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.

Forensic Evaluations for Offenders With Dementia in Taiwan's Criminal Courts.

Dementia is an increasing world-wide health problem, and the association between dementia and adjudication of crimes has rarely been studied. The data in this study are described and analyzed by gender, psychiatric diagnosis, type of crime, and the acceptance rate by the courts of opinions tendered by forensic psychiatric examiners. The source data are derived from the databank of the Judicial Yuan (Judicial Department) of the Republic of China Law and Regulations Retrieval System. There was a male predominance of 85.1 percent. Larceny (42.6%) was the most frequent crime. There was also a high judicial acceptance rate of 91.5 percent of the professional opinions received from forensic psychiatric evaluators who examined defendants at the request of the courts. Psychiatrists play an important role in providing their professional opinions for the Taiwanese courts with regard to adjudication of evaluees with dementia. Most courts accepted psychiatrists' professional opinions about offenders with dementia, and the rate of acceptance was reflected in the judicial rulings of criminal responsibility.

Diversities of behavioral traits and neuropsychological function in different substance addiction.

OBJECTIVE: There are various temperaments and personality characters that modulate the development of substance addiction. The pharmacological properties of substances would alter the homeostasis of brain function and influence the neuropsychological performance through different neurotransmissions which then facilitate diverse emotional and behavioral responses. Our goal is to assess the interaction between personality characteristics, neuropsychological performances and Stroop interference in alcoholics, heroin and amphetamine dependent persons. METHODS: Subjects with alcohol (N=95), heroin (N=82) and amphetamine (N=57) dependence were recruited. Diagnostic interview and questionnaires evaluating the psychiatric symptoms were done, followed by neuropsychological assessments of Stroop and Wisconsin card sorting tests (WCST). Differences between the study groups were analyzed by one-way ANOVA with Scheffe's test. RESULTS: The individuals with alcohol dependence had significantly higher scores of neurotic, dysphoric and impulsive traits (P<0.001) than heroin and amphetamine dependent groups. In Stroop tests, the alcohol dependent subjects also showed delayed response on incongruent naming interferences compared to both of heroin and amphetamine groups (P<0.001). Perseverative errors and responses of WCST were significantly higher in heroin than in alcoholic dependent persons (P<0.01). CONCLUSIONS: Individuals with different substance dependence have distinct behavioral traits for developing addicted behaviors and had variant deficits of neuropsychological function.

Global histone modification profiling reveals the epigenomic dynamics during malignant transformation in a four-stage breast cancer model.

BACKGROUND: Epigenetic regulation has emerged to be the critical steps for tumorigenesis and metastasis. Multiple histone methyltransferase and demethylase have been implicated as tumor suppressors or oncogenes recently. But the key epigenomic events in cancer cell transformation still remain poorly understood. METHODS: A breast cancer transformation model was established via stably expressing three oncogenes in primary breast epithelial cells. Chromatin immunoprecipitation followed by the next-generation sequencing of histone methylations was performed to determine epigenetic events during transformation. Western blot, quantitative RT-PCR, and immunostaining were used to determine gene expression in cells and tissues. RESULTS: Histones H3K9me2 and me3, two repressive marks of transcription, decrease in in vitro breast cancer cell model and in vivo clinical tissues. A survey of enzymes related with H3K9 methylation indicated that KDM3A/JMJD1A, a demethylase for H3K9me1 and me2, gradually increases during cancer transformation and is elevated in patient tissues. KDM3A/JMJD1A deficiency impairs the growth of tumors in nude mice and transformed cell lines. Genome-wide ChIP-seq analysis reveals that the boundaries of decreased H3K9me2 large organized chromatin K9 modifications (LOCKs) are enriched with cancer-related genes, such as MYC and PAX3. Further studies show that KDM3A/JMJD1A directly binds to these oncogenes and regulates their transcription by removing H3K9me2 mark. CONCLUSIONS: Our study demonstrates reduction of histones H3K9 me2 and me3, and elevation of KDM3A/JMJD1A as important events for breast cancer, and illustrates the dynamic epigenomic mechanisms during breast cancer transformation.

Ganoderma formosanum polysaccharides attenuate Th2 inflammation and airway hyperresponsiveness in a murine model of allergic asthma.

Allergic asthma is an inflammatory disease of the airways mediated by Th2 immune responses and characterized by airway hyperresponsiveness (AHR). Fungi of the genus Ganoderma are basidiomycetes that have been used in traditional Asian medicine for centuries. We recently found that PS-F2, a polysaccharide fraction purified from the submerged culture broth of Ganoderma formosanum, stimulates the activation of dendritic cells and primes a T helper 1 (Th1)-polarized adaptive immune response. This study was designed to investigate whether the Th1 adjuvant properties of PS-F2 could suppress the development of allergic asthma in a mouse model. BALB/c mice were sensitized by repeated immunization with chicken ovalbumin (OVA) and alum, followed by intranasal challenge of OVA to induce acute asthma. PS-F2 administration during the course of OVA sensitization and challenge effectively prevented AHR development, OVA-specific IgE and IgG1 production, bronchial inflammation, and Th2 cytokine production. Our data indicate that PS-F2 has a potential to be used for the prevention of allergic asthma.

Polysaccharides from Ganoderma formosanum function as a Th1 adjuvant and stimulate cytotoxic T cell response in vivo.

The fungus of Ganoderma is a basidiomycete that possesses a variety of pharmacological effects and has been used in traditional Asian medicine for centuries. Ganoderma formosanum is a native Ganoderma species isolated in Taiwan, and we have previously demonstrated that PS-F2, a polysaccharide fraction purified from the submerged culture broth of G. formosanum, exhibits immunostimulatory properties in macrophages. In this study, we further characterized the adjuvant functions of PS-F2. In vitro, PS-F2 stimulated dendritic cells (DCs) to produce proinflammatory cytokines, including TNF-α, interleukin (IL)-6, and IL-12/IL-23 p40. PS-F2 also stimulated DCs to express the maturation markers CD40, CD80, CD86, and MHC class II. In a murine splenocyte culture, PS-F2 treatment resulted in elevated expression of T-bet and interferon (IFN)-γ in T lymphocytes. When used as an adjuvant in vivo with the ovalbumin (OVA) antigen, PS-F2 stimulated OVA-specific antibody production and primed IFN-γ production in OVA-specific T lymphocytes. PS-F2-adjuvated immunization also induced OVA-specific CTLs, which protected mice from a challenge with tumor cells expressing OVA. Collectively, our data show that PS-F2 functions as an adjuvant capable of inducing a Th1-polarized adaptive immune response, which would be useful in vaccines against viruses and tumors.

Reliability and validity of outcome measures of in-hospital and at-home visits in a randomized, double-blind, placebo-controlled trial for spinal muscular atrophy.

We used at-home assessments in a clinical trial to relieve the visit burden for participants. A total of 57 patients with type II or III spinal muscular atrophy were enrolled and 10 of them (7 type II and 3 type III) received at-home assessments. The primary end points were Gross Motor Function Measure, Manual Muscle Test, and serum biomarker. The secondary endpoints were Modified Hammersmith Functional Motor Scale and forced vital capacity. The correlation coefficients and analysis of covariance showed good reliability and validity of all outcome measures. Except for Gross Motor Function Measure and Modified Hammersmith Functional Motor Scale, there were no significant differences in measures between in-hospital and at-home groups (intersubject) or among 3 patients who received both at-home and in-hospital visits (intrasubject). We concluded that at-home assessments could provide sufficient reliability in a controlled trial. This modification could help design a successful clinical trial for spinal muscular atrophy.

Correlations between change scores of measures for muscle strength and motor function in individuals with spinal muscular atrophy types 2 and 3.

OBJECTIVE: The purpose of this study was to examine the correlations between change scores on the Manual Muscle Test for muscle strength and the Gross Motor Function Measure for motor function in individuals with spinal muscular atrophy type 2 and type 3. The specific aims were to reveal any differences in correlations between younger (children) and older (adolescents and adults) individuals and also between walkers and nonwalkers with spinal muscular atrophy. DESIGN: A total of 56 individuals with spinal muscular atrophy aged 5 to 41 yrs were recruited. Muscle strength and motor function were measured three times at 6-mo intervals. The Manual Muscle Test scores of 36 muscle groups and Gross Motor Function Measure scores were obtained. Differences in these scores over time (baseline to 6 mos and baseline to 12 mos) were calculated. RESULTS: Changes in Manual Muscle Test scores correlated positively to changes in Gross Motor Function Measure scores over 12 mos (r = 0.35, P = 0.01). Significantly positive correlations between the change scores over 12 mos were evident in the older group (r = 0.48, P = 0.02) and in nonwalkers (r = 0.47, P < 0.001). CONCLUSIONS: The correlations between change scores on Manual Muscle Test and on Gross Motor Function Measure suggest that these two measures are suitable for representing concurrent changes in muscle strength and motor function in spinal muscular atrophy within 12 mos.

The Cerebral Palsy Quality of Life for Children (CP QOL-Child): evidence of construct validity.

The Cerebral Palsy Quality of Life for Children (CP QOL-Child) is the first health condition-specific questionnaire designed for measuring QOL in children with cerebral palsy (CP). However, its construct validity has not yet been confirmed by confirmatory factor analysis (CFA). Hence, this study assessed the construct validity of the caregiver proxy-report version of the Chinese version of the CP QOL-Child in children with CP using CFA. A total of 312 children with CP (mean age: 8.59 years, SD: 2.52 years) and their caregivers participated in this study. The Chinese version of the CP QOL-Child was completed by the caregivers of children with CP. Then, CFA was applied to evaluate the seven-factor measurement structure of the CP QOL-Child. The seven-factor CFA model had an adequate fit to our data as judged by χ(2) statistic and various goodness-of-fit (GOF) indices, including the root mean square error of approximation (RMSEA). This study provided empirical evidence of the construct validity of the CP QOL-Child to support its use with children with CP in the Chinese speaking society.

Item hierarchy of the Chinese version of cerebral palsy quality of life for children.

BACKGROUND: The Chinese Cerebral Palsy Quality of Life for Children (C CP QOL-Child) is the first instrument developed to measure quality of life of (QOL) children with cerebral palsy in Chinese speaking populations. OBJECTIVE: The aim of the study was to examine the psychometric properties of C CP QOL-Child using Item Response Theory Models. We were particularly interested to know how intervention strategies could be designed for individuals based on the item scores. METHODS: 145 primary caregivers (mostly mothers; mean age: 39.2) of children with cerebral palsy aged 4-12 were invited to complete the 65-item C CP QOL-Child questionnaire. Data were analyzed using Rasch analysis. RESULTS: Item difficulty estimates were aligned with person ability values, indicating that the items in the scale generally demonstrated an appropriate depth and width for measuring QOL of persons in the target population. The results also showed that after dropping the 8 items in the dimension pain and impact of disability in the 65-item scale, the revised 57-item scale exhibits unidimensionality (separation index = 4.43, r = 0.95); hence the total score computed from the 57 items adequately reflects the level of QOL of the child as perceived by the caregiver. We further found that the Rasch item difficulty estimates demonstrated an overall item hierarchy; hence therapists can expect a pattern of performance by a child with CP that is based on the established order of item difficulty. CONCLUSIONS: The hierarchical structure identified in the study may be useful for designing tailor-made interventions with an aim of improving QOL.

Relationships between task-oriented postural control and motor ability in children and adolescents with Down syndrome.

Individuals with Down syndrome (DS) have been characterized by greater postural sway in quiet stance and insufficient motor ability. However, there is a lack of studies to explore the properties of dynamic postural sway, especially under conditions of task-oriented movement. The purpose of this study was to investigate the relationships between task-oriented postural control and motor ability in children and adolescents with DS. The participants were 23 children and adolescents with DS (DS group, M±SD age, 14.4±2.8 years) and 18 age- and gender-matched peers (M±SD age, 13.8±3.6 years). A force plate was used to collect postural data represented by center of pressure (COP) parameters. Postural measurements were conducted for both groups in quiet standing with eyes open and with eyes closed, and also while throwing a ball at erect standing. Assessments of motor ability were only applied to the DS group by using two dimensions of the original version of Gross Motor Function Measure and 4 subtests of the Bruininks Oseretsky Test of Motor Proficiency, second edition. The results showed that while the participants with DS showed greater displacement and higher velocity of COP sways at quiet standing, they exhibited smaller COP displacement in anterior/posterior direction during throwing the ball. Three areas of motor ability, including standing motor skills, walk/run/jump motor skills and muscle strength, were found to make a significant contribution to the displacement and velocity of postural sway during the voluntary movement. It is suggested that future research should focus on investigating the definite underlying mechanism of postural sway during movement and the influence of increasing motor ability on the reactive postural sway in this population.