Adolescent nicotine exposure induces long-term, sex-specific disturbances in mood and anxiety-related behavioral, neuronal and molecular phenotypes in the mesocorticolimbic system.

The majority of lifetime smokers begin using nicotine during adolescence, a critical period of brain development wherein neural circuits critical for mood, affect and cognition are vulnerable to drug-related insults. Specifically, brain regions such as the medial prefrontal cortex (mPFC), the ventral tegmental area (VTA), nucleus accumbens (NAc) and hippocampus, are implicated in both nicotine dependence and pathological phenotypes linked to mood and anxiety disorders. Clinical studies report that females experience higher rates of mood/anxiety disorders and are more resistant to smoking cessation therapies, suggesting potential sex-specific responses to nicotine exposure and later-life neuropsychiatric risk. However, the potential neural and molecular mechanisms underlying such sex differences are not clear. In the present study, we compared the impacts of adolescent nicotine exposure in male vs. female rat cohorts. We performed a combination of behavioral, electrophysiological and targeted protein expression analyses along with matrix assisted laser deionization imaging (MALDI) immediately post-adolescent exposure and later in early adulthood. We report that adolescent nicotine exposure induced long-lasting anxiety/depressive-like behaviors, disrupted neuronal activity patterns in the mPFC-VTA network and molecular alterations in various neural regions linked to affect, anxiety and cognition. Remarkably, these phenotypes were only observed in males and/or were expressed in the opposite direction in females. These findings identify a series of novel, sex-selective biomarkers for adolescent nicotine-induced neuropsychiatric risk, persisting into adulthood.

Raw Inonotus obliquus polysaccharide counteracts Alzheimer's disease in a transgenic mouse model by activating the ubiquitin-proteosome system.

BACKGROUND/OBJECTIVES: Inonotus obliquus has been used as antidiabetic herb around the world, especially in the Russian and Scandinavian countries. Diabetes is widely believed to be a key factor in Alzheimer's disease (AD), which is widely considered to be type III diabetes. To investigate whether I. obliquus can also ameliorate AD, it would be interesting to identify new clues for AD treatment. We tested the anti-AD effects of raw Inonotus obliquus polysaccharide (IOP) in a mouse model of AD (3×Tg-AD transgenic mice). MATERIALS/METHODS: SPF-grade 3×Tg-AD mice were randomly divided into three groups (Control, Metformin, and raw IOP groups, n = 5 per group). ß-Amyloid deposition in the brain was analyzed using immunohistochemistry for AD characterization. Gene and protein expression of pertinent factors of the ubiquitin-proteasome system (UPS) was determined using real-time quantitative polymerase chain reaction and Western blotting. RESULTS: Raw IOP significantly reduced the accumulation of amyloid aggregates and facilitated UPS activity, resulting in a significant reduction in AD-related symptoms in an AD mouse model. The presence of raw IOP significantly enhanced the expression of ubiquitin, E1, and Parkin (E3) at both the mRNA and protein levels in the mouse hippocampus. The mRNA level of ubiquitin carboxyl-terminal hydrolase isozyme L1, a key factor involved in UPS activation, also increased by approximately 50%. CONCLUSIONS: Raw IOP could contribute to AD amelioration via the UPS pathway, which could be considered as a new potential strategy for AD treatment, although we could not exclude other mechanisms involved in counteracting AD processing.

T Cell Subsets and the Expression of Related MicroRNAs in Patients with Recurrent Early Pregnancy Loss.

This study explored the role of T cell subsets and the expression of related microRNAs in patients with recurrent early pregnancy loss (EPL). Fifty patients with EPL loss between May 2018 and May 2021 were randomly selected as the EPL group, and 50 pregnant women with normal pregnancies or normal delivery outcomes were randomly selected as the control group. The expression levels of T cell subset-related markers and T cell subset-related miRNAs, in addition to the frequencies of T cell subsets, in peripheral blood of the two groups were analyzed. In terms of T cell-related markers, the results showed that the expression levels of the transcriptional regulator TBX-21 (T-bet) and interferon regulatory factor 4 (IRF4) were significantly upregulated in peripheral blood of the patients in the EPL group (P < 0.05), whereas the expression levels of GATA binding protein 3 (GATA3) and glucocorticoid-induced tumor necrosis factor receptor (GITR) were significantly downregulated (P < 0.05). In the EPL group, the expression of mir-106b, mir-93, and mir-25 was upregulated (1.51 ± 0.129, 1.43 ± 0.132, and 1.73 ± 0.156, respectively) in regulatory T (Treg) cell-related T cell subsets, whereas the expression of miR-146a and miR-155 was downregulated (P < 0.05). The frequencies of Treg and exhausted T cells in the EPL group were significantly lower than those in the control group (P < 0.05). The cell frequencies of T helper 17 (Th17) cells and exhausted Treg cells in the EPL group were significantly higher than those in the control group (P < 0.05). In conclusion, immune cells and associated miRNA profiles can be used as prognostic biomarkers for the treatment of human reproductive disorders, such as EPL.

Adenosine 2A receptor contributes to the facilitation of post-infectious irritable bowel syndrome by γδ T cells via the PKA/CREB/NF-κB signaling pathway.

BACKGROUND: Immunological dysfunction-induced low-grade inflammation is regarded as one of the predominant pathogenetic mechanisms in post-infectious irritable bowel syndrome (PI-IBS). γδ T cells play a crucial role in innate and adaptive immunity. Adenosine receptors expressed on the surface of γδ T cells participate in intestinal inflammation and immunity regulation. AIM: To investigate the role of γδ T cell regulated by adenosine 2A receptor (A2AR) in PI-IBS. METHODS: The PI-IBS mouse model has been established with Trichinella spiralis (T. spiralis) infection. The intestinal A2AR and A2AR in γδ T cells were detected by immunohistochemistry, and the inflammatory cytokines were measured by western blot. The role of A2AR on the isolated γδ T cells, including proliferation, apoptosis, and cytokine production, were evaluated in vitro. Their A2AR expression was measured by western blot and reverse transcription polymerase chain reaction (RT-PCR). The animals were administered with A2AR agonist, or A2AR antagonist. Besides, γδ T cells were also injected back into the animals, and the parameters described above were examined, as well as the clinical features. Furthermore, the A2AR-associated signaling pathway molecules were assessed by western blot and RT-PCR. RESULTS: PI-IBS mice exhibited elevated ATP content and A2AR expression (P < 0.05), and suppression of A2AR enhanced PI-IBS clinical characteristics, indicated by the abdominal withdrawal reflex and colon transportation test. PI-IBS was associated with an increase in intestinal T cells, and cytokine levels of interleukin-1 (IL-1), IL-6, IL-17A, and interferon-α (IFN-α). Also, γδ T cells expressed A2AR in vitro and generated IL-1, IL-6, IL-17A, and IFN-α, which can be controlled by A2AR agonist and antagonist. Mechanistic studies demonstrated that the A2AR antagonist improved the function of γδ T cells through the PKA/CREB/NF-κB signaling pathway. CONCLUSION: Our results revealed that A2AR contributes to the facilitation of PI-IBS by regulating the function of γδ T cells via the PKA/CREB/NF-κB signaling pathway.

Adolescent Δ-9-tetrahydrocannabinol exposure induces differential acute and long-term neuronal and molecular disturbances in dorsal vs. ventral hippocampal subregions.

Chronic exposure to Δ-9-tetrahydrocannabinol (THC) during adolescence is associated with long-lasting cognitive impairments and enhanced susceptibility to anxiety and mood disorders. Previous evidence has revealed functional and anatomical dissociations between the posterior vs. anterior portions of the hippocampal formation, which are classified as the dorsal and ventral regions in rodents, respectively. Notably, the dorsal hippocampus is critical for cognitive and contextual processing, whereas the ventral region is critical for affective and emotional processing. While adolescent THC exposure can induce significant morphological disturbances and glutamatergic signaling abnormalities in the hippocampus, it is not currently understood how the dorsal vs. ventral hippocampal regions are affected by THC during neurodevelopment. In the present study, we used an integrative combination of behavioral, molecular, and neural assays in a neurodevelopmental rodent model of adolescent THC exposure. We report that adolescent THC exposure induces long-lasting memory deficits and anxiety like-behaviors concomitant with a wide range of differential molecular and neuronal abnormalities in dorsal vs. ventral hippocampal regions. In addition, using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS), we show for the first time that adolescent THC exposure induces significant and enduring dysregulation of GABA and glutamate levels in dorsal vs. ventral hippocampus. Finally, adolescent THC exposure induced dissociable dysregulations of hippocampal glutamatergic signaling, characterized by differential glutamatergic receptor expression markers, profound alterations in pyramidal neuronal activity and associated oscillatory patterns in dorsal vs. ventral hippocampal subregions.

Enhancing Personalized Healthcare via Capturing Disease Severity, Interaction, and Progression.

Personalized diagnosis prediction based on electronic health records (EHR) of patients is a promising yet challenging task for AI in healthcare. Existing studies typically ignore the heterogeneity of diseases across different patients. For example, diabetes can have different complications across different patients (e.g., hyperlipidemia and circulatory disorder), which requires personalized diagnoses and treatments. Specifically, existing models fail to consider 1) varying severity of the same diseases for different patients, 2) complex interactions among syndromic diseases, and 3) dynamic progression of chronic diseases. In this work, we propose to perform personalized diagnosis prediction based on EHR data via capturing disease severity, interaction, and progression. In particular, we enable personalized disease representations via severity-driven embeddings at the disease level. Then, at the visit level, we propose to capture higher-order interactions among diseases that can collectively affect patients' health status via hypergraph-based aggregation; at the patient level, we devise a personalized generative model based on neural ordinary differential equations to capture the continuous-time disease progressions underlying discrete and incomplete visits. Extensive experiments on two real-world EHR datasets show significant performance gains brought by our approach, yielding average improvements of 10.70% for diagnosis prediction over state-of-the-art competitors.

Differences Between Omicron Infections and Fever Outpatients: Comparison of Clinical Manifestations and Initial Routine Hematology Indicators.

Purpose: We evaluated the differences between patients with SARS-CoV-2 Omicron variant infections and Fever outpatients, so that prevention and control measures can be taken in time. Patients and Methods: This study retrospectively analyzed 65 patients with SARS-CoV-2 Omicron variant. Sixty-nine age- and sex-matched Fever outpatients were enrolled during the same period of time. We also reanalyzed data from 81 SARS-CoV-2 Wild-Type-infected patients. We compared the clinical characteristics and initial indexes of routine tests among the 3 groups. Results: A total of 93.8% of the patients with Omicron infections had clinical symptoms, and the major symptoms were cough, fever and pharyngalgia. Pharyngalgia was a specific manifestation in Omicron group compared to Wild-Type group. The white blood cell of the Omicron group was lower than that of the Fever group [5.0 (3.6-6.1) vs 10.1 (7.6-12.9) ×109/L, P < 0.001]. The neutrophil count in Omicron group was lower than that in Fever and Wild-Type group [2.6 (1.8-3.9) vs 8.1 (5.9-10.9), P < 0.001; 2.6 (1.8-3.9) vs 3.4 (2.5-4.7) ×109/L, P < 0.001]. The white blood cell and neutrophil counts were lower in Omicron group than in the Fever group. The top 5 major symptoms were fever, cough, pharyngalgia, headache and expectoration. Conclusion: There are differences between the patients with Omicron infections and Fever outpatients, both in clinical manifestations and initial routine hematology indicators. We hope to provide some clues for early identification combined with a history of living in the epidemic area.

Enzyme-Loaded Hemin/G-Quadruplex-Modified ZIF-90 Metal-Organic Framework Nanoparticles: Bioreactor Nanozymes for the Cascaded Oxidation of N-hydroxy-l-arginine and Sensing Applications.

Biocatalytic cascades are challenging to operate in homogeneous solution, where diffusional mass transport hinders efficient communication between the reactive components. There is great interest in developing devices to perform such transformations in confined environments, which increase the efficiency of the cascaded process by generating high local concentrations of the reactive species. Herein, a bioreactor-nanozyme assembly is introduced for the cascaded aerobic oxidation of N-hydroxy-l-arginine (NOHA) to citrulline in the presence of glucose. The reaction mimics a key step in the nitric oxide synthase oxidation of l-arginine in nature. The system consists of glucose oxidase (GOx)-loaded hemin/G-quadruplex (hemin/G4)-modified ZIF-90 metal-organic framework nanoparticles. The aerobic oxidation of glucose by GOx yields H2 O2 that fuels the hemin/G4-catalyzed oxidation of NOHA into citrulline. The process driven by the bioreactor-nanozyme system is ≈sixfold enhanced compared to the homogeneous mixture of the biocatalysts, due to its operation in the confined environment of the nanoparticles. Extension to a three-step cascade is then demonstrated using a bioreactor composed of ß-galactosidase/GOx-loaded hemin/G4-modified ZIF-90 nanoparticles activating the cascaded oxidation of NOHA to citrulline, in the presence of lactose. Moreover, the bioreactor-nanozyme hybrid is applied as a functional optical sensor of glucose, using fluorescence or chemiluminescence as readout signals.

Centromere protein U (CENPU) promotes gastric cancer cell proliferation and glycolysis by regulating high mobility group box 2 (HMGB2).

Gastric cancer is one of the most common malignancy with a leading mortality rate worldwide. Despite the progress in the diagnosis and therapeutic strategy, the associated mortality is still growing. It is of great significance to understand molecular mechanisms of the development of gastric cancer. Glycolysis is a main source of ATP provision for cancer cells including gastric cancer, and targeting glycolysis is a promising therapeutic strategy. Centromere protein U (CENPU) has been found to be overexpressed in many types of cancer. Downregulation of CENPU suppresses the proliferation and invasion of cancer cells. High mobility group box 2 (HMGB2) is identified as a biomarker to diagnose of gastric cancer. Knockdown of HMGB2 inhibits proliferation and glycolysis in gastric cancer cells. In this work, we identified that CENPU was upregulated in gastric cancer. Knockdown of CENPU was able to suppress the proliferation and glycolysis of gastric cancer cells. Further the results showed that the anti-cancer effect of CENPU was HMGB2-dependent. Taken together, CENPU is an upstream factor of HMGB2, which regulates proliferation and glycolysis of gastric cancer.

Pulmonary Rehabilitation Accelerates the Recovery of Pulmonary Function in Patients With COVID-19.

Objectives: To evaluate the effect of in-hospital pulmonary rehabilitation (PR) on short-term pulmonary functional recovery in patients with COVID-19. Methods: Patients with COVID-19 (n = 123) were divided into two groups (PR group or Control group) according to recipient of pulmonary rehabilitation. Six-min walk distance (6MW), heart rate (HR), forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), diffusing capacity of the lung for carbon monoxide (DLCO), and CT scanning were measured at the time of discharge, 1, 4, 12, and 24 weeks. Results: At week one, both PR group and Control group showed no significant changes in pulmonary function. At 4 and 12 weeks, 6MW, HR, FVC, FEV1, and DLCO improved significantly in both groups. However, the improvement in the PR group was greater than the Control group. Pulmonary function in the PR group returned to normal at 4 weeks [FVC (% predicted, PR vs. Control): 86.27 ± 9.14 vs. 78.87 ± 7.55; FEV1 (% predicted, PR vs. Control) 88.76 ± 6.22 vs. 78.96 ± 6.91; DLCO (% predicted, PR vs. Control): 87.27 ± 6.20 vs. 77.78 ± 5.85] compared to 12 weeks in the control group [FVC (% predicted, PR vs. Control): 90.61 ± 6.05 vs. 89.96 ± 4.05; FEV1 (% predicted, PR vs. Control) 94.06 ± 0.43 vs. 93.85 ± 5.61; DLCO (% predicted, PR vs. Control): 91.99 ± 8.73 vs. 88.57 ± 5.37]. Residual lesions on CT disappeared at week 4 in 49 patients in PR group and in 28 patients in control group (p = 0.0004). Conclusion: Pulmonary rehabilitation could accelerate the recovery of pulmonary function in patients with COVID-19.

Imaging of Neurotransmitters and Small Molecules in Brain Tissues Using Laser Desorption/Ionization Mass Spectrometry Assisted with Zinc Oxide Nanoparticles.

Inorganic nanostructured materials such as silicon, carbon, metals, and metal oxides have been explored as matrices of low-background signals to assist the laser desorption/ionization (LDI) mass spectrometric (MS) analysis of small molecules, but their applications for imaging of small molecules in biological tissues remain limited in the literature. Titanium dioxide is one of the known nanoparticles (NP) that can effectively assist LDI MS imaging of low molecular weight molecules (LMWM). TiO2 NP is commercially available as dispersions, which can be applied using a chemical solution sprayer. However, aggregation of NP can occur in the dispersions, and the aggregated NP can slowly clog the sprayer nozzle. In this work, the use of zinc oxide (ZnO) NP for LDI MS imaging is investigated as a superior alternative due to its dissolution in acidic pH. ZnO NP was found to deliver similar or better results in the imaging of LMWM in comparison to TiO2 NP. The regular acid washes were effective in minimizing clogging and maintaining high reproducibility. High-quality images of mouse sagittal and rat coronal tissue sections were obtained. Ions were detected predominately as Na+ or K+ adducts in the positive ion mode. The number of LMWM detected with ZnO NP was similar to that obtained with TiO2 NP, and only a small degree of specificity was observed.

An Aggregation-Induced Emission-Based Indirect Competitive Immunoassay for Fluorescence "Turn-On" Detection of Drug Residues in Foodstuffs.

A new fluorescent "turn-on" probe-based immunosensor for detecting drug residues in foodstuffs was established by combining the mechanism of aggregation-induced emission (AIE) and an indirect competitive enzyme-linked immunosorbent assay (ELISA). In this study, a luminogen, with negligible fluorescence emission (TPE-HPro), aggregated in the presence of H2O2, and exhibited astrong yellow emission based on its AIE characteristics. This AIE process was further configured into an immunoassay for analyzing drug residues in foodstuffs. In this approach, glucose oxidase (GOx) was used as an enzyme label for the immunoassay and triggered GOx/glucose-mediated H2O2 generation, which caused oxidation of TPE-HPro and a "turn-on" fluorescence response at 540 nm. To quantitatively analyze the drug residues in foodstuffs, we used amantadine (AMD) as an assay model. By combining the AIE-active "turn-on" fluorescent signal generation mechanism with conventional ELISAs, quantifying AMD concentrations in chicken muscle samples was realized with an IC50 (50% inhibitory concentration) value of 0.38 ng/mL in buffer and a limited detection of 0.06 µg/kg in chicken samples. Overall, the conceptual integration of AIE with ELISA represents a potent and sensitive strategy that broadens the applicability of the AIE-based fluorometric assays.

[Fluorescence immunosorbent assay for quantitative detection of Listeria monocytogenes].

OBJECTIVE: Establishment of fuorescence immunosorbent assay for quantitative detection of Listeria monocytogenes. METHODS: The coupled mAbs named 10 E7 H6 and 10 A11 were screened from seven strains of anti-L. monocytogenes mAbs using a sandwich enzyme-linked immunosorbent assay(ELISA). The fluorescent immunoassay was established for L. monocytogenes detection by using biotinylated 10 A11 mAbs as detection antibody, 10 E7 H6 mAbs as capture antibody, and streptavidin-labeled fluorescent microspheres as detection probes, respectively. RESULTS: The optimum concentrations of capture and detection antibodies were 10 µg/mL and 5 µg/mL, respectively, and the optimum reaction pH was 7. 4. Under these conditions, the limit of detection of the proposed method for L. monocytogenes detection was 10~5 CFU/mL, which improved by two orders of magnitude compared to conventional ELISA; and it has a certain cross-reaction with several other Listeria but no significant cross-reactivity with other pathogenic bacteria. CONCLUSION: The method can be used for the detection of L. monocytogenes in pure culture solution, and can also be used for rapid immunological screening test of several other Listeria species in the genus Listeria.

Side-Slither Data-Based Vignetting Correction of High-Resolution Spaceborne Camera with Optical Focal Plane Assembly.

Optical focal plane assemblies are increasingly being used in high-resolution optical satellite systems to enhance the width of the image using linear push-broom imaging. With this system, vignetting occurs in the area of overlap, affecting image quality. In this paper, using the characteristics of the side-slither data, we propose side-slither data-based vignetting correction of a high-resolution spaceborne camera with an optical focal plane assembly. First, the raw side-slither data standardization is used to ensure that each row has the same features. Then, with the spatial correlation of a gray-level co-occurrence matrix, the gray-level co-occurrence matrix is proposed to identify the uniform regions, to extract the sample points. Finally, due to the characteristics of compatible linear response and non-linear response, the power-law model was used to fit, and the Levenberg⁻Marquardt algorithm was used to fit the model. In the experiment, polynomial fitting, laboratory coefficients and on-orbit coefficients were used for comparison with the proposed method. The side-slither data can be treated as a uniform scene due to their characteristics, and the side-slither image that was corrected using the proposed method showed less than 1% change in mean value, a root-mean-square deviation value better than 0.1%, and the average streaking metrics were superior to 0.02. The results showed that the proposed method performs significantly better in the vignetting area.

[Vancomycin-based fluorescent enzyme-linked immunoabsorbent assay for detection of Staphylococcus aureus].

In the study, fluorescent enzyme-linked immnoabsorbent assay for detection of Staphylococcus aureus was established with IgG from pig as capture antibody and quantum dot nanobeads (QBs) labeled vancomycin (QB-Vans) as testing antibody. Quantum dot of about 100 nm partical size nanobeads were prepared and linked with vancomycin. The optimum concentrations of salt ions were 0.01 mol/L, and the optimum pH was 6.0. Under the optimum conditions, the detection sensitivity for S. aureus was 104 CFU/mL, and there was no cross-reaction with other pathogenic bacteria. Thus, the method could be used for rapid screening of S. aureus, for the clinical monitoring and foodborne pathogens detection.

Highly sensitive visual detection of amantadine residues in poultry at the ppb level: A colorimetric immunoassay based on a Fenton reaction and gold nanoparticles aggregation.

Colorimetric biosensors for the on-site visual detection of veterinary drug residues are required for food control in developing countries and other resource-constrained areas, where sophisticated instruments may not be available. In this study, we developed a highly sensitive immunoassay for amantadine residues in poultry. By introducing a novel signal generation strategy into an indirect competitive immunoassay, a highly sensitive assay for amantadine residues in chicken was achieved for naked eye readout at the part per billion (ppb) level. Signal amplification was achieved in the designed immunoassay by combining conventional indirect competitive enzyme-linked immunosorbent assay, Fenton reaction-regulated oxidation of cysteine, and gold nanoparticle aggregation. Therefore, the cascade reaction remarkably enhanced the assay sensitivity and led to a pronounced color change from red to dark purple in the solution, which could be easily distinguished with the naked eye even at approximately 1 µg kg-1 in poultry muscle. Moreover, the color change can be quantitatively assayed with a classic high-throughput plate reader for contaminated poultry samples. The limit of detection (LOD) was 0.51 nM (0.095 ng mL-1). The recovery rates for spiked chicken samples ranged from 78% to 84% with relative standard deviations <15%. Therefore, we propose that this immunoassay could be generally applicable for on-site detection in the field of food control.

Molecularly Imprinted Polymer as an Antibody Substitution in Pseudo-immunoassays for Chemical Contaminants in Food and Environmental Samples.

The chemical contaminants in food and the environment are quite harmful to food safety and human health. Rapid, accurate, and cheap detection can effectively control the potential risks derived from these chemical contaminants. Among all detection methods, the immunoassay based on the specific interaction of antibody-analyte is one of the most widely used techniques in the field. However, biological antibodies employed in the immunoassay usually cannot tolerate extreme conditions, resulting in an unstable state in both physical and chemical profiles. Molecularly imprinted polymers (MIPs) are a class of polymers with specific molecular recognition abilities, which are highly robust, showing excellent operational stability under a wide variety of conditions. Recently, MIPs have been used in biomimetic immunoassays for chemical contaminants as an antibody substitute in food and the environment. Here, we reviewed these applications of MIPs incorporated in different analytical platforms, such as enzyme-linked immunosorbent assay, fluorescent immunoassay, chemiluminescent immunoassay, electrochemical immunoassay, microfluidic paper-based immunoassay, and homogeneous immunoassay, and discussed current challenges and future trends in the use of MIPs in biomimetic immunoassays.

Two-step large-volume magnetic separation combined with PCR assay for sensitive detection of Listeria monocytogenes in pasteurized milk.

Immunomagnetic separation (IMS) is an effective tool for the preconcentration and purification of food-borne pathogens from complex food samples because of its high capture efficiency (CE). In conventional IMS, antibodies are usually conjugated on the surface of magnetic beads (MB); the random orientation and conformation changes of antibodies on the MB surface can decrease their bioactivity. Moreover, the Brownian motion of immobilized antibodies is weakened, thereby rendering their binding efficiency with bacteria lower than that of free antibodies. Thus, abundant antibodies are commonly required to ensure high CE for IMS, particularly for large volumes. In this study, a 2-step large-volume magnetic separation (10 mL) was proposed to preconcentrate Listeria monocytogenes from pasteurized milk. First, the biotinylated anti-L. monocytogenes monoclonal antibodies (mAb) were bound with L. monocytogenes in 10 mL of diluted milk through an antigen-antibody interaction, and then streptavidin-labeled MB were used to capture biotin-mAb coated with L. monocytogenes by biotin and streptavidin interaction. Under optimal conditions, the CE of 2-step magnetic separation was >90% with L. monocytogenes concentrations ranging from 8 × 100 to 8 × 104 cfu/mL, whereas the amount of biotin-mAb was 14 fold lower than that of the conventional IMS method. Coupled with a PCR assay, the detection limit of the proposed method was 8 × 100 cfu/mL in pure culture and 8 × 101 cfu/mL in pasteurized milk without any pre-enrichment process. Moreover, the overall detection time, including sample preparation, large-volume magnetic separation, and PCR, took less than 7 h. In summary, the developed 2-step large-volume IMS combined with PCR was highly sensitive and low cost and, thus, has considerable potential for the rapid screening of food-borne pathogenic bacteria.

Phage-free peptide ELISA for ochratoxin A detection based on biotinylated mimotope as a competing antigen.

To perform the biopanning of a mimotope peptide with reduced affinity to anti-ochratoxin A (OTA) monoclonal antibodies (mAbs), we executed two improved biopanning approaches with a commercial 7-mer peptide library. In the first approach, anti-mouse IgG antibodies were used to erect the anti-OTA mAbs; in the second approach, an ultralow OTA concentration (0.1 ng/mL) was used to perform the competitive elution of phage particles. After the fourth round of biopanning was completed, 30 identified clones were positive phage particles; of these phage particles, 16 exhibited strong competitive inhibition with a low OTA concentration of 0.1 ng/mL. DNA sequencing results revealed that the 16 phage particles represented six different peptide sequences. Among these particles, the phage particle with a peptide sequence of "GMVQTIF" showed the highest sensitivity to OTA detection. The biotinylated 12-mer peptide "GMVQTIF-GGGSK-biotin" was designed as a competing antigen to develop a competitive peptide ELISA. Under the optimal parameters, the proposed peptide ELISA with the biotinylated 12-mer peptide as a competing antigen exhibited good dynamic linear detection for OTA in the range of 0.005 ng/mL-0.2 ng/mL with a detection limit of 0.001 ng/mL. The median inhibition concentration of OTA was 0.024 ng/mL (n=6), which is approximately fivefold more efficient as a competing antigen than the OTA-HRP conjugates. Reaction kinetics revealed that the biotinylated 12-mer peptide exhibited lower affinity to anti-OTA mAbs than the conventional chemical OTA antigen. The practicality of the proposed peptide ELISA was compared with a conventional ELISA method. In summary, this study demonstrated a novel concept of the development of phage-free peptide ELISA for the detection of OTA by using a biotinylated mimotope peptide as a competing antigen. This novel strategy can be applied to sensitively detect other toxic small molecules during food safety monitoring.

Different downstream signalling of CCK1 receptors regulates distinct functions of CCK in pancreatic beta cells.

BACKGROUND AND PURPOSE: Cholecystokinin (CCK) is secreted by intestinal I cells and regulates important metabolic functions. In pancreatic islets, CCK controls beta cell functions primarily through CCK1 receptors, but the signalling pathways downstream of these receptors in pancreatic beta cells are not well defined. EXPERIMENTAL APPROACH: Apoptosis in pancreatic beta cell apoptosis was evaluated using Hoechst-33342 staining, TUNEL assays and Annexin-V-FITC/PI staining. Insulin secretion and second messenger production were monitored using ELISAs. Protein and phospho-protein levels were determined by Western blotting. A glucose tolerance test was carried out to examine the functions of CCK-8s in streptozotocin-induced diabetic mice. KEY RESULTS: The sulfated carboxy-terminal octapeptide CCK26-33 amide (CCK-8s) activated CCK1 receptors and induced accumulation of both IP3 and cAMP. Whereas Gq -PLC-IP3 signalling was required for the CCK-8s-induced insulin secretion under low-glucose conditions, Gs -PKA/Epac signalling contributed more strongly to the CCK-8s-mediated insulin secretion in high-glucose conditions. CCK-8s also promoted formation of the CCK1 receptor/ß-arrestin-1 complex in pancreatic beta cells. Using ß-arrestin-1 knockout mice, we demonstrated that ß-arrestin-1 is a key mediator of both CCK-8s-mediated insulin secretion and of its the protective effect against apoptosis in pancreatic beta cells. The anti-apoptotic effects of ß-arrestin-1 occurred through cytoplasmic late-phase ERK activation, which activates the 90-kDa ribosomal S6 kinase-phospho-Bcl-2-family protein pathway. CONCLUSIONS AND IMPLICATIONS: Knowledge of different CCK1 receptor-activated downstream signalling pathways in the regulation of distinct functions of pancreatic beta cells could be used to identify biased CCK1 receptor ligands for the development of new anti-diabetic drugs.