A universal all-in-one RPA-Cas12a strategy with de novo autodesigner and its application in on-site ultrasensitive detection of DNA and RNA viruses.

Revolutionary all-in-one RPA-CRISPR assays are rapidly becoming the most sought-after tools for point-of-care testing (POCT) due to their high sensitivity and ease of use. Despite the availability of one-pot methods for specific targets, the development of more efficient methods for new targets remains a significant challenge. In this study, we present a rapid and universal approach to establishing an all-in-one RPA-Cas12a method CORDSv2 based on rational balancing amplification and Cas12a cleavage, which achieves ultrasensitive detection of several targets, including SARS-CoV-2, ASFV, HPV16, and HPV18. CORDSv2 demonstrates a limit of detection (LOD) of 0.6 cp/µL and 100% sensitivity for SARS-CoV-2, comparable to qPCR. Combining with our portable device(hippo-CORDS), it has a visual detection LOD of 6 cp/µL and a sensitivity up to 100% for SARS-CoV-2 and 97% for Ct<35 ASFV samples, surpassing most one-pot visual methods. To simplify and accelerate the process for new targets, we also develop a de novo autodesigner by which the optimal couples of primers and crRNA can be selected rapidly. As a universal all-in-one RPA-CRISPR method for on-site testing, CORDSv2 becomes an attractive choice for rapid and accurate diagnosis in resource-limited settings.

A Novel Method for Pose and Position Calibration of Laser Displacement Sensors.

Laser displacement sensors are widely used in the aviation industry for the purpose of surface normal measurements. The measurement of a surface normal depends on prior knowledge of the poses and positions of the sensors, which are obtained through calibration. This paper introduces a new parameter to the traditional calibration procedure, to reduce the calibration error, and explores the factors affecting calibration using the Monte Carlo method. In the experiment, the normal measurement error of the probe consisted of four sensors after calibration was less than 0.1∘, which satisfied the established requirements. This paper indicates the boundary conditions for a successful calibration and validates the proposed method, which provides a new method for the pose and position calibration of laser displacement sensors and other similar sensors.

Efficient generation of a CYP3A4-T2A-luciferase knock-in HepaRG subclone and its optimized differentiation.

CRISPR/Cas9 has allowed development of better and easier-to-use ADME models than traditional methods by complete knockout or knock-in of genes. However, gene editing in HepaRG cells remains challenging because long-term monoclonal cultivation may alter their differentiation capacity to a large extent. Here, CRISPR/Cas9 was used to generate a CYP3A4-T2A-luciferase knock-in HepaRG subclone by Cas9-mediated homologous recombination and monoclonal cultivation. The knock-in HepaRG-#9 subclone retained a similar differentiation potential to wildtype HepaRG cells (HepaRG-WT). To further improve differentiation and expand the applications of knock-in HepaRG cells, two optimized differentiation procedures were evaluated by comparison with the standard differentiation procedure using the knock-in HepaRG-#9 subclone and HepaRG-WT. The results indicated that addition of forskolin (an adenylate cyclase activator) and SB431542 (a TGF-ß pathway inhibitor) to the first optimized differentiation procedure led to better differentiation consequence in terms of not only the initiation time for differentiation and morphological characterization, but also the mRNA levels of hepatocyte-specific genes. These data may contribute to more extensive applications of genetically modified HepaRG cells in ADME studies.

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform.

The increasing prevalence of SARS-CoV-2 variants with spike mutations has raised concerns owing to higher transmission rates, disease severity, and escape from neutralizing antibodies. Rapid and accurate detection of SARS-CoV-2 variants provides crucial information concerning the outbreaks of SARS-CoV-2 variants and possible lines of transmission. This information is vital for infection prevention and control. We used a Cas12a-based RT-PCR combined with CRISPR on-site rapid detection system (RT-CORDS) platform to detect the key mutations in SARS-CoV-2 variants, such as 69/70 deletion, N501Y, and D614G. We used type-specific CRISPR RNAs (crRNAs) to identify wild-type (crRNA-W) and mutant (crRNA-M) sequences of SARS-CoV-2. We successfully differentiated mutant variants from wild-type SARS-CoV-2 with a sensitivity of 10-17 M (approximately 6 copies/µL). The assay took just 10 min with the Cas12a/crRNA reaction after a simple RT-PCR using a fluorescence reporting system. In addition, a sensitivity of 10-16 M could be achieved when lateral flow strips were used as readouts. The accuracy of RT-CORDS for SARS-CoV-2 variant detection was 100% consistent with the sequencing data. In conclusion, using the RT-CORDS platform, we accurately, sensitively, specifically, and rapidly detected SARS-CoV-2 variants. This method may be used in clinical diagnosis.

A Survey of Application of Complementary and Alternative Medicine in Chinese Patients with Parkinson's Disease: A Pilot Study.

OBJECTIVE: To investigate the prevalence and spectrum of complementary and alternative medicine (CAM) application and its related-factors in Parkinson's disease (PD) patients in China. METHODS: A structured questionnaire regarding the use of CAMs was administered to PD patients by face-to-face interview. Demographic and social variables, clinical characteristics and treatments, and the related factors of PD were also surveyed. RESULTS: Ninety out of 113 patients (79.6%) reported that they had received CAMs treatment, of which 48.6% (55/113) patients had received CAMs treatment for PD. The top 3 kinds of CAMs for PD were Chinese materia medica, rehabilitation exercise, and acupuncture ordinally. The source of information regarding CAMs treatment mainly came from relatives and friends (51.8%, 73 cases), followed by physicians (31.9%, 45 cases), and medias such as television, newspapers or the internet (16.3%, 28 cases). Fifty percent of CAM users reported a good therapeutic efficacy, whereas 39% patients reported that they felt neither better nor worse after the treatment, while 11% patients claimed worsening of symptoms. Multiple correspondence analyses survey found the main factors affecting the selectivity of CAMs ranked in the following order: income, education, occupation, habitation, age at onset, Hoehn and Yahr staging, PD duration, and gender. CONCLUSION: CAMs, especially Chinese materia medica, rehabilitation exercise, and acupuncture, are widely used for PD patients in China.

Vibration Detection and Motion Compensation for Multi-Frequency Phase-Shifting-Based 3D Sensors.

Phase-shifting profilometry, especially employing the multi-frequency phase-shifting method, is increasingly used for in situ 3D metrology and for the inspection of industrial parts. However, environmental vibrations cause fatal measurement errors and are inevitable in such applications. To this end, an effective and fast vibration detection and motion compensation method for multi-frequency phase-shifting-based 3D sensors is presented. The proposed method quantitatively indicates the strength of the vibration and compensates for the motion error by revising the wrapped phase without accessing neighboring pixels. Different vibration intensities were simulated using an industrial robot moving to test the feasibility of the method. According to experiments, this method is valid and capable for 3D inspection systems affected by inevitable vibrations.

High-Accuracy Globally Consistent Surface Reconstruction Using Fringe Projection Profilometry.

This paper presents a high-accuracy method for globally consistent surface reconstruction using a single fringe projection profilometry (FPP) sensor. To solve the accumulated sensor pose estimation error problem encountered in a long scanning trajectory, we first present a novel 3D registration method which fuses both dense geometric and curvature consistency constraints to improve the accuracy of relative sensor pose estimation. Then we perform global sensor pose optimization by modeling the surface consistency information as a pre-computed covariance matrix and formulating the multi-view point cloud registration problem in a pose graph optimization framework. Experiments on reconstructing a 1300 mm × 400 mm workpiece with a FPP sensor is performed, verifying that our method can substantially reduce the accumulated error and achieve industrial-level surface model reconstruction without any external positional assistance but only using a single FPP sensor.

A Robot-Driven 3D Shape Measurement System for Automatic Quality Inspection of Thermal Objects on a Forging Production Line.

The three-dimensional (3D) geometric evaluation of large thermal forging parts online is critical to quality control and energy conservation. However, this online 3D measurement task is extremely challenging for commercially available 3D sensors because of the enormous amount of heat radiation and complexity of the online environment. To this end, an automatic and accurate 3D shape measurement system integrated with a fringe projection-based 3D scanner and an industrial robot is presented. To resist thermal radiation, a double filter set and an intelligent temperature control loop are employed in the system. In addition, a time-division-multiplexing trigger is implemented in the system to accelerate pattern projection and capture, and an improved multi-frequency phase-shifting method is proposed to reduce the number of patterns required for 3D reconstruction. Thus, the 3D measurement efficiency is drastically improved and the exposure to the thermal environment is reduced. To perform data alignment in a complex online environment, a view integration method is used in the system to align non-overlapping 3D data from different views based on the repeatability of the robot motion. Meanwhile, a robust 3D registration algorithm is used to align 3D data accurately in the presence of irrelevant background data. These components and algorithms were evaluated by experiments. The system was deployed in a forging factory on a production line and performed a stable online 3D quality inspection for thermal axles.

The Relationship of Methylenetetrahydrofolate Reductase Gene C677T Polymorphism and Ischemic Stroke in Chinese Han Population.

BACKGROUND: The purpose of this study was to investigate the relationship of methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism and ischemic stroke Large-artery atherosclerosis (LAA) and Small-artery occlusion (SAO) subtypes in Chinese Han Population. MATERIALS AND METHODS: One hundred and thirty LAA patients and one hundred and fifty-nine SAO patients data were prospectively collected. The control group was one hundred and ninety-eight subjects from the Medical Examination Center in the same period. Plasma homocysteine (Hcy) levels were determined by a double reagent enzymatic cycling method. The genotypes of MTHFR polymorphism (C677T) were determined using polymerase chain reaction-restriction fragment length polymorphism. RESULTS: As compared with the controls, hypertension prevalence, smoking consumption ratio were significantly higher, plasma Hcy levels increased significantly, plasma high-density lipoprotein cholesterol levels were significantly reduced in LAA and SAO groups (P<0.05 for each). Elevated plasma Hcy levels were associated with MTHFR C677T polymorphism (P<0.01). The frequency of C/T+T/T genotypes and T allele in LAA and SAO groups was significantly higher than those in control groups (P<0.01). CONCLUSIONS: The present study suggests that hypertension, smoking, decreasing plasma high-density lipoprotein cholesterol levels and hyperhomocysteinemia (HHcy) are independent risk factors for LAA and SAO subtypes of ischemic stroke in Chinese Han Population. The MTHFR gene C677T C/T+T/T genotypes and T allele increases the risk of LAA and SAO subtypes of ischemic stroke.

Identification and Documentation of Auricle Defects using Three-dimensional Optical Measurements.

Auricle defects are important and common occurrences in forensic medicine. The accurate measurement and assessment of auricle defects is key to identifying and evaluating injury, and the currently available methods are known to be labor intensive and inaccurate. In this paper, we introduce an identification and documentation of auricle defects solution, which consists of an optical three-dimensional (3D) method and an effective algorithm to calculate the maximum projection area and identify auricle defects. In this study, three separate examiners measured 40 auricles of 20 adults using 3D optical measurement and two other commonly used methods to investigate the validity and representative reliability of 3D optical measurement for auricle defect identification. Based on the statistical analysis, the 3D measurement method is valid and showed a better reliability than the reference methods. We also present a representative auricle defect identification case using the proposed 3D optical measurement method. The study concludes that the optical 3D measurement method is a reliable and effective tool for auricle defect identification.

Association of matrix metalloproteinase-3 gene 5A/6A polymorphism with the recurrence of ischemic stroke: A prospective observational study.

Studies have demonstrated that matrix metalloproteinase-3 (MMP-3) is involved in the development and progression of atherosclerosis. However, there is no information available on the association of MMP-3 5A/6A polymorphism with recurrent ischemic stroke (IS) in different IS subtypes. We investigated the potential associations between MMP-3 serum level and -1171 5A/6A polymorphism and the recurrence of IS in a Chinese population. Consecutive acute first-ever IS patients were enrolled between August 2008 and October 2013. The genotypes of MMP-3 5A/6A polymorphism were determined using polymerase chain reaction-restriction fragment length polymorphism. IS recurrence was monitored after the index event and multivariate Cox proportional hazards model was constructed to identify factors related to future IS recurrence. A total of 1282 eligible patients were enrolled. During a 2-year follow-up period, 157 (12.25%) patients had recurrent events. MMP-3 level was significantly higher in patients with 5A/6A or 5A/5A genotype (22.72±7.29ng/ul) than in patients with 6A/6A genotype (20.48±7.58ng/ul), P<0.001. No interaction between MMP-3 5A/6A polymorphism and the risk of recurrence in total IS patients was found. The variant 5A/6A+5A/5A genotype and the 5A allele were significantly associated with a high risk of recurrence for large-artery atherosclerosis (LAA) (multivariate-adjusted, P=0.002, 0.001, respectively), but not for small-artery occlusion and cardioembolism. Our finding showed that MMP-3 5A/6A may be a useful biomarker for predicting recurrence for LAA stroke patients and 5A allele carrier may bear a higher risk of recurrence among patients with the subtype of LAA.

Association of Matrix Metalloproteinase-1 and Matrix Metalloproteinase-3 Gene Variants with Ischemic Stroke and Its Subtype.

BACKGROUND: Genetic variations in the genes of matrix metalloproteinases (MMPs) may play an important role in the pathogenesis of ischemic stroke (IS). Here we investigate the association between MMP-1 -1607 1G/2G and MMP-3 -1171 5A/6A genetic polymorphisms and etiological subtypes of IS in the Han Chinese population. METHODS: A total of 640 eligible patients with IS and 637 age- and gender-matched apparently healthy volunteers were enrolled. Subtypes of IS were classified by Trial of Org 10172 in Acute Stroke Treatment criteria. MMP-1 (-1607 1G/2G) and MMP-3 (-1171 5A/6A) polymorphisms were evaluated using polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The frequencies of the 5A/6A + 5A/5A genotypes and 5A allele were significantly higher in patients with IS than in controls (P <.001, P <.001, respectively). No association was found between MMP-1 1G/2G polymorphism and overall IS. In subgroup analyses, MMP-1 1G/2G and 2G/2G genotypes increased the risk of small-artery occlusion (SAO) subtype (multivariate-adjusted, P <.001, P = .002, respectively), and MMP-3 5A/6A + 5A/5A genotypes were related with large-artery atherosclerosis (LAA) subtype (multivariate-adjusted, P <.001). Haplotype analyses indicated that 2G-6A and 1G-5A increased the risk of SAO (multivariate-adjusted, P = .029) and LAA (multivariate-adjusted, P <.001), respectively. CONCLUSIONS: MMP-1 (-1607 1G/2G) and MMP-3 (-1171 5A/6A) polymorphisms may contribute to different subtypes of IS susceptibility.

Impact of 5A/6A polymorphism of matrix metalloproteinase-3 on recurrent atherosclerotic ischemic stroke in Chinese.

Little is known about the impact of the 5A/6A polymorphism of matrix metalloproteinase-3 (MMP-3) on recurrence of atherosclerotic ischemic stroke in Chinese. The aim of this study was to investigate the association of MMP-3 serum level and 5A/6A genetic polymorphism with the recurrence of atherosclerotic ischemic stroke in the Chinese Han population. We analyzed 106 large artery atherosclerosis (LAA) recurrent ischemic stroke patients and 545 LAA first onset ischemic stroke patients from January 2009 to June 2014. Serum MMP-3 concentrations were measured with an enzyme-linked immunosorbent assay. The genotypes of MMP-3 promoter polymorphism (-1171 5A/6A) were determined using polymerase chain reaction-restriction fragment length polymorphism. The frequencies of MMP-3 5A/6A+5A/5A (32.08% vs. 21.47%, p = 0.02) genotype and 5A (16.98% vs. 11.01%, p = 0.01) allele in the recurrent group was significantly higher than those in the first onset group. After adjustment for vascular risk factors, multivariate logistic regression analysis suggested that the MMP-3 5A/6A+5A/5A genotype was an independent risk factor for LAA recurrent ischemic stroke (odds ratio [OR], 1.74; 95% confidence interval [CI], 1.09-2.79, p = 0.021). No significant difference was observed for the MMP-3 serum concentrations between the recurrent group and the first onset group (22.23 ± 8.31 vs. 21.49 ± 7.89 ng/ul, t = 0.88, p = 0.38). The MMP-3 (-1171 5A/6A) polymorphism may contribute to LAA recurrent ischemic stroke susceptibility. Analysis of 5A/6A polymorphism in MMP-3 may identify patients at higher risk for LAA ischemic stroke recurrence, who may be selected for intensive preventive therapy.

Lipophilisation of Caffeic Acid through Esterification with Propanol Using Water-tolerable Acidic Ionic Liquid as Catalyst.

Propyl caffeate was synthesized to produce lipophilic antioxidant, which used caffeic acid and propanol as starting materials, acidic ionic liquid as catalyst. The highest yield of propyl caffeate (98.7±0.8%) have been achieved under the optimum as follows: 1-butylsulfonic-3-methylimidazolium tosylate showed the best catalytic performance, molar ratio of caffeic acid to propanol was 1:20, reaction temperature was 90°C and the amount of acidic ionic liquid was 40%. The relationship between temperature and the forward rate constant gave the activation energy of 33.6 kJ mol(-1), which indicated that acidic ionic liquid possesses high catalytic activity in the synthesis of PC. And the activity of acidic ionic liquid was not inhibited by the water produced during the esterification process. More importantly, this reaction system can even proceed smoothly when initial water content was 5%.

Differential expression profiles of poplar MAP kinase kinases in response to abiotic stresses and plant hormones, and overexpression of PtMKK4 improves the drought tolerance of poplar.

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play essential roles in plant growth, development and stress response. MAPK kinases (MAPKKs), which link MAPKs and MAPKK kinases (MAPKKKs), are integral in mediating various stress responses in plants. However, to date few data about the roles of poplar MAPKKs in stress signal transduction are available. In this study, we performed a systemic analysis of poplar MAPKK gene family expression profiles in response to several abiotic stresses and stress-associated hormones. Furthermore, Populus trichocarpa MAPKK4 (PtMKK4) was chosen for functional characterization. Transgenic analysis showed that overexpression of the PtMKK4 gene remarkably enhanced drought stress tolerance in the transgenic poplar plants. The PtMKK4-overexpressing plants also exhibited much lower levels of H2O2 and higher antioxidant enzyme activity after exposure to drought stress compared to the wide type lines. Besides, some drought marker genes including PtP5CS, PtSUS3, PtLTP3 and PtDREB8 exhibited higher expression levels in the transgenic lines than in the wide type under drought conditions. This study provided valuable information for understanding the putative functions of poplar MAPKKs involved in important signaling pathways under different stress conditions.

Chaenomeles speciosa: A review of chemistry and pharmacology.

Chaenomeles speciosa (Sweet) Nakai (C. speciosa, Rosaceae family) is an effective medicinal plant, which has long been used in China to treat various diseases, such as rheumatism, cholera, dysentery, enteritis, beriberi and vitamin C deficiency syndrome. A series of chemical constituents, including triterpenoid, phenolic and phenylpropionic acids, flavonoids, saccharides, essential oils and alkaloids, have been isolated from this plant and some have already been evaluated for their biological activities. Pharmacological investigations demonstrated that C. speciosa possesses anti-inflammatory, antinociceptive, antimicrobial, antioxidant, immunoregulatory, antiparkinsonian, hepatoprotective and antitumor properties. The objective of this review was to summarise available up-to-date and comprehensive information on C. speciosa and provide a relevant reference for further investigations.