Mesophilic Argonaute-Mediated Polydisperse Droplet Biosensor for Amplification-Free, One-Pot, and Multiplexed Nucleic Acid Detection Using Deep Learning.

Detection of nucleic acids from a single multiplexed and amplification-free test is critical for ensuring food safety, clinical diagnostics, and environmental monitoring. In this study, we introduced a mesophilic Argonaute protein from Clostridium butyricum (CbAgo), which exhibits nucleic acid endonuclease activity, to achieve a programmable, amplification-free system (PASS) for rapid nucleic acid quantification at ambient temperatures in one pot. By using CbAgo-mediated binding with specific guide DNA (gDNA) and subsequent targeted cleavage of wild-type target DNAs complementary to gDNA, PASS can detect multiple foodborne pathogen DNA (<102 CFU/mL) simultaneously. The fluorescence signals were then transferred to polydisperse emulsions and analyzed by using deep learning. This simplifies the process and increases the suitability of polydisperse emulsions compared to traditional digital PCR, which requires homogeneous droplets for accurate detection. We believe that PASS has the potential to become a next-generation point-of-care digital nucleic acid detection method.

Dual-Track Multifunctional Bimetallic Metal-Organic Frameworks for Antibiotic Enrichment and Detection.

The improper use and overuse of antibiotics have led to significant burdens and detrimental effects on the environment, food supply, and human health. Herein, a magnetic solid-phase extraction program and an optical immunosensor based on bimetallic Ce/Zr-UiO 66 for the detection of antibiotics are developed. A magnetic Fe3O4@SiO2@Ce/Zr-UiO 66 metal-organic framework (MOF) is prepared to extract and enrich chloramphenicol from fish, wastewater, and urine samples, and a horseradish peroxidase (HRP)-Ce/Zr-UiO 66@bovine serum protein-chloramphenicol probe is used for the sensitive detection of chloramphenicol based on the dual-effect catalysis of Ce and HRP. In this manner, the application of Ce/Zr-UiO 66 in integrating sample pretreatment and antibiotic detection is systematically investigated and the associated mechanisms are explored. It is concluded that Ce/Zr-UiO 66 is a versatile dual-track material exhibiting high enrichment efficiency (6.37 mg g-1) and high sensitivity (limit of detection of 51.3 pg mL-1) for chloramphenicol detection and serving as a multifunctional MOF for safeguarding public health and hygiene.

Artificial Intelligence-Based Imaging Transcoding System for Multiplex Screening of Viable Foodborne Pathogens.

Multiplex detection of viable foodborne pathogens is critical for food safety and public health, yet current assays suffer trade-offs between cost, assay complexity, sensitivities, and the specificity between live and dead bacteria. We herein developed a sensing method using artificial intelligence transcoding (SMART) for rapid, sensitive, and multiplex profiling of foodborne pathogens. The assay utilizes the programmable polystyrene (PS) microspheres to encode different pathogens, inducing subsequent visible signals under conventional microscopy that can be analyzed using a customized, artificial intelligence-computer vision, which was trained to decode the intrinsic properties of PS microspheres to reveal the numbers and types of pathogens. Our approach enabled the rapid and simultaneous detection of multiple bacteria from egg samples of <102 CFU/mL without DNA amplification and showed strong consistency with the standard microbiologic and genotypic methods. We adopted our assay through phage-guided targeting to enable the discrimination between live and dead bacteria.

Click Chemistry-Mediated Particle Counting Sensing via Cu(II)-Polyglutamic Acid Coordination Chemistry and Enzymatic Reaction.

An electrical resistance-based particle counter (ERPC) with simple operation and high resolution has proved to be a promising biosensing toolkit, whereas amplification-free ERPC biosensors are incapable of analyzing trace small molecules due to their relatively low sensitivity. In this work, click chemistry-mediated particle counting sensing of small-molecule hazards in food samples with high sensitivity was developed. In this strategy, unbound alkyne-functionalized polystyrene microspheres were collected by magnetic separation from the copper-ion-mediated click reaction between alkyne-functionalized polystyrene microspheres and azido-functionalized magnetic beads, which could be used as signal probes for the readout. This click chemistry-mediated ERPC biosensor converts the detection of targets to the quantification of copper ions or ascorbic acid by performing competitive immunoassay-based coordination chemistry and enzymatic reaction, respectively. The sensitivity of the ERPC biosensor has been improved by an order of magnitude due to the signal amplification effects of click chemistry, coordination adsorption, and enzyme catalysis. Furthermore, because of the efficient separation and enrichment of immunomagnetic beads and the robustness of click chemistry, the interference from food matrixes and immunoassay is effectively reduced, and thus, our strategy is exceedingly suitable for detecting trace targets in complex samples.

Label-Free Microchannel Immunosensor Based on Antibody-Antigen Biorecognition-Induced Charge Quenching.

Pursuing convenient operations and precise testing have become an urgent requirement in clinical diagnosis, treatment, and prognosis. Label-free detection is desirable for obviating the labeling process while maintaining high sensitivity and efficiency. Here, we used the dual properties of highly selective antibody-antigen recognition and potential signaling of biomolecules to construct a label-free electroosmotic flow-driven microchannel (LF-EMB) biosensor based on an antibody-antigen biorecognition-induced charge quenching theory proposed herein. The LF-EMB consists of a one-step immune-reaction, one-button portable device, and supporting microfluidic chip, providing a high-powered tool for rapid on-site testing. The LF-EMB quantified interleukin-6 and kanamycin levels down to 1 pg/mL and 5 pg/mL, respectively, with the whole analysis completed within 35 min. The outstanding sensitivity and detection speed of the constructed LF-EMB provide a convenient option for the quantitative detection of inflammatory markers and antibiotics.

Magilock: a reliable control triggering method in multi-channel eye-control systems.

Eye-tracking technology brings a different human-computer interaction experience to users because of its intuitive, natural, and hands-free operation characteristics. Avoiding the Midas touch problem and improving the accuracy of interaction are among the main goals of the research and development of eye-control systems. This study reviews the methods and limitations of research on avoiding the Midas touch problem. For typical control clicking operations with low fault tolerance, such as mode switching and state selection in an eye-control system, this study proposes Magilock, a more reliable control triggering method with a high success rate in multi-channel eye-control systems. Magilock adds a control pre-locked mechanism between the two interactive steps of eye-control channel positioning control and other interactive channel triggering controls in the multi-channel eye-control system. This effectively avoids incorrect control triggering caused by multi-channel coordination disorder and gaze-point drift. This study also conducted ergonomic experiments to explore the lock and unlock times of the control pre-locked mechanism in Magilock. Taking into account the experimental data and subjective evaluation of the participants, we recommend setting the lock time and the unlock time of Magilock to 200 ms.

Polystyrene microsphere-mediated optical sensing strategy for ultrasensitive determination of aflatoxin M1 in milk.

Aflatoxin M1 (AFM1) contamination poses a serious threat to human health globally. Hence, it is necessary to develop reliable and ultrasensitive methods for the determination of AFM1 residue in food products at low levels. In this study, a novel polystyrene microsphere-mediated optical sensing (PSM-OS) strategy was constructed to solve the problems of low sensitivity and susceptibility to interference from the matrix in AFM1 determination. Polystyrene (PS) microspheres have the advantages of low cost, high stability, and controllable particle size. They can be useful optical signal probes for qualitative and quantitative analyses attributed to the fact that they have strong ultraviolet-visible (UV-vis) characteristic absorption peaks. Briefly, magnetic nanoparticles were modified with the complex of bovine serum protein and AFM1 (MNP150-BSA-AFM1), and biotinylated antibodies of AFM1 (AFM1-Ab-Bio). Meanwhile, PS microspheres were also functionalized with streptavidin (SA-PS950). In the presence of AFM1, a competitive immune reaction was triggered leading to the changes in AFM1-Ab-Bio concentrations on the surface of MNP150-BSA-AFM1. The complex of MNP150-BSA-AFM1-Ab-Bio binds with SA-PS950 to form the immune complexes due to the special binding of biotin and streptavidin. The remaining SA-PS950 in the supernatant was determined by UV-Vis spectrophotometer after magnetic separation, which positively correlated with the concentration of AFM1. This strategy allows for ultrasensitive determination of AFM1 with limits of detection as low as 3.2 pg/mL. It was also successfully validated for AFM1 determination in milk samples, and a high consistency was found with the chemiluminescence immunoassay. Overall, the proposed PSM-OS strategy can be used for the rapid, ultrasensitive, and convenient determination of AFM1, as well as other biochemical analytes.

Omega-3 polyunsaturated fatty acids alleviates lung injury mediated by post-hemorrhagic shock mesenteric lymph.

Severe hemorrhage-induced acute lung injury (ALI) remains the major contributor to critical patient mortality and is associated with posthemorrhagic shock mesenteric lymph (PHSML) return. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) play overall protection on acute hemorrhage, but a reliable mechanism needs to be identified. The aims of this study were to investigate the role of ω-3 PUFAs in alleviating ALI and whether is related to the endotoxin contained in PHSML. Mesenteric lymph was harvested from rats subjected to hemorrhagic shock (hemorrhage-induced hypotension of 40 ± 2 mmHg for 90 min plus by resuscitation) or sham shock. The effect of ω-3 PUFAs on pulmonary function, water content, morphology, and LBP, CD14, TNF-α, and IL-6 levels were observed in rats subjected to hemorrhagic shock, while the effect of PHSML intravenous infusion on the beneficial effect of ω-3 PUFAs also was investigated. In addition, the effect of ω-3 PUFAs on the endotoxin contents in mesenteric lymph were detected. Hemorrhagic shock-induced ALI was characterized by increased functional residual capacity (FRC), lung resistance (RI), inspiratory capacity (IC), respiratory frequency, water contents and structural damage, along with increases in LBP, IL-6, and TNF-α. ω-3 PUFAs treatment reduced FRC, RI, IC, frequency, water contents, LBP, IL-6, TNF-α, and alleviated morphological damage. In contrast, PHSML infusion abolished the advantageous effects of ω-3 PUFAs on the above indices and CD14. Furthermore, the endotoxin level of PHSML was significantly enhanced, but declined following ω-3 PUFAs administration. These findings together suggested that treatment with ω-3 PUFAs ameliorates hemorrhagic shock-induced ALI, which is associated with reduced endotoxin contained in PHSML.

Resveratrol enhances vascular reactivity in mice following lipopolysaccharide challenge via the RhoA-ROCK-MLCP pathway.

The aim of the present study was to identify whether sepsis-induced vascular hyporeactivity is associated with microcirculation disturbance and multiple organ injuries. The current study assessed the impact of resveratrol (Res) treatment on lipopolysaccharide (LPS) challenge mediated vascular hyporeactivity. Effects of Res treatment (30 mg/kg; i.m.) at 1 h following LPS stimulation (5 mg/kg; i.v.) on the survival time, mean arterial pressure (MAP), and maximal difference of MAP (ΔMAP) to norepinephrine (NE; 4.2 µg/kg) in mice were observed. The reactivity to gradient NE of isolated mesenteric arterioles and the association with the RhoA-RhoA kinase (ROCK)-myosin light chain phosphatase (MLCP) pathway were investigated by myography, and the signaling molecule protein levels were assessed using ELISA. Res treatment prolonged the survival time of mice subjected to LPS challenge, but did not prevent the LPS-induced hypotension and increase in ΔMAP. Res treatment and RhoA agonist U-46619 incubation prevented LPS-induced vascular hyporeactivity ex vivo, which were suppressed by incubation with ROCK inhibitor Y-27632. LPS-induced vascular hyporeactivity was not affected by the MLCP inhibitor okadaic acid incubation, but was further downregulated by the co-incubation of OA plus Y-27632. The inhibiting effect of Y-27632 on Res treatment was eradicated by incubation with U-46619. Furthermore, RhoA inhibitor C3 transferase did not significantly inhibit the enhancing role of Res treatment, which was further increased by U-46619 plus C3 transferase co-incubation. In addition, Res treatment eradicated the LPS-induced decreases in p-RhoA and p-Mypt1 levels and increases in MLCP levels. The results of the present study indicate that post-treatment of Res significantly ameliorates LPS-induced vascular hyporeactivity, which is associated with the activation of the RhoA-ROCK-MLCP pathway.

[Application of three-diinensional CT in the treatment of oblique facial clefts with mandibular outer cortex].

OBJECTIVE: To investigate the application of three-dimensional CT(3D-CT) in the treatment of oblique facial clefts with mandibular outer cortex, including the surgical design and results assessment. METHODS: From Jan. 2003 to Dec. 2013, 22 cases with oblique facial cleft, who underwent mandibular outer cortex onlay bone graft were retrospectively studied. 3D images from CT data were reconstructed before operation for design. Then the mandibular outer cortex onlay bone transplant was performed to reconstruct the bone defect and cleft. 3D CT was performed 5-10 days postoperatively and 6- 12 months postoperatively to assess the facial symmetry. RESULTS: According to the results of CT measurement, the average volume of the orbital bone defects on the affected side decreased by(64. 6 ± 14. 4)% 5 to 10 days after operation. The average volume of the maxillary and zygomatic bone defects on the affected side decreased by(71.4 ± 15.7)% after surgery. After 6 to 12 months,the average recovery of the mandibular donor site was (57. 9 ± 13. 9)% of the removed mandibular outer cortex. The average absorption of grafted bones was(24.7 ± 25.6 )%. The average height difference between the centre of pupils on both sides before surgery was(3.76 ± 1.27) mm,which decreased to( 1. 15 ± 1.00) mm 5 to 10 days after surgery(P =0. 000) , and( 1.35 ± 1. 13) mm 6 to 12 months after surgery(P = 0. 003). The relapse may be caused by the absorption of the grafted bones. CONCLUSIONS: 3D-CT can be used for preoperative design and postoperative assessment in the treatment of oblique facial cleft with mandibular outer cortex.

A selective inhibitor of Na+/Ca2+ exchanger, SEA0400, preserves cardiac function and high-energy phosphates against ischemia/reperfusion injury.

The Ca2+ overload by Ca2+ influx via Na+/Ca2+ exchanger (NCX) is a critical mechanism in myocardial ischemia/reperfusion injury. We investigated protective effects of a novel selective inhibitor of NCX, SEA0400, on cardiac function and energy metabolism during ischemia and reperfusion. Langendorff-perfused rat hearts were exposed to 35 minutes global ischemia and 40 minutes reperfusion. Using 31P nuclear magnetic resonance spectroscopy, cardiac phosphocreatine (PCr), ATP, and pHi were monitored. SEA0400 did not change the basic cardiac function, but improved the recovery of left ventricular developed pressure (LVDP) after reperfusion (27.6 +/- 4.9 mm Hg in control, 101.2 +/- 19.3 mm Hg in 0.1 microM, and 115.5 +/- 13.3 mm Hg in 1 microM SEA0400, means +/- SE, n = 6, P < 0.05). SEA0400 reduced left ventricular end-diastolic pressure and increased coronary flow after reperfusion. SEA0400 improved the recoveries of cardiac phosphocreatine and ATP after reperfusion, but did not affect pHi. There were significant linear correlations between left ventricular developed pressure and cardiac phosphocreatine (r = 0.79, P < 0.05), and left ventricular developed pressure and ATP (r = 0.80, P < 0.05). However, SEA0400 increased the incidence and duration of reperfusion ventricular arrhythmias. SEA0400 added only after reperfusion also improved both the contractile function and energy metabolism. It is concluded that the selective inhibition of NCX may be effective to preserve high-energy phosphates and to improve cardiac function after reperfusion, but may not be able to prevent fatal arrhythmias.