Ultrasensitive Point-of-Care Detection of Protein Markers Using an Aptamer-CRISPR/Cas12a-Regulated Liquid Crystal Sensor (ALICS).

Despite extensive efforts, point-of-care testing (POCT) of protein markers with high sensitivity and specificity and at a low cost remains challenging. In this work, we developed an aptamer-CRISPR/Cas12a-regulated liquid crystal sensor (ALICS), which achieved ultrasensitive protein detection using a smartphone-coupled portable device. Specifically, a DNA probe that contained an aptamer sequence for the protein target and an activation sequence for the Cas12a-crRNA complex was prefixed on a substrate and was released in the presence of target. The activation sequence of the DNA probe then bound to the Cas12a-crRNA complex to activate the collateral cleavage reaction, producing a bright-to-dark optical change in a DNA-functionalized liquid crystal interface. The optical image was captured by a smartphone for quantification of the target concentration. For the two model proteins, SARS-CoV-2 nucleocapsid protein (N protein) and carcino-embryonic antigen (CEA), ALICS achieved detection limits of 0.4 and 20 pg/mL, respectively, which are higher than the typical sensitivity of the SARS-CoV-2 test and the clinical CEA test. In the clinical sample tests, ALICS also exhibited superior performances compared to those of the commercial ELISA and lateral flow test kits. Overall, ALICS represents an ultrasensitive and cost-effective platform for POCT, showing a great potential for pathogen detection and disease monitoring under resource-limited conditions.

Monodispersed and Monofunctionalized DNA-Caged Au Nano-Clusters with Enhanced Optical Properties for STED Imaging.

The performance of Stimulated Emission Depletion (STED) microscopy depends critically on the fluorescent probe. Ultrasmall Au nanoclusters (Au NCs) exhibit large Stokes shift, and good stimulated emission response, which are potentially useful for STED imaging. However, Au NCs are polydispersed in size, sensitive to the surrounding environment, and difficult to control surface functional group stoichiometry, which results in reduced density and high heterogeneity in the labeling of biological structures. Here, this limitation is overcome by developing a method to encapsulate ultrasmall Au NCs with DNA cages, which yielded monodispersed, and monofunctionalized Au NCs that are long-term stable. Moreover, the DNA-caging also greatly improved the fluorescence quantum yield and photostability of Au NCs. In STED imaging, the DNA-caged Au NCs yielded ≈40 nm spatial resolution and are able to resolve microtubule line shapes with good labeling density and homogeneity. In contrast, without caging, the Au NCs-DNA conjugates only achieved ≈55 nm resolution and yielded spotted, poorly resolved microtubule structures, due to the presence of aggregates. Overall, a method is developed to achieve precise surface functionalization and greatly improve the monodispersity, stability, as well as optical properties of Au NCs, providing a promising class of fluorescent probes for STED imaging.

DNA Molecular Glue Assisted Bacterial Conjugative Transfer.

Bacterial conjugation, a commonly used method to horizontally transfer functional genes from donor to recipient strains, plays an important role in the genetic manipulation of bacteria for basic research and industrial production. Successful conjugation depends on the donor-recipient cell recognition and a tight mating junction formation. However, the efficiency of conjugative transfer is usually very low. In this work, we developed a new technique that employed DNA molecule "glue" to increase the match frequency and the interaction stability between the donor and recipient cells. We used two E. coli strains, ETZ and BL21, as a model system, and modified them with the complementary ssDNA oligonucleotides by click chemistry. The conjugation efficiency of the modified bacteria was improved more than 4 times from 10% to 46%. This technique is simple and generalizable as it only relies on the active amino groups on the bacterial surface. It is expected to have broad applications in constructing engineered bacteria.

Multi-parameter Inputted Logic-Gating on Aptamer-Encoded Extracellular Vesicles for Colorectal Cancer Diagnosis.

Extracellular vesicles (EVs) have emerged as a potential biomarker in liquid biopsy. However, cancer heterogeneity poses significant challenge to precise molecular diagnosis based on single-parameter input. Hence, strategies for analyzing multiple inputs with molecular computing were developed with the aim of improving diagnostic accuracy in liquid biopsy. In the present study, based on the surface of aptamer-encoded EVs, three toe-hold extended DNA aptamers served as specific inputs to perform AND-logic-gating to distinguish between healthy and cancerous EVs. In addition, this strategy has been successfully employed to analyze circulating EVs in clinical samples from colorectal cancer patients and healthy donors. The developed method has a promising future in the analysis of multiplex EV membrane proteins and the identification of early cancer.

Bioactive Isoquinoline Alkaloids with Diverse Skeletons from Fissistigma polyanthum.

Six new isoquinoline alkaloids, including aporphine alkaloids (2, 3, 9, and 10), a benzylisoquinoline alkaloid (13), and a protoberberine alkaloid (17), were isolated from the roots of Fissistigma polyanthum, along with a new furanone (20) and 13 known isoquinoline alkaloids (1, 4-8, 11, 12, 14-16, 18, and 19). The structures of the new compounds were elucidated by the analysis of spectroscopic data. Compounds 1 and 2 are rare oxalyl-fused dehydroaporphine alkaloids. Compound 12 presented the most potent dual-target activities on AChE inhibition and Aß aggregation inhibition, while compounds 13 and 19 simultaneously exhibited discernible AChE and BChE inhibitions with antioxidant activities. The activity results indicate that F. polyanthum alkaloids have a potential of inhibition and prevention of Alzheimer's disease mainly through both ChEs and ß-amyloid pathways in addition to antioxidant activity.

Chicoric acid supplementation prevents systemic inflammation-induced memory impairment and amyloidogenesis via inhibition of NF-κB.

Chicoric acid (CA), a natural phenolic acid extracted from chicory and the echinacea (purple coneflower) plant (Echinacea purpurea), has been regarded as a nutraceutical that has powerful antioxidant and antiobesity activities. We investigated the inhibitory effects of CA on systemic inflammation-induced neuroinflammation, amyloidogenesis, and cognitive impairment. C57BL/6J mice were treated with 0.05% CA in the drinking water for 45 d. The mice were then treated by intraperitoneal injection of lipopolysaccharide (LPS). It was found that CA prevented LPS-induced memory impairment and neuronal loss through behavioral tests and histological examination. Furthermore, amyloidogenesis in the CNS was detected. The results showed that CA prevented LPS-induced increases in amyloid ß (1-42 specific) (Aß1-42) accumulation, levels of amyloid precursor protein, and neuronal ß-secretase 1 (BACE1), as well as the equilibrium cholinergic system in mouse brain. Moreover, CA down-regulated LPS-induced glial overactivation by inhibiting the MAPK and NF-κB pathway. Consequently, CA reduced the levels of NF-κB transcriptionally regulated inflammatory mediators and cytokines such as iNOS, cyclooxygenase-2 (COX-2), IL-1ß, and TNF-α in both mouse brain and BV2 microglial cells. These results demonstrated that CA alleviated memory impairment and amyloidogenesis triggered by LPS through suppressing NF-κB transcriptional pathway, suggesting that CA might be a plausible therapeutic intervention for neuroinflammation-related diseases such as Alzheimer disease.-Liu, Q., Chen, Y., Shen, C., Xiao, Y., Wang, Y., Liu, Z., Liu, X. Chicoric acid supplementation prevents systemic inflammation-induced memory impairment and amyloidogenesis via inhibition of NF-κB.

Mirror-Image Thymidine Discriminates against Incorporation of Deoxyribonucleotide Triphosphate into DNA and Repairs Itself by DNA Polymerases.

DNA polymerases are known to recognize preferably d-nucleotides over l-nucleotides during DNA synthesis. Here, we report that several general DNA polymerases catalyze polymerization reactions of nucleotides directed by the DNA template containing an l-thymidine (l-T). The results display that the 5'-3' primer extension of natural nucleotides get to the end at chiral modification site with Taq and Phanta Max DNA polymerases, but the primer extension proceeds to the end of the template catalyzed by Deep Vent (exo-), Vent (exo-), and Therminator DNA polymerases. Furthermore, templating l-nucleoside displays a lag in the deoxyribonucleotide triphosphate (dNTP) incorporation rates relative to natural template by kinetics analysis, and polymerase chain reactions were inhibited with the DNA template containing two or three consecutive l-Ts. Most interestingly, no single base mutation or mismatch mixture corresponding to the location of l-T in the template was found, which is physiologically significant because they provide a theoretical basis on the involvement of DNA polymerase in the effective repair of l-T that may lead to cytotoxicity.

The study of the role of purified anti-mouse CD193 (CCR3) antibody in allergic rhinitis mouse animal models.

The pathogenesis of allergic asthma is similar to that of allergic rhinitis, with inflammation cells producing and releasing inflammatory mediators and cytokines closely related to CCR3.Based on the theory of "one airway, one disease", the use of CCR3 monoclonal antibody may have a similar effect on allergic rhinitis. However, there are few studies on CCR3 monoclonal antibody in allergic rhinitis. Therefore, the aim of this study was to investigate the effective concentration of CCR3 monoclonal antibody, to compare the effects of different methods of administration, and to examine the lung condition of allergic mice to investigate whether antibody treatment protects the lungs. In this study, we constructed a mouse model of allergic rhinitis and intraperitoneally injected different doses of CCR3 monoclonal antibody (5, 10, and 20 uL/mg) to observe its therapeutic effect: observing changes in tissue morphology of nasal mucosa, infiltration of inflammation, and using ELISA to detect changes in relevant inflammatory mediators and cytokines, studying the role of CCR3 mAb in inhibiting CCR3-related actions on the nasal mucosa of allergic rhinitis mice. Furthermore, In addition, the therapeutic effects of intraperitoneal injection (i.p.) and intranasal administration (i.n.) were studied on the basis of effective concentrations.

Liver resection for large and huge hepatocellular carcinoma: Predictors of failure, recurrence patterns, and prognoses.

BACKGROUND: Characteristic symptoms and signs are often absent in patients with hepatocellular carcinoma (HCC). As a result, many patients are not diagnosed until their tumors have grown to large (> 5cm) or huge sizes (> 10cm). Liver resection has traditionally been reserved for patients with small HCC, but more recently it is being used for patients with large and huge tumors. The aim of this study was to determine risk predictors of recurrence, patterns of recurrence, and survival rates for large and huge HCC patients who underwent curative liver resection. MATERIALS AND METHODS: We retrospectively identified a subgroup of patients who underwent liver resection for HCC with diameters 5 cm or larger. Overall survival (OS) and recurrence-free survival (RFS) rates were calculated using the Kaplan-Meier method. Univariate and multivariate Cox regression analyses were performed to investigate potential risk factors for recurrence and death. RESULTS: Among 897 patients, the median follow-up was 48 (range, 5-140) months. The 1-, 3-, and 5-year RFS rates were 51.6%, 36.1%, and 30.1%, respectively, and OS rates were 80.2%, 55.4%, and 47.7%, respectively. Significant independent predictors of recurrence were preoperative satellite nodule (HR = 2.25; 95% CI, 1.17-4.31; p = .02), preoperative AFP levels above 400 ng/ml (HR = 1.23; 95% CI, 1.04-1.45; p = .01), resection margins of 1 cm or less (HR = 1.21; 95% CI, 1.00-1.46; p = .047), cirrhosis (HR = 2.64; 95% CI, 2.13-3.28; p < .001), and microvascular invasion (HR = 1.71; 95% CI, 1.45-2.20; p < .001). All of these except narrow resection margin were also independent risk factors of OS. CONCLUSIONS: Hepatic resection for patients with large and huge HCC without hepatic vascular invasion, extrahepatic metastases, or severe chronic liver disease results in acceptable long-term outcomes.

RuCu Nanosheets with Ultrahigh Nanozyme Activity for Chemodynamic Therapy.

Nanoenzymes have been widely explored for chemodynamic therapy (CDT) in cancer treatment. However, poor catalytic efficiency of nanoenzymes, especially in the tumor microenvironment with insufficient H2 O2 and mild acidity, limits the effect of CDT. Herein, a new ultrathin RuCu nanosheet (NS) based nanoenzyme which has a large specific surface area and abundant channels and defects is developed. The RuCu NSs show superb catalytic efficiency for the oxidation of peroxidase substrate H2 O2 at a wide range of pH and their catalytic efficiency (kcat /Km = 177.2 m-1  s-1 ) is about 14.9 times higher than that of the single-atom catalyst FeN3 P. Besides being an efficient nanozyme as peroxidase, the RuCu NSs possess other two enzyme activities, not only disproportionating superoxide anion to produce H2 O2 but also consuming glutathione to keep a high concentration of H2 O2 in the tumor microenvironment for Fenton reaction. With these advantages, the RuCu NSs exhibit good performance to kill cancer cells and inhibit tumor growth in mice, demonstrating a promising potential as new CDT reagent.

Experimental study of ß-TCP scaffold loaded with VAN/PLGA microspheres in the treatment of infectious bone defects.

Antibiotic bone cement filling technology has been widely used in the treatment of infectious bone defects for decades. However, the current treatment requires multiple complicated procedures, which would lead to pain and financial burden for patients. Repairing bone defects and control infection at the same time is the pain spot of orthopaedic area. In this study, we develop a composite scaffold that aiming at effectively repair infectious bone defects simultaneously. Vancomycin hydrochloride(Van) /Poly(lactic-co-glycolic) acid(PLGA) microspheres prepared by double emulsion method were successfully loaded into ß-tricalcium phosphate scaffold through electrostatic and physical crosslinking. Full characterization, including mechanical properties, biocompatibility, in vitro release profile and antibacterial properties of the composite scaffolds(CPSFs) were performed. The rabbit osteomyelitis model based on big hole and small hole methods was established. Pharmacodynamics study, including the local bacteriostatic and osteogenic ability were evaluated by X-ray, Micro-CT and histopathology at 4 months after surgery. These findings indicate that a reliable rabbit model of local bone defect infection successfully established by big hole approach. The CPSFs with significant histocompatibility and biocompatibility could sustained release vancomycin for extended duration. It exhibited great application potential in clinical aim at the indication of local infectious bone defects.

Real-Life Experience of Regorafenib in Patients With Advanced Hepatocellular Carcinoma.

Background: The RESORCE trial reported that regorafenib was effective as the second-line treatment for patients with hepatocellular carcinoma (HCC) after progression on sorafenib. Real-world data are needed to assess clinical outcomes and adverse events in the setting of daily practice. Objective: We aimed to evaluate the efficacy and safety of regorafenib after disease progression with sorafenib in Chinese patients with advanced HCC. Patients and Methods: A total of 41 patients with advanced HCC who did not respond to sorafenib and followed a regorafenib regimen were enrolled in this retrospective study. Overall survival (OS), progression-free survival (PFS), radiological responses, and adverse events (AEs) were evaluated. Survival curves were compared by using the log-rank test and constructed with the Kaplan-Meier method. Results: The median PFS with regorafenib was 6.6 months (range: 5.0-8.2 months), and the median OS with regorafenib was not reached. The 1-year OS rate of regorafenib was 66.4%. The median OS of sequential sorafenib to regorafenib treatment was 35.3 months [95% confidence interval (CI), 24.3-46.3], and the 2-year OS rate of sequential sorafenib to regorafenib treatment was 74.4%. The most common AEs of regorafenib treatment were elevated aspartate aminotransferase [17/41 patients (41.5%)], elevated alanine aminotransferase [16/41 patients (39%)] and hand-foot syndrome [14/41 patients (34.1%)]. Conclusion: Regorafenib appears to be safe and clinically effective in patients with advanced HCC who progressed on first-line sorafenib.

Reverse anti-breast cancer drug resistance effects by a novel two-step assembled nano-celastrol medicine.

Multidrug resistance (MDR) has become one of the most intractable problems in clinics as it would cause failure in chemotherapy. In this study, we demonstrated that a nanoscale self-assembled nanomedicine, which almost consisted of a pure chemo-drug, could efficiently overcome MDR. Celastrol (CST) was directly assembled into a discrete nanomedicine by precipitation, and then CST nanoparticles (CNPs) inhibited drug efflux pumps by activating HSF-1 expression and promoting HSF-1 translocation into nucleus to suppress the Pgp expression. The more drug accumulated in cells could activate apoptosis signals simultaneously and realize drug resistance reversal. CNPs significantly increased the level of ROS to regulate ERK/JNK signaling, which would further induce resistant cell apoptosis. The tandem apoptosis strategy used the same concentration of CST but achieved a higher antitumor effect. Overall, our study provides a new translational and alternative strategy using conventional natural products to overcome MDR with high efficacy.

Diagnosis of paediatric tuberculosis by optically detecting two virulence factors on extracellular vesicles in blood samples.

Sensitive and specific blood-based assays for the detection of pulmonary and extrapulmonary tuberculosis would reduce mortality associated with missed diagnoses, particularly in children. Here we report a nanoparticle-enhanced immunoassay read by dark-field microscopy that detects two Mycobacterium tuberculosis virulence factors (the glycolipid lipoarabinomannan and its carrier protein) on the surface of circulating extracellular vesicles. In a cohort study of 147 hospitalized and severely immunosuppressed children living with HIV, the assay detected 58 of the 78 (74%) cases of paediatric tuberculosis, 48 of the 66 (73%) cases that were missed by microbiological assays, and 8 out of 10 (80%) cases undiagnosed during the study. It also distinguished tuberculosis from latent-tuberculosis infections in non-human primates. We adapted the assay to make it portable and operable by a smartphone. With further development, the assay may facilitate the detection of tuberculosis at the point of care, particularly in resource-limited settings.

Transcriptional Regulator YqeI, Locating at ETT2 Locus, Affects the Pathogenicity of Avian Pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is the leading cause of systemic infections in poultry worldwide and has a hidden threat to public health. Escherichia coli type three secretion system 2 (ETT2), similar to the Salmonella pathogenicity island SPI1, is widely distributed in APEC and associated with virulence. The function of YqeI, which is one of the hypothetical transcriptional regulators locating at the ETT2 locus of APEC, is unknown. In this study, we successfully obtained the mutant strain AE81ΔyqeI of the wild type strain AE81 and performed the transcriptional profiling assays. Additionally, the transcriptional sequencing results revealed that YqeI influenced localization, locomotion and biological adhesion and so on. The transmission electron microscope observation showed that the wild type strain AE81 possessed long curved flagella, whereas the mutant strain AE81ΔyqeI hardly had any. The strain AE81ΔyqeI exhibited lower motility than AE81 after culturing the dilute bacterial suspension on a semisolid medium. It was also found that the survival ability of AE81ΔyqeI weakened significantly when AE81ΔyqeI was cultured with 0%, 10%, 20%, 30%, 40% and 50% SPF serum in PBS, and AE81ΔyqeI had decreased adherence to DF-1 cells compared with AE81 in the bacterial adhesion assay. The bacterial colonization assay indicated that the virulence of AE81ΔyqeI was reduced in the heart, liver, spleen, and lung. These results confirmed that the transcription regulator YqeI is involved in APEC's pathogenicity, and this study provides clues for future research.

Immobilized Ferrous Ion and Glucose Oxidase on Graphdiyne and Its Application on One-Step Glucose Detection.

Graphdiyne (GDY) is a novel two-dimensional (2D) carbon allotrope with sp-hybridized carbon atoms and hexagonal rings. Because of its unique structure and electronic property, GDY was reported as a promising candidate applied in energy storage, catalysis, biosensing and so on. However, using GDY as a platform to immobilize metal ion or enzyme was still not reported. Here, we presented a GDY-based composite with dual-enzyme activity by immobilizing ferrous ion and glucose oxidase onto GDY sheet. GDY showed great adsorption capacity and maintained the high catalytic activity of ferrous ion. The ferrous ion preferred to adsorb in between the neighboring two C-C triple bonds of GDY with lower adsorption energy (-5.64 eV) if compared to graphene (-1.69 eV). Meanwhile, GDY exhibited the ability of adsorbing glucose oxidase while did not obviously influence the structure and catalytic activity of the enzyme. The as-prepared composite was successfully used in one-step blood glucose detection. This work provides a new insight on ion and enzyme immobilization by 2D material.

Gd-Metallofullerenol nanoparticles cause intracellular accumulation of PDGFR-α and morphology alteration of fibroblasts.

Gadolinium-metallofullerenols (Gd@C82(OH)22) are a promising agent for cancer therapy and have shown beneficial effects in regulating the tumor microenvironment with low toxicity. However, the underlying mechanism by which Gd@C82(OH)22 interacts with fibroblasts remains unclear. In order to explore the critical role that activated fibroblasts play in tumorigenesis and fibrosis, we investigated the regulatory effect of Gd@C82(OH)22 in fibroblast activation and oncogenic transformation, and found that the PDGFR-α is an essential molecule in modulating the morphology and functional changes in fibroblasts after Gd@C82(OH)22 treatment. Apart from increasing the PDGFR-α protein level, Gd@C82(OH)22 nanoparticles also significantly increased the protein level of Rab5, which is required for regulating PDGFR-α endosomal recycling. The Rab5-mediated recycling of PDGFR-α maybe attributed to the Gd@C82(OH)22 regulated inhibition of fibroblast activation. Overall, our work demonstrated that Gd@C82(OH)22 nanoparticles can attenuate the PDGF-stimulated phosphorylation of PDGFR-α in fibroblasts and suppress the fibroblast activation by interrupting endosomal recycling. These findings may be contributed to the collagen accumulation for encaging cancer.

Poly(lactic-co-glycolic acid)/Polycaprolactone Nanofibrous Membranes for High-Efficient Capture of Nano- and Microsized Particulate Matter.

The incidence of many diseases is closely related to air pollution. Suspended particulate matter of different sizes represents a major source of environmental pollution. Fine particles, especially ultrafine particles smaller than 2.5 µm, might be more harmful to human health because of their extremely small size, which enables them to penetrate human lungs and bronchi and makes them difficult to filter out. Therefore, the fatal risks associated with PM call for the development of air purification materials with high efficiency and low resistance. In this study, poly(lactic-co-glycolic acid) and polycaprolactone were used to prepare nanofibrous membranes suitable for the efficient capture of particulate matter formed in haze-fog episodes, especially particles smaller than 0.5 µm. The present nanofibrous membranes exhibit superior filtration efficiency for particulate matter, with a much lower pressure drop compared to typical commercial microfiber air filters. Thanks to the combination of small pore size, high porosity, and robust mechanical properties, the poly(lactic-co-glycolic acid)/polycaprolactone (6:4) composite membrane exhibits a high filtration efficiency of 97.81% and a low pressure drop of 181 Pa. These favorable features, combined with the easy availability and biocompatibility of the component materials, highlight the promising potential of the present nanofibrous membranes for the development of personal wearable air purifiers.

Synergistic combination chemotherapy using carrier-free celastrol and doxorubicin nanocrystals for overcoming drug resistance.

A key challenge of chemotherapy in clinical treatments is multidrug resistance (MDR), which mainly arises from drug efflux-induced tumor cell survival. Thus, it is necessary to provide biocompatible chemotherapeutics to improve drug accumulation in MDR cells. Herein, two clinical small molecular drugs, celastrol (CST) and doxorubicin (DOX), were self-assembled into carrier-free and biocompatible nanoparticles (CST/DOX NPs) via a simple and green precipitation method for synergistic combination chemotherapy to overcome DOX resistance. These spherical CST/DOX NPs can improve the water-solubility of CST, reduce the dosage of DOX, and therefore significantly enhance cellular drug accumulation by activating heat shock factor 1 (HSF-1) and inhibiting NF-κB to depress P-gp expression, which results in apoptosis and autophagy of DOX resistant cells through the ROS/JNK signaling pathway. Finally, synergistic combination chemotherapy was attained in both MCF-7/MDR cells and 3D multicellular tumor spheroids. Thus, CST/DOX NPs provide an alternative for overcoming drug resistance in future clinical applications.

Direct site-specific treatment of skin cancer using doxorubicin-loaded nanofibrous membranes.

Doxorubicin (DOX) is widely used in cancer therapy. However, its application is sometimes limited by its adverse cardiotoxicity and delivery pathways. In our study, we prepared a topical implantable delivery device for controlled drug release and site-specific treatment. The core region consisted of poly (lactic co-glycolic acid) and poly-caprolactone, whereas the shell region was composed of cross-linked gelatin. DOX was enclosed in the core region of a core-shell nanofiber obtained by electrospinning. This implantable delivery device was implanted on the top of the melanoma in a mouse model, which had shown a DOX-controlled release profile with sustained and sufficient local concentration against melanoma growth in mice with negligible side effects. Compared with the traditional intravenous administration, the implantable device allows precisely localized treatment and therefore can reduce the dose, decrease the injection frequency, and ensure antitumor efficacy associated with lower side effects to normal tissues. Using a coaxial electrospinning process, it is promising to deliver different hydrophobic or hydrophilic drugs for direct tumor site-specific therapy without large systemic doses and minimized systemic toxicity.