Automatic Microfluidic Harmonized RAA-CRISPR Diagnostic System for Rapid and Accurate Identification of Bacterial Respiratory Tract Infections.

Respiratory tract infections (RTIs) pose a grave threat to human health, with bacterial pathogens being the primary culprits behind severe illness and mortality. In response to the pressing issue, we developed a centrifugal microfluidic chip integrated with a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats (CRISPR) system to achieve rapid detection of respiratory pathogens. The limitations of conventional two-step CRISPR-mediated systems were effectively addressed by employing the all-in-one RAA-CRISPR detection method, thereby enhancing the accuracy and sensitivity of bacterial detection. Moreover, the integration of a centrifugal microfluidic chip led to reduced sample consumption and significantly improved the detection throughput, enabling the simultaneous detection of multiple respiratory pathogens. Furthermore, the incorporation of Chelex-100 in the sample pretreatment enabled a sample-to-answer capability. This pivotal addition facilitated the deployment of the system in real clinical sample testing, enabling the accurate detection of 12 common respiratory bacteria within a set of 60 clinical samples. The system offers rapid and reliable results that are crucial for clinical diagnosis, enabling healthcare professionals to administer timely and accurate treatment interventions to patients.

Immunomagnetic Separation Combined with RCA-CRISPR/Cas12a for the Detection of Salmonella typhimurium on a Figure-Actuated Microfluidic Biosensor.

A figure-actuated microfluidic biosensor was developed for the rapid and sensitive detection of Salmonella typhimurium using immunomagnetic separation to separate target bacteria and rolling circle amplification (RCA) combined with CRISPR/Cas12a to amplify the detection signal. The magnetic nanoparticles (MNPs) modified with the capture antibodies (MNPs@Ab1) and RCA primer linked with recognized antibodies (primer@Ab2) were first used to react with S. typhimurium, resulting in the formation of MNPs@Ab1-S. typhimurium-primer@Ab2 complexes. Then, the RCA and CRISPR/Cas12a reagents were successively pumped into the chamber and incubated at the appropriate conditions. With the help of a 3D-printed signal detector, the fluorescence signal was collected and analyzed using the smartphone APP for the determination of bacterial concentration. This biosensor exhibited a wide linear range for the detection of S. typhimurium with a low limit of detection of 1.93 × 102 CFU/mL and a mean recovery of about 106% in the spiked milk sample.

The polymorphic analysis of cox1 and cob genes of Echinococcus granulosus in the Ngari region of Tibet in China.

Cystic echinococcosis (CE) is an important zoonotic parasitic disease caused by Echinococcus granulosus (E. granulosus). CE seriously threatens human health and the development of animal husbandry. The Ngari region is one of the world's highest endemic regions for CE, while genetic polymorphisms of E. granulosus were unclear. Paraffin slices of liver Cyst were collected from seventy-nine surgical patients with echinococcosis in the Ngari region. DNA was extracted from samples. The cox1 and cob genes of mitochondrial DNA of E. granulosus were simultaneously amplified and sequenced. The sequencing results were compared with the standard sequence (KU925397.1and HF947574.1). Phylogenetic trees and the haplotype network of cob and cox1 genes were constructed and analyzed genotypes of E. granulosus isolated from humans in the Ngari Region of Tibet. Out of 79 hydatid cyst samples collected from surgery patients, 60 isolates were identified as G1/ G3, and two isolates were identified as G6/ G7. Analysis of the cob/ cox1 genes revealed 9/7 mutations resulting in 8/6 haplotypes, respectively. The cob and cox1 neutrality indices computed by Tajima's D and Fu's Fs tests showed high negative values in Echinococcus granulosus sensu stricto (E. granulosus s. s.). The result suggested that E. granulosus in the Ngari region experienced population expansion or a negative selection. We found that G1/ G3 was still the main genotype, and G6/ G7 was found occasionally in humans of the Ngari region. Therefore, we recommend future surveys and control efforts to investigate G1/ G3 and G6/ G7 transmission in the Ngari region.

Unveiling a novel clinical risk assessment model for identifying non-suicidal self-injury risks in depressed Chinese adolescents amidst the COVID-19 pandemic: insights from low self-esteem, internet use, and sleep disturbance.

Background: Non-suicidal self-injury (NSSI) is a highly prevalent behavioral problem among depression adolescent patients that can result in numerous adverse outcomes. This study endeavors to bridge this knowledge gap by creating a comprehensive model that incorporates multiple aspects of NSSI to accurately evaluate its risk in adolescents with depression, thereby enhancing our ability to prevent and address this challenging issue. Method: Using a cross-sectional design, we recruited 302 adolescents with depressive disorders who visited or were hospitalized at Shandong Mental Health Center from December 2021 to June 2022. The participants completed several self-report questionnaires, including the Chinese version of the Internet Addiction Test, the Pittsburgh Sleep Quality Index questionnaire, the Defeat Scale, the Social Avoidance and Distress Scale and the Children's Depression Inventory. Logistic regression analysis was performed to identify the diagnostic factors, which were further used to establish clinical risk assessment models. A receiver operating characteristic curve (ROC) to identify the best model. An external validating team was introduced to verify the assessing efficiency. Results: Based on a logistic regression analysis, three variables have been identified as significant risk factors. Specifically, adolescents with depression who experience low self-esteem, internet use, or suffer from sleep disturbance face an increased risk of NSSI. An integrated risk index for NSSI exhibits excellent accuracy in identifying depressed adolescents at risk of NSSI (area under the curve = 0.86, sensitivity = 0.88, specificity = 0.69). In the validation cohort, the identification performance remains strong (area under the curve = 0.84, sensitivity = 0.72, specificity = 0.81). Conclusion: This study highlighted the role of self-esteem, internet use and sleep disturbance in the development of NSSI. The risk index diagnosing NSSI onset may help to guide the design and application of novel interventions to minimize this risky behavior in future depressed adolescents.

Functional Inhibition of Natural Killer Cells in a BALB/c Mouse Model of Liver Fibrosis Induced by Schistosoma japonicum Infection.

Background and Aims: Schistosomiasis japonica is a widespread human zoonotic disease, and in China, there are many patients with schistosomiasis suffering from liver fibrosis. Many studies have shown that natural killer (NK) cells could reduce the progression of hepatic fibrosis by directly killing hepatic stellate cells (HSCs). However, NK cells could not inhibit the progress of liver fibrosis induced by Schistosoma japonicum infection. We aimed to investigate the function of NK cells in schistosomiasis. Methods: BALB/c mice were infected with S. japonicum cercariae. The receptors and their proportions expressed on NK cells in the liver and spleen from infected mice were detected using flow cytometry. Levels of IFN-γ, perforin, and granzyme of NK cells, and collagen I, III, and α-SMA of hepatic tissue, were detected using quantitative real-time PCR. Changes in cytokine levels in sera were detected using a cytometric bead array. Liver fibrosis was evaluated using hematoxylin and eosin and Masson staining. NK function in the schistosomiasis model was analyzed. Results: From 2 to 4 weeks post-infection, NK cells were activated, with significantly increased levels of effector molecules (IFN-γ, perforin, and granzyme) that peaked at 4 weeks after infection. The proportion of NK cells increased in the liver and spleen from 6 to 10 weeks post-infection. However, the function of NK cells was inhibited from 6 to 10 weeks post-infection with significantly decreased levels of activated receptors (AR), inhibitory receptors (IR), and effector molecules. The levels of IFN-γ, IL-12, and IL-6 in mouse serum peaked at 6 weeks post-infection, and IL-10 and IL-21 levels peaked at 8 weeks post-infection. Hepatic fibrosis markers increased significantly at 6 weeks after infection. Conclusion: Our study suggested that NK cells were activated from 2 to 4 weeks post-infection and participated in inflammation in the mouse model. After the S. japonicum laid their eggs, NK cells became inhibited, with decreased levels of both activating and inhibitory NK cell receptors, as well as cytotoxic molecules. In addition, liver fibrosis formed. In mice infected with S. japonicum, the process of liver fibrosis might be alleviated by removing the functional inhibition of NK cells.