A Colorimetric RT-LAMP Assay for Rapid Detection of Soybean mosaic Virus SC15.

Soybean mosaic virus (SMV) represents one of the most devastating viral diseases affecting soybeans worldwide. Among its strains, SMV-SC15 is notable for its virulence, predominance, and widespread occurrence. Rapid and on-site diagnosis is important for controlling the spread of SMV-SC15. In this study, we proposed a colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SMV-SC15 using three color indicators for visual interpretation: Neutral Red (N-Red), Bromothymol Blue (BTB), and SYBR Green I. The SMV-SC15 in the soybean tissue was detected with remarkable sensitivity and specificity within 30 min, achieving a detection limit as low as 10-4 ng/µL. 200 soybean leaf samples from the field were analyzed by the colorimetric RT-LAMP assays, holding significant potential for rapid screening of SMV-SC15-resistant cultivars, thereby contributing to effective SMV control.

Choline Phosphate-Grafted Nanozymes as Universal Extracellular Vesicle Probes for Bladder Cancer Detection.

Urinary extracellular vesicles (uEVs) are regarded as highly promising liquid-biopsy biomarkers for the early diagnosis and prognosis of bladder cancer (BC). However, detection of uEVs remains technically challenging owing to their huge heterogeneity and ultralow abundance in real samples. We herein present a choline phosphate-grafted platinum nanozyme (Pt@CP) that acts as a universal EV probe for the construction of a high-throughput and high-sensitivity immunoassay, which allowed multiplex profiling of uEV protein markers for BC detection. With the Pt@CP-based immunoassays, three uEV protein markers (MUC-1, CCDC25, and GLUT1) were identified for BC, by which the BC cases (n = 48), cystitis patients (n = 27), and healthy donors (n = 24) were discriminated with high clinical sensitivity and specificity (area under curve = 98.3%). For the BC cases (n = 9) after surgery, the Pt@CP-based immunoassay could report the postoperative residual tumor that cannot be observed by cystoscopy, which is clinically significant for assessing BC recurrence. This work provides generally high sensitivity for EV detection, facilitating the discovery and clinical use of EV-based biomarkers.

Multihierarchical Regulation To Achieve Quantum Dot Nanospheres with a Photoluminescence Quantum Yield Close to 100.

Quantum dots (QDs) exhibit superior brightness and photochemical stability, making them the preferred option for highly sensitive single-molecule detection compared with fluorescent dyes or proteins. Nevertheless, their high surface energy leads to nonspecific adsorption and poor colloidal stability. In the past decades, we have found that QD-based fluorescent nanoparticles (FNs) can not only address these limitations but also enhance detection sensitivity. However, the photoluminescence quantum yield (PLQY) of FNs is significantly lower compared with that of original QDs. It is urgent to develop a strategy to solve the issue, aiming to further enhance detection sensitivity. In this study, we found that the decrease of PLQY of FNs prepared by free radical polymerization was attributed to two factors: (1) generation of defects that can cause nonradiative transitions resulting from QD-ligands desorption and QD-shell oxidation induced by free radicals; (2) self-absorption resulting from aggregation caused by incompatibility of QDs with polymers. Based on these, we proposed a multihierarchical regulation strategy that includes: (1) regulating QD-ligands; (2) precisely controlling free radical concentration; and (3) constructing cross-linked structures of polymer to improve compatibility and to reduce the formation of surface defects. It is crucial to emphasize that the simultaneous coordination of multiple factors is essential. Consequently, a world-record PLQY of 97.6% for FNs was achieved, breaking through the current bottleneck at 65%. The flexible application of this regulatory concept paves the way for the large-scale production of high-brightness QD-polymer complexes, enhancing their potential applications in sensitive biomedical detection.

Culture-free detection of ß-lactamase-Producing bacteria in urinary tract infections using a paper sensor.

Developing simple, inexpensive, fast, sensitive, and specific probes for antibiotic-resistant bacteria is crucial for the management of urinary tract infections (UTIs). We here propose a paper-based sensor for the rapid detection of ß-lactamase-producing bacteria in the urine samples of UTI patients. By conjugating a strongly electronegative group -N+(CH3)3 with the core structures of cephalosporin and carbapenem antibiotics, two visual probes were achieved to respectively target the extended-spectrum/AmpC ß-lactamases (ESBL/AmpC) and carbapenemase, the two most prevalent factors causing antibiotic resistance. By integrating these probes into a portable paper sensor, we confirmed 10 and 8 cases out of 30 clinical urine samples as ESBL/AmpC- and carbapenemase-positive, respectively, demonstrating 100% clinical sensitivity and specificity. This paper sensor can be easily conducted on-site, without resorting to bacterial culture, providing a solution to the challenge of rapid detection of ß-lactamase-producing bacteria, particularly in resource-limited settings.

In Vivo Fluorescent Labeling of Foam Cell-Derived Extracellular Vesicles as Circulating Biomarkers for In Vitro Detection of Atherosclerosis.

Real-time monitoring of the development of atherosclerosis (AS) is key to the management of cardiovascular disease (CVD). However, existing laboratory approaches lack sensitivity and specificity, mostly due to the dearth of reliable AS biomarkers. Herein, we developed an in vivo fluorescent labeling strategy that allows specific staining of the foam cell-derived extracellular vesicles (EVs) in atherosclerotic plaques, which are released into the blood as circulating biomarkers for in vitro detection of AS. This strategy relies on a self-assembled nanoprobe that could recognize foam cells specifically, where the probe is degraded by the intracellular HClO to produce a trifluoromethyl-bearing boron-dipyrromethene fluorophore (termed B-CF3), a lipophilic dye that can be transferred to the exosomal membranes. These circulating B-CF3-stained EVs can be detected directly on a fluorescence spectrometer or microplate reader without resorting to any sophisticated analytical method. This liquid-biopsy format enables early detection and real-time differentiation of lesion vulnerability during AS progression, facilitating effective CVD management.

Rapid and visual identification of ß-lactamase subtypes for precision antibiotic therapy.

The abuse of antibiotics urgently requires rapid identification of drug-resistant bacteria at the point of care (POC). Here we report a visual paper sensor that allows rapid (0.25-3 h) discrimination of the subtypes of ß-lactamase (the major cause of bacterial resistance) for precision antibiotic therapy. The sensor exhibits high performance in identifying antibiotic-resistant bacteria with 100 real samples from patients with diverse bacterial infections, demonstrating 100% clinical sensitivity and specificity. Further, this sensor can enhance the accuracy of antibiotic use from 48% empirically to 83%, and further from 50.6% to 97.6% after eliminating fungal infection cases. Our work provides a POC testing platform for guiding effective management of bacterial infections in both hospital and community settings.

Python-assisted detection and photothermal inactivation of Salmonella typhimurium and Staphylococcus aureus on a background-free SERS chip.

In this study, a background-free surface-enhanced Raman scattering (SERS) chip with a sandwich configuration was fabricated to enable reliable detection and photothermal inactivation of multiple bacteria. The SERS chip consists of a graphene-coated, phenylboronic-modified plasmonic gold substrate (pAu/G/PBA), and two aptamer-functionalized core (gold)-shell (Prussian blue/Poly-L-lysine and 4-mercaptobenzonitrile/polydopamine) SERS tags (Au@PB@PLL@Apt and Au@MB@PDA@Apt). The detection signals rely on the characteristic and nonoverlapping Raman bands of the SERS tags within the Raman-silent region (1800-2800 cm-1), where no background signals from the sample matrix are observed, leading to improved detection sensitivity and accuracy. Considering the relatively large size of bacteria (e.g., micron level), a rapid Raman mapping technique was chosen over conventional point-scan methods to achieve more reliable quantitative analysis of bacteria. This technique involves collecting and analyzing intensity signals of SERS tags from all the scattering points with an average ensemble effect, which is facilitated by the use of Python. As a proof-of-concept, model bacterium of Salmonella typhimurium and Staphylococcus aureus were successfully detected using the SERS chip with a dynamic range of 10-107 CFU/mL. Additionally, the SERS chip demonstrated successful detection of these bacteria in whole blood samples. Moreover, the photothermal effect of pAu/G led to efficient bacteria elimination, achieving approximately 100% eradication. This study integrated a background-free SERS chip with a Python-assisted rapid Raman mapping technique, resulting in a reliable, rapid and accurate method for detecting and eliminating multiple bacteria, which may provide a promising alternative for multiple screening of bacteria in real samples.

GEO data mining identifies potential immune-related genes in hypertrophic scar and verities in a rabbit model.

Objective: Hypertrophic scar (HTS), the secondary major abnormal tissue after wound healing, is the most frequent and severe type of skin scar. Dysregulated immune response plays an important role in HTS formation. In this study, we identified the potential immune-related genes in HTS and explored their potential therapeutic significance. Methods: We first screened out the potential immune-related genes in HTS microarrays via bioinformatics analysis using public datasets. We then constructed a rabbit model of ear scar to investigate the morphological features of HTS and verify the basic expression of potential immune-related genes in HTS tissue. Finally, we used AlphaFold to determine the protein homology between human and rabbit scar tissues. Results: Bioinformatics analysis revealed 22 differentially expressed genes (DEGs) and a single differential infiltration of immune cells (naïve B cells) in HTS and normal tissues. Six of the DEGs were correlated with naïve B cell numerically. CCL2, PLXDC2 and FOXF2 were expressed in rabbit ear scar model. PLXDC2 and FOXF2 showed relatively high homology between human and rabbit scar tissues. Conclusions: PLXDC2 and FOXF2, both closely related to immune cell infiltration and specifically expressed in HTS, represent potential therapeutic targets in HTS.

Peripheral origin exosomal microRNAs aggravate glymphatic system dysfunction in diabetic cognitive impairment.

Cognitive dysfunction is one of the common central nervous systems (CNS) complications of diabetes mellitus, which seriously affects the quality of life of patients and results in a huge economic burden. The glymphatic system dysfunction mediated by aquaporin-4 (AQP4) loss or redistribution in perivascular astrocyte endfeet plays a crucial role in diabetes-induced cognitive impairment (DCI). However, the mechanism of AQP4 loss or redistribution in the diabetic states remains unclear. Accumulating evidence suggests that peripheral insulin resistance target tissues and CNS communication affect brain homeostasis and that exosomal miRNAs are key mediators. Glucose and lipid metabolism disorder is an important pathological feature of diabetes mellitus, and skeletal muscle, liver and adipose tissue are the key target insulin resistance organs. In this review, the changes in exosomal miRNAs induced by peripheral metabolism disorders in diabetes mellitus were systematically reviewed. We focused on exosomal miRNAs that could induce low AQP4 expression and redistribution in perivascular astrocyte endfeet, which could provide an interorgan communication pathway to illustrate the pathogenesis of DCI. Furthermore, the mechanisms of exosome secretion from peripheral insulin resistance target tissue and absorption to the CNS were summarized, which will be beneficial for proposing novel and feasible strategies to optimize DCI prevention and/or treatment in diabetic patients.

Prognostic value of coagulation and fibrinolysis function indexes and NETs for sepsis patients.

OBJECTIVE: To clarify the role of coagulation and fibrinolysis as well as the level of neutrophil extracellular traps (NETs) in patients with sepsis, and to explore their clinical significance in identifying the disease and predicting the prognosis. METHODS: In this retrospective study, the clinical data from 120 patients with sepsis admitted to People's Hospital of Changshou from January 2019 to December 2021 were analyzed. The patients were divided into a survival group and a death group according to the survival of patients within 28 days of admission. Another 120 patients with common bacterial infection were selected as the bacterial group and 120 healthy subjects who underwent physical examination in our hospital during the same period were selected as the healthy group. NETs, coagulation and fibrinolysis indexes, prothrombin time (PT), fibrinogen (FIB), D-dimer level, International Normalized Ratio (INR), Acute Physiology and Chronic Health Evaluation (APACHE) II score, and sequential organ failure assessment (SOFA) score of the patients with sepsis were compared with those of bacterial group and healthy group. Correlations between these measures were analyzed, and the predictive value of NETs for survival in patients with sepsis was assessed. RESULTS: Compared with bacterial group and healthy group, the levels of serum NETs, PT, FIB, D-dimer, and INR value in sepsis patients were significantly increased. The level of NETs was positively associated with APACHE II score, SOFA score, PT, FIB, D-dimer, and INR. INR showed good performance in predicting death within 28 days after admission in sepsis patients. CONCLUSION: The NETs and coagulation indexes have high predictive value for the prognosis of patients with sepsis.

Reversible zwitterionic coordination enables rapid, high-yield, and high-purity isolation of extracellular vesicles from biofluids.

Extracellular vesicles (EVs) hold great clinical value as promising diagnostic biomarkers and therapeutic agents. This field, however, is hindered by technical challenges in the isolation of EVs from biofluids for downstream purposes. We here report a rapid (<30 min) isolation method for EV extraction from diverse biofluids with yield and purity exceeding 90%. These high performances are ascribed to the reversible zwitterionic coordination between the phosphatidylcholine (PC) on EV membranes and the "PC-inverse" choline phosphate (CP) decorated on magnetic beads. By coupling this isolation method with proteomics, a set of differentially expressed proteins on the EVs were identified as potential colon cancer biomarkers. Last, we demonstrated that the EVs in various clinically relevant biofluids, such as blood serum, urine, and saliva, can also be isolated efficiently, outperforming the conventional approaches in terms of simplicity, speed, yield, and purity.

Engineered Cancer-Derived Small Extracellular Vesicle-Liposome Hybrid Delivery System for Targeted Treatment of Breast Cancer.

Cancer-derived small extracellular vesicles (sEVs) may be a promising drug delivery system that targets cancer cells due to their unique features, such as native homing ability, biological barrier crossing capability, and low immune response. However, the oncogenic cargos within them pose safety concerns, hence limiting their application thus far. We proposed using an electroporation-based strategy to extract the endogenous cargos from cancer-derived sEVs and demonstrated that their homing ability was still retained. A membrane fusion technique was used to fuse these sEVs with liposomes to form hybrid particles, which possessed both benefits of sEVs and liposomes. Anti-EGFR monoclonal antibodies were modified on the hybrid particles to improve their targeting ability further. The engineered hybrid particles showed higher drug loading ability that is 33.75 and 43.88% higher than that of liposomes and sEVs, respectively, and improved targeting ability by 52.23% higher than hybrid particles without modification. This delivery system showed >90% cell viability and enhanced treatment efficiency with 91.58 and 79.26% cell migration inhibition rates for the miR-21 inhibitor and gemcitabine, respectively.

GelMA Hydrogel Loaded with Extracellular Vesicles Derived from Umbilical Cord Mesenchymal Stem Cells for Promoting Cutaneous Diabetic Wound Healing.

Chronic diabetic wounds have become a significant cause of disability worldwide. It is highly desired to develop effective therapies that can promote the rapid healing of diabetic wounds. Owing to the outstanding hydrophilic and water-retaining properties, hydrogels could accelerate the healing process. Extracellular vesicles (EVs) have shown the ability to promote cell regeneration and angiogenesis. In this study, we chose a gelatin methacryloyl (GelMA) hydrogel, a kind of biomaterial characteristic of good biocompatibility, to load the EVs derived from umbilical cord mesenchymal stem cells (UCMSCs) in order to have a long-lasting effect by consistent release of EVs. Then, the hydrogel with EVs was used to treat diabetic wounds in rat models. Nuclear magnetic resonance spectroscopy and scanning electron microscopy were used to characterize the synthesis of the hydrogel; cell experiments, animal experiments, and histological staining were used to evaluate the function of the hydrogel with EVs. The results show that the GelMA hydrogel incorporated with the UCMSC-derived EVs exhibits unique physicochemical properties, excellent biocompatibility, and much enhanced therapeutic effects for diabetic wounds.

A Novel Diagnostic Model for Early Hepatitis B Virus-Related Hepatocellular Carcinoma Diagnosis.

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is closely related to the diagnostic stage. Due to the difficulty diagnosing early-stage HCC, most patients with HCC are diagnosed at the advanced stage. In this study, we used protein induced by vitamin K absence or antagonist-II (PIVKA-II) and alpha-fetoprotein (AFP) combined with aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (T-Bil) to establish a novel diagnostic model for early-stage hepatitis B virus (HBV)-related HCC. METHODS: The serum levels of PIVKA-II, AFP, AST, ALT, and T-Bil were measured in 148 patients with early-stage HBV-related HCC and 940 patients with chronic hepatitis B. The receiver operating characteristic (ROC) curves were used to verify the diagnostic efficacy of the novel diagnostic model for early-stage HBV-related HCC. RESULTS: The mathematical model of [1.5 x PIVKA-II/(AST x T-Bil) + AFP/(ALT x T-Bil)] was selected as the novel diagnostic model. The areas under ROC curves (AUROCs) of the novel diagnostic model for detecting early-stage HBV-related HCC were significantly higher than those of PIVKA-II, AFP, and PIVKA-II combined with AFP (HCC ≤ 5 cm: 0.925 vs. 0.826, 0.666, and 0.821; HCC < 3 cm: 0.896 vs. 0.741, 0.651, and 0.765, respectively) (all p < 0.001). Using serum levels of AFP ≥ 20 ng/mL, the diagnostic model had the highest AUROC values of 0.960 and 0.933 for HCC ≤ 5 cm (89 cases) and HCC < 3 cm (40 cases), respectively, with a sensitivity of 83.15%, and 77.50% and specificity of 95.34% and 90.69%, respectively. CONCLUSIONS: The novel diagnostic model is superior to PIVKA-II and AFP for diagnosing early-stage HBV-related HCC, especially in patients with abnormal serum AFP levels.

Identification of faecal extracellular vesicles as novel biomarkers for the non-invasive diagnosis and prognosis of colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignancies that is usually detected late in the clinic. The currently available diagnostic tools for CRC are either invasive or insensitive to early lesions due to the dearth of reliable biomarkers. In this study, we discovered that the extracellular vesicles (EVs) in the faeces of CRC patients can act as a potent biomarker for the non-invasive diagnosis and prognosis of CRC. This finding is based on the identification of two transmembrane proteins-CD147 and A33-on faeces-derived EVs (fEVs) that are intrinsically associated with CRC. The detection results show that the levels of CD147 and A33 on fEVs were upregulated in the CRC patients (n = 48), dramatically distinguishing them from the healthy donors (n = 16). The CD147/A33-enriched EVs offer a clinical sensitivity of 89%, much higher than that (40%) of carcinoembryonic antigen (CEA), a clinically-established serum biomarker for CRC diagnosis. In addition, the analysis of longitudinal faeces samples (n = 29) demonstrated that the CD147/A33-enriched fEVs can be utilized to track the prognosis of CRC. Due to the high compliance of faeces-based detection, the CD147/A33-enriched fEVs could serve as new-generation CRC biomarkers for large-scale, non-invasive CRC screening as well as real-time monitoring of patient outcomes during clinical interventions.

A double-switch pHLIP system enables selective enrichment of circulating tumor microenvironment-derived extracellular vesicles.

Circulating tumor microenvironment-derived extracellular vesicles (cTME-EVs) are gaining considerable traction in cancer research and liquid biopsy. However, the study of cTME-EVs is largely limited by the dearth of a general isolation technique to selectively enrich cTME-EVs from biological fluids for downstream analysis. In this work, we broke through this dilemma by presenting a double-switch pH-low insertion peptide (D-S pHLIP) system to exclusively harvest cTME-EVs from the blood serum of tumor mouse models. This D-S pHLIP system consists of a highly sensitive pH-driven conformational switch (pKa ≈ 6.8) that allows specific installation of D-S pHLIP on the EV membranes in TME (pH 6.5 to 6.8) and a unique hook-like switch to "lock" the peptide securely on the cTME-EVs during the systemic circulation. The D-S pHLIP-anchored cTME-EVs were magnetically enriched and then analyzed with high-resolution messenger RNA sequencing, by which more than 18 times the number of TME-related differentially expressed genes and 10 times the number of hub genes were identified, compared with those achieved by the gold-standard ultracentrifugation. This work could revolutionize basic TME research as well as clinical liquid biopsy for cancer.

Achieving Ultrasensitive Chromogenic Probes for Rapid, Direct Detection of Carbapenemase-Producing Bacteria in Sputum.

Carbapenemase-producing bacteria (CPB) stand as the most dangerous "superbugs" in the clinic. Rapid point-of-care (POC) detection of CPB in clinical samples is key to timely and effective infection management. We herein report the first ultrasensitive chromogenic probe that allows direct POC detection of CPB in clinical sputum samples at a sample-to-result time of less than 15 min. This chromogenic probe is modularly designed by conjugating the carbapenem core with a benzene derivative bearing an electronegativity-tunable substituent. Unexpectedly high sensitivity was achieved simply by choosing strong electron-withdrawing substituents, such as -N+(CH3)3, without resorting to complex molecular design. Through integrating the probes with a portable paper chip, 24 out of 80 clinical sputum samples from sepsis patients with lung infections were quickly diagnosed as CPB-positive, exhibiting 100% clinical sensitivity and specificity. This low-cost paper chip assay can be readily performed on-site, breaking through the dilemma of rapid CPB detection, especially in resource-limited settings.

Self-Referenced Synthetic Urinary Biomarker for Quantitative Monitoring of Cancer Development.

Urinary monitoring of diseases has gained considerable attention due to its simple and non-invasive sampling. However, urinalysis remains limited by the dearth of reliable urinary biomarkers and the intrinsically enormous heterogeneity of urine samples. Herein, we report, to our knowledge, the first renal-clearable Raman probe encoded by an internal standard (IS)-conjugated reporter that acts as a quantifiable urinary biomarker for reliable monitoring of cancer development, simultaneously eliminating the impact of sample heterogeneity. Upon delivery of the probes into tumor microenvironments, the endogenously overexpressed ß-glucuronidase (GUSB) can cleave the target-responsive residues of the probes to produce IS-retained gold nanoclusters, which were excreted into host urine and analyzed by Au growth-based surface-enhanced Raman spectroscopy. As a result, the in vivo GUSB activity was transformed into in vitro quantitative urinary signals. Based on this IS-encoded synthetic biomarker, both the cancer progression and therapy efficacy were quantitatively monitored, potentiating clinical implications.

Cancer-derived small extracellular vesicles: emerging biomarkers and therapies for pancreatic ductal adenocarcinoma diagnosis/prognosis and treatment.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal cancers worldwide with high mortality, which is mainly due to the lack of reliable biomarkers for PDAC diagnosis/prognosis in the early stages and effective therapeutic strategies for the treatment. Cancer-derived small extracellular vesicles (sEVs), which carry various messages and signal biomolecules (e.g. RNAs, DNAs, proteins, lipids, and glycans) to constitute the key features (e.g. genetic and phenotypic status) of cancer cells, are regarded as highly competitive non-invasive biomarkers for PDAC diagnosis/prognosis. Additionally, new insights on the biogenesis and molecular functions of cancer-derived sEVs pave the way for novel therapeutic strategies based on cancer-derived sEVs for PDAC treatment such as inhibition of the formation or secretion of cancer-derived sEVs, using cancer-derived sEVs as drug carriers and for immunotherapy. This review provides a comprehensive overview of the most recent scientific and clinical research on the discovery and involvement of key molecules in cancer-derived sEVs for PDAC diagnosis/prognosis and strategies using cancer-derived sEVs for PDAC treatment. The current limitations and emerging trends toward clinical application of cancer-derived sEVs in PDAC diagnosis/prognosis and treatment have also been discussed.

Trojan Horse Delivery of Spherical Nucleic Acid Probes into the Cytoplasm for High-Fidelity Imaging of MicroRNAs.

We report a Trojan horse strategy to efficiently deliver the spherical nucleic acid probes (namely, nanoflares) into the cytoplasm for microRNA (miRNA) imaging with high fidelity, breaking through the cytoplasmic transport dilemma of RNA probes in living cells. The nanoflare is encapsulated into a "Trojan horse" consisting of zwitterionic choline phosphates (CPs) and acid-degradable crosslinkers; the former effectively promotes cell uptake and the latter triggers instantaneous liberation of the nanoflare probes from the lysosome to the cytoplasm. The exposed nanoflares in the cytoplasm can be lightened up by the target miRNAs specifically. Compared with the conventional nanoflares as well as the improved ones in previous reports, the "Trojan horse" nanoflares avoid nuclease degradation and thiol displacement during the delivery process, providing unprecedentedly high accuracy for intracellular miRNA imaging.