An Aggregation-Induced Emission-Based Dual Emitting Nanoprobe for Detecting Intracellular pH and Unravelling Metabolic Variations in Differentiating Lymphocytes.

Monitoring T lymphocyte differentiation is essential for understanding T cell fate regulation and advancing adoptive T cell immunotherapy. However, current biomarker analysis methods necessitate cell lysis, leading to source depletion. Intracellular pH (pHi) can be affected by the presence of lactic acid (LA), a metabolic mediator of T cell activity such as glycolysis during T cell activation; therefore, it is a potentially a good biomarker of T cell state. In this work, a dual emitting enhancement-based nanoprobe, namely, AIEgen@F127-AptCD8, was developed to accurately detect the pHi of T cells to "read" the T cell differentiation process. The nanocore of this probe comprises a pair of AIE dyes, TPE-AMC (pH-sensitive moiety) and TPE-TCF, that form a donor-acceptor pair for sensitive detection of pHi by dual emitting enhancement analysis. The nanoprobe exhibits a distinctly sensitive narrow range of pHi values (from 6.0 to 7.4) that can precisely distinguish the differentiated lymphocytes from naïve ones based on their distinct pHi profiles. Activated CD8+ T cells demonstrate lower pHi (6.49 ± 0.09) than the naïve cells (7.26 ± 0.11); Jurkat cells exhibit lower pHi (6.43 ± 0.06) compared to that of nonactivated ones (7.29 ± 0.09) on 7 days post-activation. The glycolytic product profiles in T cells strongly correlate with their pHi profiles, ascertaining the reliability of probing pHi for predicting T cell states. The specificity and dynamic detection capabilities of this nanoprobe make it a promising tool for indirectly and noninvasively monitoring T cell activation and differentiation states.

[Comparison of effectiveness of three surgical methods in treatment of Pauwels type â ¢ femoral neck fracture in young and middle-aged patients].

Objective: To compare the effectiveness of three surgical methods in the treatment of Pauwels type Ⅲ femoral neck fracture in young and middle-aged patients, in order to provide reference for clinical selection of appropriate surgical methods. Methods: The clinical data of 103 patients with Pauwels type Ⅲ femoral neck fracture who met the selection criteria between June 2018 and December 2021 were retrospectively analyzed. The fractures were fixed with hollow screws in an inverted triangular shape (37 cases, hollow screw group), hollow screws in an inverted triangular shape combined with eccentric shaft screw (34 cases, eccentric shaft screw group), and hollow screws in an inverted triangular shape combined with medial support plate (32 cases, support plate group). There was no significant difference in age, gender, cause of injury, body mass index, time from injury to operation, side of the fracture, and Garden classification, whether they were in traction preoperatively, and other baseline data between groups ( P>0.05). The operation time, intraoperative blood loss, the number of fluoroscopy, the length of hospital stay, early postoperative complication and postoperative weight-bearing time of the three groups were recorded. Harris score was used to evaluate joint function at 6 and 12 months after operation, and the difference between the two time points (change value) was calculated for comparison between groups. X-ray films were reviewed to evaluate the quality of fracture reduction (Garden index) and healing, as well as the occurrence of internal fixation failure and femoral head necrosis. Results: The patients of the three groups were successfully completed. Compared with the hollow screw group and the eccentric shaft screw group, the operation time and intraoperative blood loss of the support plate group significantly increased, the number of fluoroscopy reduced, and the quality of fracture reduction was better, the differences were significant ( P<0.05). The operation time, intraoperative blood loss, and the number of fluoroscopy of the hollow screw group were less than those of the eccentric shaft screw group, the differences were significant ( P<0.05). There was no significant difference in the length of hospital stay between groups ( P>0.05). All patients in the three groups were followed up 21-52 months, with an average follow-up time of 36.0 months, and there was no significant difference between groups ( P>0.05). The incisions of all patients healed by first intention. Imaging reexamination showed that there was no significant difference in the incidence of fracture nonunion between groups ( P>0.05). The fracture healing, partial weight-bearing, and full weight-bearing were significantly earlier in the eccentric shaft screw group and the support plate group than in the hollow screw group ( P<0.05). There was no significant difference in change value of Harris score, the incidence of postoperative deep venous thrombosis and femoral head necrosis between groups ( P>0.05); however, the incidence of internal fixation failure in the support plate group and the eccentric shaft screw group was significantly lower than that in the hollow screw group ( P<0.05). The incidence of postoperative lateral thigh irritation in the support plate group was significantly lower than that in the hollow screw group ( P<0.05); there was no significant difference between the eccentric shaft screw group and the other two groups ( P>0.05). The overall incidences of postoperative complications in the eccentric shaft screw group and the support plate group were significantly lower than that in the hollow screw group ( P<0.05). Conclusion: For young and middle-aged patients with Pauwels type Ⅲ femoral neck fracture, compared with simple hollow screw fixation in an inverted triangular shape, combined with medial support plate or eccentric shaft screw internal fixation can shorten the fracture healing time, reduce the incidences of postoperative complication, more conducive to early functional exercise of the affected limb; at the same time, the operation time and blood loss of combined eccentric shaft screw internal fixation are less than those of combined medial support plate internal fixation, so the hollow screw in an inverted triangular shape combined with eccentric shaft screw fixation may be a better choice.

Magnetic-responsive upconversion luminescence resonance energy transfer (LRET) biosensor for ultrasensitive detection of SARS-CoV-2 spike protein.

Upconversion nanoparticles (UCNPs) are ideal donors for luminescence resonance energy transfer (LRET)-based biosensors due to their excellent upconversion luminescence properties. However, the relatively large size of antibodies and proteins limits the application of UCNPs-based LRET biosensors in protein detection because the large steric hindrance of proteins leads to low energy transfer efficiency between UCNPs and receptors. Herein, we developed a magnetic responsive UCNPs-based LRET biosensor to control the coupling distance between antibody-functionalized UCNPs (Ab-UCNPs) as donors and antibody-PEG linker-magnetic gold nanoparticles (Ab-PEG-MGNs) as acceptors for ultrasensitive and highly selective detection of SARS-CoV-2 spike proteins. Our results showed that this platform reversibly shortened the coupling distance between UCNPs and MGNs and enhanced the LRET signal with a 10-fold increase in the limit of detection (LOD) from 20.6 pg/mL without magnetic modulation to 2.1 pg/mL with magnetic modulation within 1 h. The finite-difference time-domain (FDTD) simulation with cyclic distance change confirmed the distance-dependent LRET efficiency under magnetic modulation, which supported the experimental results. Moreover, the applications of this magnetic-responsive UCNP-based LRET biosensor could be extended to other large-size biomolecule detection.

Nanomanipulation of Ligand Nanogeometry Modulates Integrin/Clathrin-Mediated Adhesion and Endocytosis of Stem Cells.

Nanosubstrate engineering can be a biomechanical approach for modulating stem cell differentiation in tissue engineering. However, the study of the effect of clathrin-mediated processes on manipulating this behavior is unexplored. Herein, we develop integrin-binding nanosubstrates with confined nanogeometries that regulate clathrin-mediated adhesion- or endocytosis-active signaling pathways for modulating stem fates. Isotropically presenting ligands on the nanoscale enhances the expression of clathrin in cells, thereby facilitating uptake of dexamethasone-loaded nanoparticles (NPs) to boost osteogenesis of stem cells. In contrast, anisotropic ligand nanogeometry suppresses this clathrin-mediated NP entry by strengthening the association between clathrin and adhesion spots to reinforce mechanotransduced signaling, which can be abrogated by the pharmacological inhibition of clathrin. Meanwhile, inhibiting focal adhesion formation hinders cell spreading and enables a higher endocytosis efficiency. Our findings reveal the crucial roles of clathrin in both endocytosis and mechanotransduction of stem cells and provide the parameter of ligand nanogeometry for the rational design of biomaterials for tissue engineering.

A dual "turn-on" biosensor based on AIE effect and FRET for in situ detection of miR-125b biomarker in early Alzheimer's disease.

MicroRNA-125b (miR-125b) is highly associated with synaptic dysfunction and tau hyperphosphorylation in the early pathogenesis of Alzheimer's disease (AD), making it a promising biomarker for early AD diagnosis. Hence, there is an urgent need for a reliable sensing platform to assist in situ miR-125b detection. In this work, we report a dual "turn-on" fluorescence biosensor based on the nanocomposite of aggregation-induced emission fluorogen (AIEgen)-labeled oligonucleotide (TPET-DNA) probes immobilized on the surface of cationic dextran modified molybdenum disulfide (TPET-DNA@Dex-MoS2). In the presence of the target, TEPT-DNA can hybridize with miR-125b to form a DNA/RNA duplex, causing TPET-DNA to detach from the surface of Dex-MoS2 that simultaneously activates the dual fluorescence enhancement processes: (1) recovery of TPET-DNA signal and (2) strong fluorescent emission from AIEgen triggered by restriction of the intramolecular rotation. The sensing performance of TPET-DNA@Dex-MoS2 was demonstrated by detecting miR-125b in vitro with good sensitivity at the picomolar level and rapid response (≤1 h) without amplification procedures. Furthermore, our nanoprobes exhibited excellent imaging capabilities to aid real-time monitoring of the endogenous miR-125b in PC12 cells and brain tissues of mice AD model induced by local administration of okadaic acid (OA). The fluorescence signals of the nanoprobes indicated miR-125b was spatially associated with phosphorylated tau protein (p-tau) in vitro and in vivo. Therefore, TPET-DNA@Dex-MoS2 could be a promising tool for in situ and real-time monitoring of the AD-related microRNAs and also provide mechanistic insight into the early prognosis of AD.

A CRISPR-Cas12a integrated SERS nanoplatform with chimeric DNA/RNA hairpin guide for ultrasensitive nucleic acid detection.

Background: CRISPR-Cas12a has been integrated with nanomaterial-based optical techniques, such as surface-enhanced Raman scattering (SERS), to formulate a powerful amplification-free nucleic acid detection system. However, nanomaterials impose steric hindrance to limit the accessibility of CRISPR-Cas12a to the narrow gaps (SERS hot spots) among nanoparticles (NPs) for producing a significant change in signals after nucleic acid detection. Methods: To overcome this restriction, we specifically design chimeric DNA/RNA hairpins (displacers) that can be destabilized by activated CRISPR-Cas12a in the presence of target DNA, liberating excessive RNA that can disintegrate a core-satellite nanocluster via toehold-mediated strand displacement for orchestrating a promising "on-off" nucleic acid biosensor. The core-satellite nanocluster comprises a large gold nanoparticle (AuNP) core surrounded by small AuNPs with Raman tags via DNA hybridization as an ultrabright Raman reporter, and its disassembly leads to a drastic decrease of SERS intensity as signal readouts. We further introduce a magnetic core to the large AuNPs that can facilitate their separation from the disassembled nanostructures to suppress the background for improving detection sensitivity. Results: As a proof-of-concept study, our findings showed that the application of displacers was more effective in decreasing the SERS intensity of the system and attained a better limit of detection (LOD, 10 aM) than that by directly using activated CRISPR-Cas12a, with high selectivity and stability for nucleic acid detection. Introducing magnetic-responsive functionality to our system further improves the LOD to 1 aM. Conclusion: Our work not only offers a platform to sensitively and selectively probe nucleic acids without pre-amplification but also provides new insights into the design of the CRISPR-Cas12a/SERS integrated system to resolve the steric hindrance of nanomaterials for constructing biosensors.

Magnetic-Responsive Surface-Enhanced Raman Scattering Platform with Tunable Hot Spot for Ultrasensitive Virus Nucleic Acid Detection.

Surface-enhanced Raman scattering (SERS)-based biosensors are promising tools for virus nucleic acid detection. However, it remains challenging for SERS-based biosensors using a sandwiching strategy to detect long-chain nucleic acids such as nucleocapsid (N) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) because the extension of the coupling distance (CD) between the two tethered metallic nanostructures weakens electric field and SERS signals. Herein, we report a magnetic-responsive substrate consisting of heteoronanostructures that controls the CD for ultrasensitive and highly selective detection of the N gene of SARS-CoV-2. Significantly, our findings show that this platform reversibly shortens the CD and enhances SERS signals with a 10-fold increase in the detection limit from 1 fM to 100 aM, compared to those without magnetic modulation. The optical simulation that emulates the CD shortening process confirms the CD-dependent electric field strength and further supports the experimental results. Our study provides new insights into designing a stimuli-responsive SERS-based platform with tunable hot spots for long-chain nucleic acid detection.

Predictive Performance and Optimal Cut-Off Points of Blood Pressure for Identifying Arteriosclerosis among Adults in Eastern China.

This study aimed to assess the predictive performance and establish optimal cut-off points of blood pressure for identifying arteriosclerosis in eastern Chinese adults. Brachial-ankle pulse wave velocity (baPWV) was utilized to evaluate arteriosclerosis. The predictive performance of blood pressure for arteriosclerosis was determined by the area under the curve (AUC) of receiver operating characteristics; the optimal blood pressure cut-off points were determined by Youden's index. A logistic regression model was used to acquire the odds ratio (OR) of blood pressure for arteriosclerosis. The AUCs of blood pressure for identifying arteriosclerosis were 0.868 (95%CI: 0.860-0.875) for systolic blood pressure (SBP) and 0.835 (95%CI: 0.827-0.843) for diastolic blood pressure (DBP), both p < 0.01. The AUCs of women were higher than that of men (0.903 vs. 0.819 for SBP; 0.847 vs. 0.806 for DBP; Z test p < 0.05). The AUCs in the 18-39.9-years group were higher than that in the 40-59.9-years and 60-84-years groups (0.894 vs. 0.842 and 0.818 for SBP; 0.889 vs. 0.818 and 0.759 for DBP; Z test p < 0.05). The total optimal cut-off points of blood pressure for predicting arteriosclerosis were 123.5/73.5 mmHg (SBP/DBP) overall; 123.5/73.5 and 126.5/79.5 mmHg for women and men, respectively; and 120.5/73.5, 123.5/76.5, and 126.5/75.5 mmHg for 18-39.9-years, 40-59.9-years, and 60-84-years groups, respectively. Blood pressure indexes had a high predictive performance for identifying arteriosclerosis with the optimal cut-off point of 123.5/73.5 mmHg (SBP/DBP) in eastern Chinese adults. Women or the younger population have a higher predictive performance and lower cut-off points to identify arteriosclerosis.

The Interplay Between Epigenetic Regulation and CD8+ T Cell Differentiation/Exhaustion for T Cell Immunotherapy.

Effective immunotherapy treats cancers by eradicating tumourigenic cells by activated tumour antigen-specific and bystander CD8+ T-cells. However, T-cells can gradually lose cytotoxicity in the tumour microenvironment, known as exhaustion. Recently, DNA methylation, histone modification, and chromatin architecture have provided novel insights into epigenetic regulations of T-cell differentiation/exhaustion, thereby controlling the translational potential of the T-cells. Thus, developing strategies to govern epigenetic switches of T-cells dynamically is critical to maintaining the effector function of antigen-specific T-cells. In this mini-review, we 1) describe the correlation between epigenetic states and T cell phenotypes; 2) discuss the enzymatic factors and intracellular/extracellular microRNA imprinting T-cell epigenomes that drive T-cell exhaustion; 3) highlight recent advances in epigenetic interventions to rescue CD8+ T-cell functions from exhaustion. Finally, we express our perspective that regulating the interplay between epigenetic changes and transcriptional programs provides translational implications of current immunotherapy for cancer treatments.

Biosynthetic Mycotoxin Conjugate Mimetics-Mediated Green Strategy for Multiplex Mycotoxin Immunochromatographic Assay.

Various mycotoxins widely co-exist in agro-products, and their combined effects cause toxicity and potential carcinogenicity to humans and animals. In this work, we developed an economical and sensitive quantum dots (QDs)/QD microbead (QDs/QB)-based multiplex immunochromatographic assay (mICA) for the rapid detection of fumonisin B1 (FB1), zearalenone (ZEN), and ochratoxin A (OTA) without the building-up process of mycotoxin conjugates. QDs and QBs were selected as fluorescent reporters and conjugated with antimycotoxin monoclonal antibodies for improving sensitivity. Furthermore, phage-displayed FB1, ZEN, and OTA mimotope peptide-based soluble and monovalent fusions to maltose-binding protein (MBP) were applied onto the test line of the mICA as the mimetic coating antigen. Under the optimized conditions, the visual detection limits (vLODs) of peptide-MBP-based mICA could be obtained as 0.25 ng/mL for FB1, 3.0 ng/mL for ZEN, and 0.5 ng/mL for OTA within 10 min. The results for spiked real sample detection indicate good accuracy, reproducibility, and practicability. In addition, the proposed mICA was comparable with ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in terms of reliability in detecting FB1, ZEN, and OTA using natural samples. From the point of promoting commercial production, these time-saving and low-cost peptide-MBP antigens applied in ICA might provide promising potential for promoting productivity and decreasing the cost of production.

[Feasibility study of Kirschner wire-fixation-cortical bone technique in treatment of intertrochanteric fracture].

OBJECTIVE: To explore the feasibility and effectiveness of maintaining the reduction of unstable intertrochanteric fractures by Kirschner wire-fixation-cortical bone technique. METHODS: Forty patients with intertrochanteric fracture [AO/Orthopaedic Trauma Association (AO/OTA) type 31-A2.2] admitted between May 2015 and January 2017 and requiring closed reduction and proximal femoral nail antirotation (PFNA) were randomly divided into trial group (intraoperative Kirschner wire-fixation-cortical bone technique group, 20 cases) and control group (conventional treatment group, 20 cases). There was no significant difference in general data of gender, age, side, body mass index, cause of injury, time from injury to operation between the two groups ( P>0.05). The operation time, intraoperative blood loss, and intraoperative fluoroscopy times of the two groups were recorded; the reduction quality of fracture was observed according to the corresponding relationship between medial and anterior cortex (positive, neutral, and negative support) of intraoperative fluoroscopy proposed by ZHANG Shimin, and the stability of internal fixation and fracture healing were observed; Harris score was used to evaluate the recovery of hip function at 12 months after operation. RESULTS: In the trial group, 6 cases (30%) had 2 Kirschner wires implanted less than 4 times, 7 cases (35%) had 5-8 times, and 7 cases (35%) had 9 times or more. There was no significant difference in operation time and intraoperative blood loss between the two groups ( P>0.05), but the blood transfusion volume and intraoperative fluoroscopy times in the trial group were significantly less than those in the control group ( P<0.05). Both groups were followed up 13-21 months, with an average of 17 months. There was no complications such as wound infection, deep venous thrombosis of lower extremities, refracture, and internal fixation-related complications. The quality of intraoperative reduction in the trial group was significantly better than that in the control group ( Z=-2.794, P=0.024). The Harris score of the trial group was significantly better than that of the control group at 12 months after operation ( t=2.98, P=0.01). CONCLUSION: The use of Kirschner wire-fixation-cortical bone technique during intertrochanteric fracture closed reduction and PFNA internal fixation surgery can effectively maintain the reduction effect, reduce the number of fluoroscopy, improve the reduction quality, reduce allogeneic blood input, obtain better hip function, and do not increase the operation time and intraoperative blood loss.

Phage displayed mimotope peptide-based immunosensor for green and ultrasensitive detection of mycotoxin deoxynivalenol.

Immunoassays play an important role in the monitoring and detection of mycotoxins in food. However, mycotoxin conjugates in the immunoassay system synthesized with toxic mycotoxin as raw material has seriously harmed the environment and human health during and after the performance. In this work, using mycotoxin deoxynivalenol (DON) as a model happen, a selected DON mimotope (phage-displayed peptide) that specifically binds to anti-DON antibody was served as a mimetic competing antigen in electrochemical immunosensor for ultrasensitive and green detection of DON. Under the optimized conditions, the mimotope peptide-based electrochemical immunosensor showed a dynamic range of 0.1-10,000 pg/ml and the limit detection (LOD) of 0.07 pg/ml. In addition, the accuracy and reproducibility of the established electrochemical immunosensor were evaluated by testing cross-reactivity and spiking DON negative samples. The result showed that mimotope peptide-based immunosensor has negligible cross-reactivity with other mycotoxins, and the average recovery of corn and wheat sample was 90.4%-118% and 101.3%-111%, respectively. Furthermore, the novel concept might provide potential applications to a general method for the immunosensor of various toxic small molecules.

An ultrasensitive electrochemical immunosensor for Cry1Ab based on phage displayed peptides.

The rapid and widespread adoption of Bacillus thuringiensis (Bt) proteins in genetically modified (GM) crops has raised concerns about the impact of GM crops on environment and food safety. A sensitive and specific method for detecting Bt proteins in GM crops is of great significance for environment and food risk assessment. In this study, using Cry1Ab as a model Bt protein, an ultrasensitive electrochemical immunosensor for Cry1Ab protein has been developed based on phage displayed peptide. Phage displayed peptides against Cry1Ab protein were obtained from a phage displayed peptide library without animal immunization process through biopanning-elution strategy. After modification of electrode with gold nanoparticles, selected phage displayed peptide was applied to electrochemical immunosensor for Cry1Ab. Under the optimized conditions, the peptide-based immunosensor showed a dynamic range of 0.01-100ng/mL and a limit detection of 7pg/mL. Specific measurement of this established method was conducted by testing cross-reactivity of Cry1Ac (88% amino acid sequence homology to Cry1Ab protein), and the result showed that peptide-based immunosensor has negligible cross-reactivity with analogue. In addition, the accuracy and reproducibility of this established immunosensor was evaluated by testing the recovery of spiked samples and assay coefficients of variation, respectively. The results showed that the average recovery of corn and wheat sample was 90-120% and 86.7-120%, respectively; the intra-assay coefficient of variation was 7.4% (n = 6), and the inter-assay coefficient of variation was 6.9% (n = 6) at 1ng/mL Cry1Ab solution. Furthermore, the novel concept of peptide-based immunosensor may provide a potential application in general method for the ultrasensitive detection of various Bt proteins.