. These fragments, known as the N-terminal (IntN) and C-terminal (IntC) split inteins, associate through non-covalent interactions including hydrophobic forces, hydrogen bonds, and van der Waals forces to assemble into an active three-dimensional structure, which then drives efficient extein ligation and enables protein trans-splicing. Protein trans-splicing mediated by split inteins has become a cornerstone for traceless protein ligation owing to its high specificity and irreversibility, fundamentally reshaping strategies for protein modification, assembly, and functional regulation. Compared with traditional chemical ligation methods, split intein systems require no complex chemical derivatization of peptide fragments and can operate efficiently at micromolar concentrations under physiological conditions, thus avoiding structural and functional damage caused by organic reagents. In contrast to enzymatic ligation tools such as sortase, split inteins eliminate the need for additional enzymes or cofactors, simplifying reaction systems, reducing costs, and minimizing non-specific side products. These distinctive advantages render split inteins highly promising for applications in chemical biology, synthetic biology, and biopharmaceutical development. In recent years, deepened mechanistic understanding has established structure-guided rational design as the primary approach to overcoming key limitations of split inteins, including intrinsic aggregation propensity, strict extein sequence dependence, and limited splicing efficiency. Bioinformatic tools have been used to identify aggregation-prone regions in the IntN fragment, and site-directed mutagenesis of hydrophobic residues, relocation of split sites, or removal of misfolding-prone sequences has substantially reduced in vitro aggregation and improved soluble expression and assembly activity. Rational engineering of catalytic residues and adjacent flexible loops has relaxed strict amino acid preferences at extein junctions, enhancing sequence tolerance and reducing the risk of functional impairment in target proteins. Consensus design based on multiple sequence alignments has yielded ultra-fast splicing variants such as Cfa DnaE and Cat-TerL, which exhibit significantly accelerated kinetics and improved tolerance to denaturing conditions. Meanwhile, advances in structural biology have further clarified the conformational dynamics and catalytic mechanisms of splicing, supporting the precise design of high-performance intein modules. On this basis, electrostatic interaction tuning and metagenomic screening have yielded multiple mutually orthogonal split intein pairs, enabling selective multi-fragment protein ligation and providing new routes for the efficient synthesis of large multi-domain functional proteins. With these engineered split inteins offering continuously improved performance and expanded applicability, protein trans-splicing has been widely applied in numerous cutting-edge areas of protein research and biomedicine. In gene delivery, split intein-based systems overcome the packaging limit of adeno-associated viral vectors, enabling the accurate reconstitution of large therapeutic proteins and base editors in target cells, thereby enhancing the efficacy and scope of gene therapy for genetic diseases. In internal protein sequence editing, split inteins mediate precise sequence replacement and modification in flexible regions or loops of target proteins, without the need for complex multi-step ligation and protein refolding involved in traditional protein semisynthesis. In protein-protein interaction studies, intein-mediated splicing covalently captures transient and weak intracellular complexes, enabling sensitive, high-throughput interaction detection and drug screening. In synthetic biology, conditionally controllable splicing systems support the construction of diverse intracellular and cell-surface biological logic gates for the precise regulation of cellular behavior. In mechanistic biochemical research, split inteins enable photocatalytic proximity labeling and site-specific tagging, allowing the preparation of homogeneous protein samples carrying precise post-translational modifications such as ubiquitination and polyglutamylation for chromatin interactome analysis and epigenetic studies. Moreover, covalent trapping strategies using split inteins stabilize transient enzymatic intermediates, providing unprecedented insights into molecular mechanisms such as nucleosome ubiquitination that are difficult to elucidate using conventional methods. This review systematically summarizes key technological advances in split inteins over the past decade, highlighting engineering strategies, mechanistic insights, and the development of orthogonal components. It comprehensively surveys emerging applications at the frontiers of protein research, analyzes current core challenges, and proposes future directions, particularly emphasizing artificial intelligence-driven de novo design and novel splicing pathways to break existing technical bottlenecks. By enabling traceless, efficient, and versatile protein manipulation, split inteins continue to serve as indispensable tools that drive innovation in protein engineering and fundamental life science research.

Acute kidney injury (AKI) is a prevalent and life-threatening clinical syndrome characterised by a rapid decline in renal function and diverse pathological etiologies. The condition has been demonstrated to be associated with elevated mortality rates and an increased risk of progression to chronic kidney disease. At present, clinicians depend heavily on conventional functional markers, such as serum creatinine and urine output, for the diagnosis and staging of the disease. It is evident that these conventional indicators characteristically manifest a considerable temporal delay and only undergo modification subsequent to considerable tissue damage. This severely restricts the timeframe for early detection and timely therapeutic intervention. Furthermore, standard markers fail to provide specific biological information regarding the underlying cellular injury mechanisms. The utilisation of advanced probe technologies in molecular imaging offers a robust alternative to overcome these inherent diagnostic limitations.This comprehensive review systematically evaluates recent progress in the design and application of two primary categories of molecular imaging tools for acute kidney disease, specifically reactive probes and enzyme-activated probes. Reactive probes are engineered to specifically interact with redox-active chemical species, including hydrogen peroxide, peroxynitrite, hypochlorous acid, and sulfur dioxide. Because oxidative stress constitutes a primary early event in acute renal tubular damage, these probes enable researchers and clinicians to visualize early cellular injury and radical accumulation well before global renal functional decline becomes evident. We discuss the application of these reactive probes across multiple imaging modalities including fluorescence imaging, magnetic resonance imaging (MRI), positron emission tomography (PET), and photoacoustic techniques. Photoacoustic imaging combines high spatial resolution with deep tissue penetration and has successfully demonstrated the ability to provide diagnostic alerts up to 12 h before any detectable rise in serum creatinine levels. Additionally, specific reactive probes have shown promising translational potential when tested by high-throughput screening in clinical human urine samples. Enzyme-activated probes target the specific catalytic activity of disease-relevant enzymes. These include well-documented renal tubular structural biomarkers such as NAG, GGT, and ALP, along with apoptosis-related caspases and specific nitroreductases. By responding only to enzymatic cleavage, these tools provide highly specific and pathology-directed imaging readouts. Recent structural design strategies in this field have advanced significantly beyond single-enzyme detection. Researchers are now focusing on sophisticated dual-target recognition to minimize background noise, multimodal integration to cross-validate imaging signals, and theranostic applications where probes simultaneously deliver diagnostic feedback and therapeutic agents to injured tissues. Nanotechnology serves as a fundamental enabler for realizing these advanced probe functions. By precisely optimizing nanoparticle parameters such as hydrodynamic size, surface charge, and targeting ligands, researchers can achieve amplified signal output, highly precise kidney delivery, and protection against premature degradation in the systemic circulation. For example, modifying surface charges can significantly enhance the active uptake of nanoprobes by damaged renal tubular epithelial cells.While preclinical probe development has progressed rapidly, moving these technologies into routine clinical practice remains a major challenge. We analyze the translational feasibility and current obstacles from biological, technological, and regulatory perspectives. Although biological targets such as KIM-1, FAP, and ALP have been validated in extensive patient cohorts, practical barriers severely limit their immediate clinical application. These obstacles involve complex changes in in vivo pharmacokinetics. During an acute injury episode, the extreme drop in the glomerular filtration rate alters probe clearance and can cause unwanted systemic accumulation or confusing background imaging signals. Other major hurdles include a lack of comprehensive long-term toxicity data and the absence of standardized manufacturing protocols to ensure batch-to-batch consistency. Future successful translation will require rigorous multi-center clinical studies to confirm the true diagnostic value of these probes over traditional markers. Researchers must also establish strict standardization of imaging procedures and comprehensive safety evaluations. Ultimately, this review provides a thorough reference framework for designing clinically translatable molecular probes and building a precision diagnostic imaging system for acute kidney injury.

Hepatocellular carcinoma(HCC),which is essentially primary liver cancer,is closely related to CD8+T cell immune infiltration and immune suppression.We constructed a CD8+T cells related risk score model to pre-dict the prognosis of HCC patients and provided therapeutic guidance based on the risk score.Using integrated bulk RNA sequencing(RNA-seq)and single-cell RNA sequencing(scRNA-seq)datasets,we identified stable CD8+T cell signatures.Based on these signatures,a 3-gene risk score model,comprised of KLRB1,RGS2,and TN-FRSF1B was constructed.The risk score model was well validated through an independent external validation co-hort.We divided patients into high-risk and low-risk groups according to the risk score and compared the differ-ences in immune microenvironment between these two groups.Compared with low-risk patients,high-risk patients have higher M2-type macrophage content(P＜0.0001)and lower CD8+T cells infiltration(P＜0.0001).High-risk patients predict worse response to immunotherapy treatment than low-risk patients(P＜0.01).Drug sensitivity a-nalysis shows that PI3K-β inhibitor AZD6482 and TGFβRII inhibitor SB505124 may be suitable therapies for high-risk patients,while the IGF-1R inhibitor BMS-754807 or the novel pyrimidine-based anti-tumor metabolic drug Gemcitabine could be potential therapeutic choices for low-risk patients.Moreover,expression of these 3-gene mod-el was verified by immunohistochemistry.In summary,the establishment and validation of a CD8+T cell-derived risk model can more accurately predict the prognosis of HCC patients and guide the construction of personalized treatment plans.

Objective:To investigate the differences between high-frequency ultrasound-guided manual detorsion combined with surgery(MD+S)and surgery alone in the treatment of testicular torsion,and to provide some new evidence for the timely diagno-sis and treatment of the disease.Methods:We retrospectively analyzed the clinical data on 134 cases of unilateral testicular torsion within 48 hours treated in our hospital by MD+S or by surgery alone from January 2015 to May 2022.We statistically analyzed the age distribution,and duration and degrees of testicular torsion,followed by comparison between the two groups.Results:In the 134 ca-ses,the median age of onset was 15(13-19)years old,the median onset-to-visit time was 15(8-25)hours,and the median de-gree of torsion was 360°(180°-1080°).Of the total number of patients,21 underwent testicular excision and the other 113 were treated with the testis preserved,with no statistically significant difference in age distribution between the two groups(P＞0.05),and a higher rate of testis resection in those with longer duration and greater angle of torsion(P＜0.05).Totally,33 of the patients were assigned to the MD+S group and 101 to the surgery alone group.According to the actual clinical conditions and excluding those with torsion time longer than 24 hours and torsion angle greater than 720 °,28 of the patients underwent ultrasound-guided MD+S(with 1 case of testis resection,3.6％),and 68 received surgery alone(with 7 cases of testis resection,10.3％).The rate of testis resection was higher in the surgery alone than that in the MD+S group,but with no statistically significant difference between the two groups(P＞0.05),which was considered to be related to the small sample size in this study.Conclusion:The popularization of testicular torsion knowledge can shorten the onset-to-visit time,and reasonable manual detorsion before emergency surgery can reduce the rate of testis resection.

OBJECTIVE To stimulate the debridement and dressing process under the approximately real clinical scene by fluorescence labeling and understand the contamination status of the operators' bodies and their surround-ings so as to improve the strategies.METHODS A total of 41 trainees were recruited from Enterostomal Therapist Nursing Education Program of Peking University School of Medicine and International School of Wound Thera-pists of Peking University First Hospital in 2023.The examination was designed by stimulating the debridement and dressing operations with fluorescence labeling,the orange was used to simulate the wound of the patients,and the pad towel was placed under the orange to represent the contaminated surroundings of the wound.A round le-sion area with diameter of 2 cm was uniformly marked on the surface of the orange peel by the operators,the trai-nees were asked to remove the lesion tissues and take dressings.The fluorescent powders were smeared evenly on the orange and pad towel before the examination,and no fluorescence labeling for the surroundings was guaranteed.The trainees were required to wear hats,masks,isolation gowns and gloves during the operations,and take off the gloves and trace the scope of fluorescent contamination with ultraviolet radiator after the examina-tion.RESULTS The contamination rate was 92.68％for the trainees' bodies,100.00％for their surroundings.The hands and forearms were the most severe contaminated body sites,and the contamination rates were 85.37％(35/41)and 34.15％(14/41),respectively.The downside of the pad towel was the most frequently contaminated area of the surroundings,with the proportion of 90.24％(37/41);the right side of the pad towel was the area with the contamination disseminated farthest,with the median distance of dissemination 13.50 cm.In addition,the dressing change carts of 75.61％(31/41)of the trainees were contaminated.CONCLUSIONS The operations of de-bridement and dressing change may lead to varying degrees of contamination of their bodies and surroundings.It is necessary to strengthen the hand hygiene and protective isolation during the process of dressing change and pay attention to the disinfection of the dressing change carts so as to minimize the risk of hospital-associated infec-tions.

Bovine rotavirus(BRV)is an important pathogen causing diarrhea in calves.To understand the genomic charac-teristics and genetic variations in bovine rotavirus,and to further enrich data on the biological characteristics of rotavirus,we aimed to amplify 11 gene segments of the isolated and cultured G6P[1]bovine rotavirus BLL strain,perform whole genome se-quencing,and analyze the molecular characteristics.MEGA7.0 and DNAMAN software were used for homology and typing a-nalysis,and the whole genome phylogenetic tree was constructed to analyze genetic evolution relationships.The complete geno-type of the BLL strain was G6-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3.Phylogenetic analysis of the VP7 and VP4 genes of the BLL strain showed that the VP7 gene had the highest homology with RVA/Cow-wt/HB01/China/2021,and the VP4 gene of the BLL strain was in the same branch as RVA/Human-tc/ISR/Ro8059/1995.From the sequence alignment of VP8*amino acids,the sialic acid domain of the BLL strain was found to be similar to that in other P[1]strains,but different from those in other types of strains,except for residue 189,which was the same as that in Ro8059 but different from that in other strains.The results suggested that the BLL strain might potentially infect humans.Therefore,continued monitoring and study of the biological characteristics of this strain are necessary to provide more information and evidence supporting further research on the cross-species transmission of group A rotavirus in China.

Potassium-calcium activates channel subfamily N member 3(KCNN3/SK3/KCa2.3)is in-volved in regulating cellular calcium signaling,muscle contraction and neurotransmitter release.Dysregu-lation of the KCNN3 channel is associated with the development of various tumors.We use bioinformatics analysis to identify whether KCNN3 regulates the occurrence and development of stomach adenocarcinoma(STAD)as a prognostic target.By analyzing the Human Protein Atlas(HPA)database and The Cancer Genome Atlas(TCGA)database,we found that the protein and mRNA levels of KCNN3 were dramatic-ally reduced in STAD,and TCGA database showed that KCNN3 significantly correlated with the prognosis and clinical features of STAD.In addition,we found that high expression of KCNN3 in STAD reduced the IC50 of several drugs in STAD cells,suggesting that high expression of KCNN3 correlated with the drug sensitivity of STAD.To investigate the underlying biological mechanism,we identified a potential KCNN3 interaction factor,tumor necrosis factor receptor superfamily member 7(CD27/TNFRSF7),which is expressed at low levels in STAD.RT-qPCR and Western blotting confirmed that KCNN3 and CD27 positively correlated with each other at protein and mRNA levels,and co-immunoprecipitation and immunofluorescence experiments confirmed that the two proteins interact and colocalize in the cytoplasm.Moreover,we confirmed the inhibitory effect of KCNN3 on the proliferation,migration and invasion of hu-man STAD cells in vitro and in vivo through subcutaneous tumorigenesis and cellular experiments.Fur-thermore,GO/KEGG enrichment analysis showed that KCNN3 was enriched in signaling pathways regula-ting the immune response and calcium or metal ion transport.Lastly,we verified through cell co-culture,RT-qPCR and CCK8 assays that high expression of KCNN3 can promote the increase of T cell activating factor and the killing effect of T cells on STAD cells.Therefore,our results suggest that KCNN3 is a po-tential inhibitory factor affecting the occurrence and progression of STAD.

Hepatocellular carcinoma(HCC),which is essentially primary liver cancer,is closely related to CD8+T cell immune infiltration and immune suppression.We constructed a CD8+T cells related risk score model to pre-dict the prognosis of HCC patients and provided therapeutic guidance based on the risk score.Using integrated bulk RNA sequencing(RNA-seq)and single-cell RNA sequencing(scRNA-seq)datasets,we identified stable CD8+T cell signatures.Based on these signatures,a 3-gene risk score model,comprised of KLRB1,RGS2,and TN-FRSF1B was constructed.The risk score model was well validated through an independent external validation co-hort.We divided patients into high-risk and low-risk groups according to the risk score and compared the differ-ences in immune microenvironment between these two groups.Compared with low-risk patients,high-risk patients have higher M2-type macrophage content(P＜0.0001)and lower CD8+T cells infiltration(P＜0.0001).High-risk patients predict worse response to immunotherapy treatment than low-risk patients(P＜0.01).Drug sensitivity a-nalysis shows that PI3K-β inhibitor AZD6482 and TGFβRII inhibitor SB505124 may be suitable therapies for high-risk patients,while the IGF-1R inhibitor BMS-754807 or the novel pyrimidine-based anti-tumor metabolic drug Gemcitabine could be potential therapeutic choices for low-risk patients.Moreover,expression of these 3-gene mod-el was verified by immunohistochemistry.In summary,the establishment and validation of a CD8+T cell-derived risk model can more accurately predict the prognosis of HCC patients and guide the construction of personalized treatment plans.

Objective:To explore the effects of stepped exercise rehabilitation training on cardiopulmonary function and quality of life in patients with ischemic heart disease.Methods:This randomized controlled study enrolled 110 patients with ischemic heart disease admitted to Jiangsu Province Hospital between January 2021 and December 2023.Patients were randomly divided into control group(n=53,routine intervention program)and intervention group(n=52,additional stepped exercise rehabilitation training).After 3-month intervention,cardiopulmonary function[stroke volume(SV),cardiac index(CI),forced expiratory volume in the first second(FEV1),maximum voluntary ventilation(MVV)],quality of life[China questionnaire of quality of life in patients with cardiovascular diseases(CQQC)score],exercise endurance[exercise duration(ED),peak oxygen intake(VO2peak),anaerobic threshold(AT)]and incidence of major adverse cardiovascular events(MACE)were compared between the two groups.Results:We included 53 patients[28 males(52.83％),age 40～69(56.25±7.76)years old]in control group compared to 52 patients[27 males(51.92％),age 40～71(55.25±2.40)years old]in the intervention group after 3-month intervention.Com-pared to patients in the control group,those in the intervention group had significantly higher SV[(65.46±3.58)ml vs.(61.69±3.78)ml],CI[(2.82±0.12)L·min-1·m-2 vs.(2.54±0.09)L·min-1·m-2],FEV1[(2.50±0.06)L vs.(2.31±0.06)L],MVV[(79.66±0.82)L/min vs.(77.16±1.09)L/min],CQQC score[(65.23±2.84)points vs.(47.98±3.25)points],ED[(382.62±31.19)s vs.(353.37±36.32)s],VQ2peak[(20.05±2.43)ml·kg-1·min-2 vs.(16.86±1.61)ml·kg-1·min-2]and AT[(13.34±0.83)ml·kg-1·min-2 vs.(11.46±0.89)ml·kg-1·min-2](P＜0.001 all),and significantly lower total incidence of MACE(11.54％vs.32.08％,P=0.011).Conclu-sion:Stepped exercise rehabilitation training may improve cardiopulmonary function,quality of life,and exercise endurance in patients with ischemic heart disease during the rehabilitation.