A Multiplex Recombinase-Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC -2, and blaNDM -1 Genes in Klebsiella pneumoniae.

OBJECTIVE: This study aimed to establish a highly sensitive and rapid single-tube, two-stage, multiplex recombinase-aided qPCR (mRAP) assay to specifically detect the khe, blaKPC-2, and blaNDM-1 genes in Klebsiella pneumoniae. METHODS: mRAP was carried out in a qPCR instrument within 1 h. The analytical sensitivities of mRAP for khe, blaKPC-2, and blaNDM-1 genes were tested using recombinant plasmids and dilutions of reference strains. A total of 137 clinical isolates and 86 sputum samples were used to validate the clinical performance of mRAP. RESULTS: mRAP achieved the sensitivities of 10, 8, and 14 copies/reaction for khe, blaKPC-2, and blaNDM-1 genes, respectively, superior to qPCR. The Kappa value of qPCR and mRAP for detecting khe, blaKPC-2, and blaNDM-1 genes was 1, 0.855, and 1, respectively (p < 0.05). CONCLUSION: mRAP is a rapid and highly sensitive assay for potential clinical identification of khe, blaKPC-2, and blaNDM-1 genes in K. pneumoniae.

Miniaturized Lens Antenna with Enhanced Gain and Dual-Focusing for Millimeter-Wave Radar System.

This paper presents a waveguide Lens antenna at the W-band adopting dual-focusing Lens to improve the performance. The Lens antenna consisted of a waveguide slotted structure and lenses processed using NOA73 meet the demands of miniaturization for current communication systems. The antenna radome fabricated using NOA73 not only protects the antenna structure but also improves the gain of the antenna by about 9.5 dBi via electromagnetic wave dual-focusing. A prototype is fabricated using novel UV-LIGA technology. Measured results are compared with simulated values. Measured results confirmed the fabricated antenna operated in the W-band with a 10 dB fractional bandwidth (FBW) of 6.5% from 97.5 to 104 GHz and a peak gain of 22 dBi at 100 GHz in the direction perpendicular to the plane of the feed waveguide. A good agreement between simulation and measurement is obtained, demonstrating efficient radiations in the operating band.

The dropper stress characteristics under different moving load speeds for a high-speed railway.

Dropper failure seriously threatens the operation safety of a high-speed railway. In this work, for a simple chain suspension catenary, one span with five droppers is performed to establish a model and thus the effects of the moving load speed on dropper stress are investigated. First, the partial differential vibration equation of dropper is obtained through the mechanical analysis and converted into the finite difference equation. Then, we consider contact line as a beam element to obtain its motion equation. Furthermore, the boundary and initial conditions of five droppers are determined. Finally, the stresses of five droppers are numerically calculated and the effects of the moving load speed on dropper stress are investigated by writing a MATLAB code. The results suggest that the dropper location significantly affects its stress. Compared with other droppers, droppers II and IV have much more severe vibration amplitudes. Different moving load speeds could cause different stress change of each dropper. With the increasing speed, dropper experiences longer bending compression stage and the bending amplitude increases. The impact of the moving load speed on dropper stress is significant.

A Controlled Clinical Study of Accelerated High-Dose Theta Burst Stimulation in Patients with Obsessive-Compulsive Disorder.

Background: Obsessive-compulsive disorder (OCD) is frequently treated using a combination of counseling, drugs, and, more recently various transcranial stimulation protocols, but all require several weeks to months for clinically significant improvement, so there is a need for treatments with faster onset. This study investigated whether an accelerated high-dose theta burst stimulation (ahTBS) protocol significantly improves the efficacy of OCD compared to traditional 1-Hz repetitive transcranial magnetic stimulation (rTMS) in the routine clinical setting. Method: Forty-five patients with OCD were randomized into two groups and treated with ahTBS or 1-Hz rTMS for 5 days. Patients were assessed at baseline at the end of treatment using the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS). Results: After 5 days of treatment, there was a significant decrease in Y-BOCS scores in both groups (p < 0.001), and the difference between the two groups was not statistically significant (group × time interaction, F = 1.90, p=0.18). There was also no statistically significant difference in other secondary outcome indicators, including depression, anxiety symptoms, and response rate. However, the ahTBS group had a greater trend in response rate. Neuropsychological testing showed no negative cognitive side effects of either treatment. Conclusion: Accelerated high-dose TBS is as safe and has comparable short-term efficacy to traditional 1-Hz rTMS for the clinical treatment of OCD. Further research is needed to explore optimal ahTBS parameters, validate the utility of this treatment modality, and identify factors predictive of rapid clinical response to guide clinical decision-making. This trial is registered with NCT05221632.

Non-alcoholic fatty liver disease risk prediction model and health management strategies for older Chinese adults: a cross-sectional study.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver condition that affects a quarter of the global adult population. To date, only a few NAFLD risk prediction models have been developed for Chinese older adults aged ≥ 60 years. This study presented the development of a risk prediction model for NAFLD in Chinese individuals aged ≥ 60 years and proposed personalised health interventions based on key risk factors to reduce NAFLD incidence among the population. METHODS: A cross-sectional survey was carried out among 9,041 community residents in Shanghai. Three NAFLD risk prediction models (I, II, and III) were constructed using multivariate logistic regression analysis based on the least absolute shrinkage and selection operator regression analysis, and random forest model to select individual characteristics, respectively. To determine the optimal model, the three models' discrimination, calibration, clinical application, and prediction capability were evaluated using the receiver operating characteristic (ROC) curve, calibration plot, decision curve analysis, and net reclassification index (NRI), respectively. To evaluate the optimal model's effectiveness, the previously published NAFLD risk prediction models (Hepatic steatosis index [HSI] and ZJU index) were evaluated using the following five indicators: accuracy, precision, recall, F1-score, and balanced accuracy. A dynamic nomogram was constructed for the optimal model, and a Bayesian network model for predicting NAFLD risk in older adults was visually displayed using Netica software. RESULTS: The area under the ROC curve of Models I, II, and III in the training dataset was 0.810, 0.826, and 0.825, respectively, and that of the testing data was 0.777, 0.797, and 0.790, respectively. No significant difference was found in the accuracy or NRI between the models; therefore, Model III with the fewest variables was determined as the optimal model. Compared with the HSI and ZJU index, Model III had the highest accuracy (0.716), precision (0.808), recall (0.605), F1 score (0.692), and balanced accuracy (0.723). The risk threshold for Model III was 20%-80%. Model III included body mass index, alanine aminotransferase level, triglyceride level, and lymphocyte count. CONCLUSIONS: A dynamic nomogram and Bayesian network model were developed to identify NAFLD risk in older Chinese adults, providing personalized health management strategies and reducing NAFLD incidence.

Multicenter evaluation of a simple and sensitive nucleic acid self-testing for SARS-CoV-2.

A rapid and accurate COVID-19 diagnosis is a prerequisite for blocking the source of infection as soon as possible and taking the appropriate medical action. Herein, we developed GeneClick, a device for nucleic acid self-testing of SARS-CoV-2, consisting of three modules: a sampling kit, a microfluidic chip-based disposable cartridge, and an amplification reader. In addition, we evaluated the clinical performance of GeneClick using 2162 nasal swabs collected at three medical institutions, using three commercial RT-qPCR kits and an antigen self-test as references. Compared to RT-qPCR, the sensitivity and specificity of the GeneClick assay were 97.93% and 99.72%, respectively, with a kappa value of 0.979 (P â€‹< â€‹0.01). Of the 2162 samples, 2076 were also tested for SARS-CoV-2 antigens. Among the 314 positive samples identified by GeneClick assay, 63 samples were undetected by antigen tests. Overall, the GeneClick nucleic acid self-test demonstrated higher accuracy than the antigen-based detection. Based on the additional features, including simple operation, affordable price, portable device, and reliability of smartphone APP-driven sampling and result reporting, GeneClick offers a powerful tool for field-based SARS-CoV-2 detection in primary healthcare institutions or at-home use.

The impact of delayed gastric emptying as measured by gastric emptying scintigraphy on the outcome of magnetic sphincter augmentation.

INTRODUCTION: The impact of delayed gastric emptying (DGE) on the outcome of anti-reflux surgery (ARS) is controversial. There is concern that poor gastric emptying diminishes outcomes. Magnetic sphincter augmentation (MSA) may have a comparatively mild impact on gastric physiology, but the relationship between DGE and MSA outcomes is unknown. This study aims to evaluate the relationship between objective DGE and MSA outcomes over time. METHODS: Patients who completed gastric emptying scintigraphy (GES) prior to MSA between 2013 and 2021 were included. DGE was defined as a 4 h retention > 10% or half emptying time > 90 min on GES. Outcomes were compared between DGE and normal gastric emptying (NGE) groups at 6 months, 1 and 2 years. Sub-analysis of patients with severe (> 35%) DGE and correlation analysis between 4-h retention and symptom and acid-normalization were performed. RESULTS: The study population consisted of 26 (19.8%) patients with DGE and 105 with NGE. DGE was associated with more 90-days readmissions (18.5 vs 2.9%, p = 0.009). At 6 months patients with DGE had higher median (IQR) GERD-HRQL total [17.0(10-29) vs 5.5(3-16), p = 0.0013], heartburn [1(1-3) vs 0(0-1), p = 0.0010) and gas-bloat [4(2-5) vs 2(1-3), p = 0.033] scores. Outcomes at 1 and 2 years follow-up were comparable (p > 0.05). From 6 months to 1-year the gas-bloat score decreased from 4(2-5) to 3(1-3), p = 0.041. Total and heartburn scores decreased, but not significantly. Severe DGE (n = 4) patients had lower antiacid medication freedom at 6 months (75 vs 87%, p = 0.014) and 1-year (50 vs 92%, p = 0.046). There were non-significant trends for higher GERD-HRQL scores, dissatisfaction, and removal rates in severe DGE at 6 months and 1-year. There was a weak correlation between 4-h retention and 6-month GERD-HRQL total score [R = 0.253, 95%CI (0.09-0.41), p = 0.039], but not acid-normalization (p > 0.05). CONCLUSION: Outcomes after MSA are diminished early on in patients with mild-to-moderate DGE, but comparable by 1 year and durable at 2 years. Severe DGE outcomes may be suboptimal.

A rapid and highly sensitive multiple detection of human adenovirus type 3, type 7 and respiratory syncytial virus by recombinase-aided reverse transcription PCR.

BACKGROUND: Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase-aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology. METHODS: In this study, we developed and validated the multiplex reverse transcription recombinase-aided PCR (multiplex RT-RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process. RESULTS: Using recombinant plasmids, the sensitivity of multiplex RT-RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT-RAP showed no cross-reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT-RAP and the results were found to be consistent with those of corresponding RT-qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT-RAP was two to eightfold higher than that of corresponding RT-qPCR. CONCLUSION: We conclude the multiplex RT-RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load.

Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant Mycobacterium tuberculosis.

Objectives: The World Health Organization (WHO) Global tuberculosis Report 2021 stated that rifampicin-resistant tuberculosis (RR-TB) remains a major public health threat. However, the in-practice diagnostic techniques for RR-TB have a variety of limitations including longer time, lack of sensitivity, and undetectable low proportion of heterogeneous drug resistance. Methods: Here we developed a multiplex LNA probe-based RAP method (MLP-RAP) for more sensitive detection of multiple point mutations of the RR-TB and its heteroresistance. A total of 126 clinical isolates and 78 sputum samples collected from the National Tuberculosis Reference Laboratory, China CDC, were tested by MLP-RAP assay. In parallel, qPCR and Sanger sequencing of nested PCR product assay were also performed for comparison. Results: The sensitivity of the MLP-RAP assay could reach 5 copies/µl using recombinant plasmids, which is 20 times more sensitive than qPCR (100 copies/µl). In addition, the detection ability of rifampicin heteroresistance was 5%. The MLP-RAP assay had low requirements (boiling method) for nucleic acid extraction and the reaction could be completed within 1 h when placed in a fluorescent qPCR instrument. The result of the clinical evaluation showed that the MLP-RAP method could cover codons 516, 526, 531, and 533 with good specificity. 41 out of 78 boiled sputum samples were detected positive by MLP-RAP assay, which was further confirmed by Sanger sequencing of nested PCR product assay, on the contrary, qPCR was able to detect 32 samples only. Compared with Sanger sequencing of nested PCR product assay, both the specificity and sensitivity of the MLP-RAP assay were 100%. Conclusion: MLP-RAP assay can detect RR-TB infection with high sensitivity and specificity, indicating that this assay has the prospect of being applied for rapid and sensitive RR-TB detection in general laboratories where fluorescent qPCR instrument is available.

Rapid two-stage amplification in a single tube for simultaneous detection of norovirus GII and group a rotavirus.

The most prevalent viruses currently causing diarrhea are norovirus and rotavirus, and rapid and sensitive detection methods are essential for the early diagnosis of disease. The purpose of this study was to establish a sensitive single-tube two-stage nucleic acid amplification method-reverse transcription recombinase-assisted PCR (RT-RAP)-for simultaneous detection of norovirus GII and group A Rotavirus, with the first stage consisting of isothermal reverse transcription recombinase-aided amplification (RT-RAA) and the second stage consisting of qPCR (quantitative PCR). RT-RAP is more sensitive than either RT-RAA or qRT-PCR (quantitative RT-PCR) alone. And the addition of a barrier that can be disassembled after heating enabled the detection of samples within 1 h in a single closed tube. Sensitivity was 10 copies/reaction of norovirus (Novs) GII and group A rotavirus (RVA). In parallel, two hundred fecal specimens were used to evaluate the method and compare it with a commercial fluorescent quantitative RT-PCR. The data showed kappa values of 0.957 and 0.98 (p < 0.05) for detecting Novs GII and RVA by the two methods, indicating the potential of the newly established assay to be applied to clinical and laboratory testing.

Re-Emerged Genotype IV of Japanese Encephalitis Virus Is the Youngest Virus in Evolution.

An outbreak of viral encephalitis caused by a Japanese encephalitis virus (JEV) genotype IV infection occurred in Australia between 2021 and 2022. A total of 47 cases and seven deaths were reported as of November 2022. This is the first outbreak of human viral encephalitis caused by JEV GIV since it was first isolated in Indonesia in the late 1970s. Here, a comprehensive phylogenetic analysis based on the whole genome sequences of JEVs revealed it emerged 1037 years ago (95% HPD: 463 to 2100 years). The evolutionary order of JEV genotypes is as follows: GV, GIII, GII, GI, and GIV. The JEV GIV emerged 122 years ago (95% HPD: 57-233) and is the youngest viral lineage. The mean substitution rate of the JEV GIV lineage was 1.145 × 10-3 (95% HPD values, 9.55 × 10-4, 1.35 × 10-3), belonging to rapidly evolving viruses. A series of amino acid mutations with the changes of physico-chemical properties located in the functional important domains within the core and E proteins distinguished emerging GIV isolates from old ones. These results demonstrate the JEV GIV is the youngest JEV genotype at a rapid evolution stage and has good host/vector adaptability for introduction to non-endemic areas. Thus, surveillance of JEVs is highly recommended.

A Rapid and Sensitive Detection of HIV-1 with a One-Pot Two-Stage Reverse Transcription Recombinase Aided Real-Time PCR Assay.

Human immunodeficiency virus 1 (HIV-1) attacks the immune system, making people susceptible to various diseases, thus increasing their risk of death. Comprehensive detection of major HIV-1 strains circulating in China is vital for effective HIV-1 infection prevention and treatment. HIV-1 nucleic acid detection is considered effective for HIV-1 diagnosis since traditional immunological testing may fail to detect HIV-1 infection during the window period. This work demonstrates a one-pot two-stage amplification assay (RT-RAP), a combination of reverse transcription recombinase (RT- RAA), and quantitative real-time polymerase chain reaction (qRT-PCR). The turn-around time of the assay is only 50 min and can be performed with commonly available laboratory equipment, the qPCR devices. The RT-RAP assay could detect approximately 5 and 14 copies/reaction of HIV-1 DNA and RNA using recombinant plasmids and standard reference strains, respectively. Additionally, we found that the clinical performance of RT-RAP (detected 169 samples out of 170 specimens) was consistent with that of qRT-PCR. The sensitivity and specificity of RT-RAP were 100.00% (99/99) and 98.59% (70/71), respectively, while its positive and negative predictive values were 99.00% (99/100) and 100.00% (70/70), respectively. The total coincidence rate of the RT-RAP was 99.41% (169/170), with a kappa value of 0.988 (p < 0.05). We demonstrated that RT-RAP could rapidly detect the common HIV-1 subtypes commonly circulating in China with comparable sensitivity and specificity to qRT-PCR.

Reliability of foot posture index (FPI-6) for evaluating foot posture in patients with knee osteoarthritis.

Objective: To determine the reliability of FPI-6 in the assessment of foot posture in patients with knee osteoarthritis (KOA). Methods: Thirty volunteers with KOA (23 females, 7 males) were included in this study, assessed by two raters and at three different moments. Inter-rater and test-retest reliability were assessed with Cohen's Weighted Kappa (Kw) and Intraclass Correlation Coefficient (ICC). Bland-Altman plots and respective 95% limits of agreement (LOA) were used to assess both inter-rater and test-retest agreement and identify systematic bias. Moreover, the internal consistency of FPI-6 was assessed by Spearman's correlation coefficient. Results: FPI-6 total score showed a substantial inter-rater (Kw = .66) and test-retest reliability (Kw = .72). The six items of FPI-6 demonstrated inter-rater and test-retest reliability varying from fair to substantial (Kw = .33 to .76 and Kw = .40 to .78, respectively). Bland-Altman plots and respective 95% LOA indicated that there appeared no systematic bias and the acceptable agreement of FPI-6 total score for inter-rater and test-retest was excellent. There was a statistically significant positive correlation between each item and the total score of FPI-6, which indicated that FPI-6 had good internal consistency. Conclusion: In conclusion, the reliability of FPI-6 total score and the six items of FPI-6 were fair to substantial. The results can provide a reliable way for clinicians and researchers to implement the assessment of foot posture in patients with KOA.

Development of field-applicable endogenous internally controlled recombinase-aided amplification (EIC-RAA) assays for the detection of human papillomavirus genotypes 6 and 11 using sample releasing agent.

Objective: Human papillomavirus (HPV) 6 and 11 are the two most common low-risk HPV subtypes, accounting for more than 90% of condyloma acuminatum. A simple, accurate and rapid screening method to be applied in community-level hospitals is in high demand. Methods: Endogenous internally controlled recombinase-assisted amplification (EIC-RAA) assays for HPV6 and 11 were performed in a single closed-tube at 39 °C within 30 min. The sensitivity and specificity of EIC-RAA were examined using recombinant plasmids and pre-tested HPV DNA. A total of 233 clinical samples were collected, and the DNA was extracted by traditional multi-step extraction, or sample releasing agent, before analysis by EIC-RAA. For comparison, HPV detection via Quantitative real-time PCR (qPCR) was also performed. Results: The sensitivity of EIC-RAA analysis was 10 copies/reaction for HPV6, 100 copies/reaction for HPV11, and 100 copies/reaction for the human ß-globin gene. No cross-reaction was observed with other HPV subtypes. Clinical performance of the EIC-RAA assay achieved a 100% of concordance rate with the commercial HPV qPCR kit. Further, the EIC-RAA assay achieved a 100% of concordance rate when using multi-step extracted DNA and sample releasing agent-processed DNA. Summary: The EIC-RAA assay for HPV6 and 11 detection possesses the advantages of accuracy, simplicity and rapidity, and demonstrates great potential to be used in community-level hospitals for field investigation.

Establishment and evaluation of a risk-prediction model for hypertension in elderly patients with NAFLD from a health management perspective.

Elderly patients with nonalcoholic fatty liver disease are at a higher risk of developing. This study established an effective, individualised, early Hypertension risk-prediction model and proposed health management advice for patients over 60 years of age with NAFLD. Questionnaire surveys, physical examinations, and biochemical tests were conducted in 11,136 participants. The prevalence of NAFLD among 11,136 participants was 52.1%. Risk factors were screened using the least absolute shrinkage and selection operator model and random forest model. A risk-prediction model was established using logistic regression analysis and a dynamic nomogram was drawn. The model was evaluated for discrimination, calibration, and clinical applicability using receiver operating characteristic curves, calibration curves, decision curve analysis, net reclassification index (NRI), and external validation. The results suggested that the model showed moderate predictive ability. The area under curve (AUC) of internal validation was 0.707 (95% CI: 0.688-0.727) and the AUC of external validation was 0.688 (95% CI: 0.672-0.705). The calibration plots showed good calibration, the risk threshold of the decision curve was 30-56%, and the NRI value was 0.109. This Hypertension risk factor model may be used in clinical practice to predict the Hypertension risk in NAFLD patients.

Lewis acid Mg2+-doped cobalt phosphate nanosheets for enhanced electrocatalytic oxygen evolution reaction.

Cobalt-based materials are considered to be promising electrocatalysts for the oxygen evolution reaction (OER). However, their catalytic efficiencies are still limited by sluggish surface adsorption and high activation overpotentials. Herein, Lewis acid Mg2+-doped Co3(PO4)2 nanosheets were prepared through a simple two-step strategy for enhanced electrocatalytic oxygen evolution reaction. The Lewis acid Mg2+ dopants can increase the cobalt-oxygen covalency and thus facilitate the adsorption of oxygenated intermediates, charge transfer and the pre-oxidation of cobalt species during the OER.

Field Evaluation of a Duplex RT-RAA Assay for Rapid Detection of SARS-CoV-2 - Hebei Province, China, January 2021.

Introduction: Recently, a local cluster epidemic has occurred in Shijiazhuang City, Hebei Province. Failure to promptly identify patients with fever in rural areas was the major reason for this epidemic. Methods: We presented the field evaluation of a new real-time reverse transcription recombinase-aided amplification (RT-RAA) kit incorporating an endogenous internal control in a single-tube format, completed at the Hebei CDC from January 17, 2021 to January 27, 2021. Results: We evaluated the diagnostic performance of RT-RAA assay using automatic extracted RNA of 808 clinical samples. Compared with reverse transcriptase real-time quantitative PCR (qRT-PCR), RT-RAA kit achieved 92.41% sensitivity, 98.78% specificity and a 96.29% coincidence rate, demonstrating an excellent agreement between the RT-RAA assay and qRT-PCR assay. Furthermore, 58 samples were extracted using a manual extraction method within 5 minutes, but only samples with high nucleic acid concentration (cycle threshold value not higher than 32) could be stably detected. Discussion: The RT-RAA is more suitable to meet the needs of rapid, sensitive, and accurate detection in community-level medical institutions.

Detection of low-load Epstein-Barr virus in blood samples by enriched recombinase aided amplification assay.

Epstein-Barr virus (EBV), a common human γ-herpesvirus, infects more than 90% of adults worldwide. The purpose of this study was to establish a novel EBV detection method by combining the recombinase aided amplification (RAA) assay with an initial enrichment step that utilizes magnetic beads coated with a recombinant human mannan-binding lectin (rhMBL, M1 protein). An M1 protein-protein A magnetic bead complex (M1 beads) was prepared and used to achieve separation and enrichment of EBV from blood. After nucleic acid extraction, DNA was amplified by RAA. Using 388 whole blood samples and 1 serum sample, we explored the specificity, sensitivity and applicability of the newly developed detection method and compared it with commercial quantitative real-time polymerase chain reaction (qPCR) following M1 bead enrichment, traditional qPCR and traditional RAA. After enrichment, the positivity rate of EBV was increased from 15.94% to 17.74% by RAA (P < 0.05) and from 7.20% to 15.17% by qPCR (P < 0.05). The viral loads after enrichment were increased by 1.13 to 23.19-fold (P < 0.05). Our data demonstrates that an RAA assay incorporating M1 bead enrichment is a promising tool for detecting low EBV viral loads in blood samples that will facilitate an early response to EBV infection.

Development of a duplex recombinase-aided amplification assay for direct detection of Mycoplasma pneumoniae and Chlamydia trachomatis in clinical samples.

BACKGROUND: Pneumonia caused by Mycoplasma pneumoniae is common in the elderly and children, and pneumonia caused by Chlamydia trachomatis is prevalent in newborns. This study aimed to establish a rapid, sensitive, and simple method for the direct detection of M. pneumoniae and C. trachomatis in clinical samples without DNA extraction. METHODS: We established a duplex recombinase-aided amplification (RAA) assay with the RNAseP gene as an internal control for detecting the P1 gene of M. pneumoniae and the ORF8 gene of C. trachomatis, respectively. The results were obtained at 39 °C within 15-20 min. A total of 130 clinical samples suspected of M. pneumoniae or C. trachomatis infection were collected and tested by duplex RAA and PCR. DNA extracted via a commercial kit or treated with a nucleic acid-releasing agent was used and compared, respectively. Standard recombinant plasmids were used to test the sensitivity of the duplex RAA assay. In addition, other similar common pathogens were used to verify the specificity of the duplex RAA assay. RESULTS: The sensitivity of the duplex RAA assay for detecting M. pneumoniae and C. trachomatis was 10 copies/µL using recombinant plasmids. Compared with PCR, the sensitivity and specificity of duplex RAA assays for M. pneumoniae and C. trachomatis was 100% using clinical DNA samples extracted using a commercial kit and a nucleic acid-releasing agent, and the Kappa value was 1. CONCLUSION: The advantages of this duplex RAA assay include high sensitivity and specificity, short duration, and simple extraction steps, with potential for use in the on-site detection of M. pneumoniae and C. trachomatis in resource-limited settings.

Phoenixin-14 Ameliorates Cellular Senescence Against Morphine in M17 Neuronal Cells.

Drug dependence on morphine is commonly accompanied by neurodegenerative disorders. A previous study showed that prolonged exposure to morphine induces cellular senescence in neuronal cells by reducing telomere length. Phoenixin-14 is a newly discovered brain peptide with pleiotropic roles. However, it is unknown whether phoenixin-14 possesses a beneficial property against morphine-induced cellular senescence. Our results show that morphine reduced the expression of G protein-coupled receptor 173 (GPR173) in M17 neuronal cells. Therefore, we speculated that phoenixin-14, as a ligand for GPR173, may be involved in the morphine-mediated response in M17 cells. Further, we found that phoenixin-14 mitigated morphine-induced oxidative stress by reducing the reactive oxygen species (ROS) production and increasing superoxide dismutase (SOD) activity in M17 neuronal cells. The morphine-induced cellular senescence in M17 neuronal cells was prevented by phoenixin-14. Phoenixin-14 resolved the morphine-caused cell cycle arrest with significant changes in the expression levels of p21, cyclin-dependent kinases 6 (CDK6), and p-Rb. It also elevated the telomerase activity and restored the expressions of human telomerase reverse transcriptase (hTERT) and TERF2 in morphine-induced M17 neuronal cells. Furthermore, phoenixin-14 restored the yes-associated protein (YAP) expression against morphine in M17 neuronal cells. Knockdown of YAP abolished the beneficial effects of phoenixin-14 on cellular senescence against morphine induction. Taken together, these aggregate data demonstrate that phoenixin-14 prevented cellular senescence against morphine induction in M17 neuronal cells via regulating YAP expression.