A MnO2 nanosheet-mediated CRISPR/Cas12a system for the detection of organophosphorus pesticides in environmental water.

Nowadays, easy, convenient, and sensitive sensing strategies are still critical for organophosphorus pesticides in environmental water samples. Herein, a novel organophosphorus pesticide (OP) assay based on acetylcholinesterase (AChE) and a MnO2 nanosheet-mediated CRISPR/Cas12a reaction is reported. The single-strand DNA (ssDNA) activator of CRISPR/Cas12a was simply adsorbed on the MnO2 nanosheets as the nanoswitches of the assay. In the absence of target OPs, AChE hydrolyzed acetylcholine (ATCh) to thiocholine (TCh), which reduced the MnO2 nanosheets to Mn2+, resulting in the release of the activator followed by activation of the CRISPR/Cas12a system. The activated Cas12a thereafter nonspecifically cleaved the FAM/BHQ1-labeled ssDNA (FQ-reporter), producing a fluorescence signal. Upon the addition of target OPs, the hydrolysis of ATCh by AChE was inhibited owing to OPs combining with AChE, and thus effective quantification of OPs could be achieved by measuring the fluorescence changes of the system. As a proof of concept, dichlorvos (DDVP) was chosen as a model OP analyte to address the feasibility of the proposed method. Attributed to the excellent trans-cleavage activity of Cas12a, the fluorescent biosensor exhibits a satisfactory limit of detection (LOD) for DDVP at 0.135 ng mL-1. In addition, the excellent recoveries for the detection of DDVP in environmental water samples demonstrate the applicability of the proposed assay in real sample research.

The Relationship between Self-Perceived Health and Clinical Symptoms in Patients with Frozen Shoulders.

Current healthcare is centered on the perception of people's health. The purpose of this study was to investigate the relationship between self-perceived health (physical, psychological, social, and environmental dimensions) and two main clinical symptoms (shoulder pain and restricted shoulder motion) in patients with frozen shoulders. A total of 49 patients diagnosed with frozen shoulders were recruited and divided into high- and low-disability groups according to the severity of their frozen shoulders. Participants were measured for shoulder passive range of motion, pain intensity, and self-perceived health, using a brief version of the World Health Organization Quality of Life questionnaire. The results showed that the high-disability group had poorer self-perceived health (lower quality of life scores) than the low-disability group (p < 0.05). There was no significant correlation between the quality of life scores and the two clinical symptoms in either the high- or low-disability group. Our findings revealed that the multidimensional self-perceived health of frozen shoulder patients could not be inferred from the severity of shoulder pain and restricted shoulder motions. This study suggests that healthcare providers should pay more attention to patients' self-perceived health needs while addressing the clinical symptoms in patients with frozen shoulders.

Cas12a-assisted RTF-EXPAR for accurate, rapid and simple detection of SARS-CoV-2 RNA.

Developing highly accurate and simple approaches to rapidly identify and isolate SARS-CoV-2 infected patients is important for the control of the COVID-19 pandemic. We, herein, reported the performance of a Cas12a-assisted RTF-EXPAR strategy for the identification of SARS-CoV-2 RNA. This assay combined the advantages of RTF-EXPAR with CRISPR-Cas12a can detect SARS-CoV-2 within 40 min, requiring only isothermal control. Particularly, the simultaneous use of EXPAR amplification and CRISPR improved the detection sensitivity, thereby realizing ultrasensitive SARS-CoV-2 RNA detection with a detection limit of 3.77 aM (∼2 copies/µL) in an end-point fluorescence read-out fashion, and at 4.81 aM (∼3 copies/µL) level via a smartphone-assisted analysis system (RGB analysis). Moreover, Cas12a increases the specificity by intrinsic sequence-specific template recognition. Overall, this method is fast, sensitive, and accurate, needing minimal equipment, which holds great promise to meet the requirements of point-of-care molecular detection of SARS-CoV-2.

Rapid and sensitive detection of Ebola RNA in an unamplified sample based on CRISPR-Cas13a and DNA roller machine.

A fast and simple Cas13a-based assay approach for direct detecting Ebola RNA in unamplified samples is reported. The procedure (named Cas-Roller) is comprised of a 10-min Cas13a-mediated cleavage protocol, followed by a DNA roller running for 30 min. This involves Cas13a collateral cleaving a suitably designed substrate in the presence of Ebola virus RNA sequence, and the cleavage product is used for DNA roller to amplify and generate fluorescent signals. After optimization of the conditions, the assay is able to achieve a limit of detection as low as 291 aM (∼175 copies RNA/µL) along with excellent anti-interfering performance in human serum and blood detection, which is ∼310-fold improved compared with the direct CRISPR assay. The entire workflow can be completed in ∼40 min at 37 °C without any pre-amplification, transcription, or centrifugation steps, thus avoiding the generation of false-negative or positive results. In addition, the downstream roller reaction is independent of the target sequence, this method can be applied to detect any other RNA by merely redesigning the hybridization regions of the crRNA. Overall, this strategy gives a new idea for the construction of simple and accurate Cas13a-based assays for the direct detection of RNA.

Neurobehavioral Differences of Valproate and Risperidone on MK-801 Inducing Acute Hyperlocomotion in Mice.

OBJECTIVE: The glutamate system plays a major role in the development of neuropsychiatric disorders such as addiction, epilepsy, dementia, and psychosis. MK-801 (dizocilpine), an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, could increase locomotor activity and stereotyped neurobehaviors mimicking schizophrenic-like features in the mouse model. The study would explore the neuropharmacological differences of risperidone and valproic acid on the MK-801-induced neurobehavioral changes. METHODS: The subjects were male C57BL/6J mice obtained from the National Laboratory Animal Center. Drug effects were assessed using the open field with a video-tracking system and gaiting tests. After habitation, risperidone (0, 0.1 mg/kg) or valproic acid (0, 200 mg/kg) was injected and ran locomotion for 30 mins. Sequentially, mice were followed by intraperitoneal injection (i.p.) with MK-801 (0, 0.2 mg/kg) and ran locomotion for 60 mins. Gaiting behaviors such as step angles, stride lengths, and stance widths were measured following the study drugs. RESULTS: The results showed that risperidone and valproic acid alone could not alter the locomotor activities. Following the MK-801 injection, the travelled distance and speed in the entire open field dramatically increased. The dose 0.1 mg/kg of risperidone could totally inhibit the MK-801-induced hyperlocomotion compared with that of the saline-injected group (p < 0.001). The valproic acid (200 mg/kg) partially suppressed the hyperlocomotion which is induced by MK801. CONCLUSION: The more dominant effect of risperidone to rescue MK-801 induced hyperlocomotion compared with that of valproic acid. The partial suppression of valproic acid may imply the psychopharmacological evidence as adjuvant effect to treat psychotic patients through tuning glutamatergic neurotransmission.

Histone demethylase LSD1 promotes RIG-I poly-ubiquitination and anti-viral gene expression.

Under RNA virus infection, retinoic acid-inducible gene I (RIG-I) in host cells recognizes viral RNA and activates the expression of type I IFN. To investigate the roles of protein methyltransferases and demethylases in RIG-I antiviral signaling pathway, we screened all the known related enzymes with a siRNA library and identified LSD1 as a positive regulator for RIG-I signaling. Exogenous expression of LSD1 enhances RIG-I signaling activated by virus stimulation, whereas its deficiency restricts it. LSD1 interacts with RIG-I, promotes its K63-linked polyubiquitination and interaction with VISA/MAVS. Interestingly, LSD1 exerts its function in antiviral response not dependent on its demethylase activity but through enhancing the interaction between RIG-I with E3 ligases, especially TRIM25. Furthermore, we provide in vivo evidence that LSD1 increases antiviral gene expression and inhibits viral replication. Taken together, our findings demonstrate that LSD1 is a positive regulator of signaling pathway triggered by RNA-virus through mediating RIG-I polyubiquitination.

c-MYC expression in T (III/IV) stage oral squamous cell carcinoma (OSCC) patients.

Purpose: c-MYC has been noted in many tumor types, but its functional significance and clinical utility in oral squamous cell carcinoma (OSCC) are not well known. Here we studied the expression of c-MYC in correlation to clinical outcome in patients with oral squamous cell carcinoma. Methods: The current study, using immunohistochemical staining, first examined c-MYC expression in OSCC patients and further correlated its expression with clinicopathological parameters. Results: c-MYC was expressed in the majority of OSCC patients (n=133). The c-MYC expression is associated with histological grade (P=0.0205) of patients with oral squamous cell carcinoma. Multivariate Cox regression analysis revealed that TN stage (P<0.001), American Joint Committee on Cancer (AJCC) stage (P<0.0001), and tumor differentiation (P=0.0025) were independent factors for overall survival in patients with OSCC except for c-MYC expression (P>0.05). Multiplicative-scale interaction between T stage (III/IV) and low c-MYC expression on mortality risk was identified (P=0.0233). Kaplan-Meier survival analysis demonstrated that oral cancer patients (T III/IV stage) with high c-MYC expression had better survival than those with low and medium c-MYC expression (P=0.0270). Conclusion: Our data indicate that c-MYC is a potential biomarker that can be used as a therapeutic target for treating OSCC patients with T stage (III/IV).

Memantine Rescues Neurosyphilis-Related Schizophrenic-like Features and Cognitive Deficit.

OBJECTIVES: Neurosyphilis, an infectious neuroinflammatory disorder, could cause diverse neuropsychiatric symptoms mimicking disorders of schizophrenia and dementia; hence, it is known as the "chameleon of psychiatry." Here, we present a subject with neurosyphilis with schizophrenic features and share the treatment outcome. METHODS: A 42-year-old single man had schizophrenic-like features and cognitive dysfunction for 1 year. Neurosyphilis was confirmed by a cerebral spinal fluid study. The brain image revealed multiple punctuated white matter gliosis in the bilateral frontal lobes and old lacunar infarctions in the bilateral basal hippocampus. The neuropsychiatric functions were declined until adjunctive memantine therapy. RESULTS: With the add-on therapy of memantine 10 mg daily, the psychotic and dementic symptoms markedly improved, and the patient recovered to the premorbid state in the 2-year follow-up course. CONCLUSIONS: Memantine has an adjunctive effect on neurosyphilis-related neuropsychiatric disorder via modulation of the glutamatergic neurotransmission and microglia-induced neuroinflammation.

Histone demethylase KDM3A is required for enhancer activation of hippo target genes in colorectal cancer.

Hippo pathway is involved in tumorigenesis, and its regulation in cytosol has been extensively studied, but its regulatory mechanisms in the nuclear are not clear. In the current study, using a FBS-inducing model following serum starvation, we identified KDM3A, a demethylase of histone H3K9me1/2, as a positive regulator for hippo target genes. KDM3A promotes gene expression through two mechanisms, one is to upregulate YAP1 expression, and the other is to facilitate H3K27ac on the enhancers of hippo target genes. H3K27ac upregulation is more relevant with gene activation, but not H3K4me3; and KDM3A depletion caused H3K9me2 upregulation mainly on TEAD1-binding enhancers rather than gene bodies, further resulting in H3K27ac decrease, less TEAD1 binding on enhancers and impaired transcription. Moreover, KDM3A is associated with p300 and required for p300 recruitment to enhancers. KDM3A deficiency delayed cancer cell growth and migration, which was rescued by YAP1 expression. KDM3A expression is correlated with YAP1 and hippo target genes in colorectal cancer patient tissues, and may serve as a potential prognosis mark. Taken together, our study reveals novel mechanisms for hippo signaling and enhancer activation, which is critical for tumorigenesis of colorectal cancer.