Ratiometric CRISPR/Cas12a-Triggered CHA System Coupling with the MSRE to Detect Site-Specific DNA Methylation.

The precise determination of DNA methylation at specific sites is critical for the timely detection of cancer, as DNA methylation is closely associated with the initiation and progression of cancer. Herein, a novel ratiometric fluorescence method based on the methylation-sensitive restriction enzyme (MSRE), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) amplification were developed to detect site-specific methylation with high sensitivity and specificity. In detail, AciI, one of the commonly used MSREs, was employed to distinguish the methylated target from nonmethylated targets. The CRISPR/Cas12a system was utilized to recognize the site-specific target. In this process, the protospacer adjacent motif and crRNA-dependent identification, the single-base resolution of Cas12a, can effectively ensure detection specificity. The trans-cleavage ability of Cas12a can convert one target into abundant activators and can then trigger the CHA reaction, leading to the accomplishment of cascaded signal amplification. Moreover, with the structural change of the hairpin probe during CHA, two labeled dyes can be spatially separated, generating a change of the Förster resonance energy transfer signal. In general, the proposed strategy of tandem CHA after the CRISPR/Cas12a reaction not only avoids the generation of false-positive signals caused by the target-similar nucleic acid but can also improve the sensitivity. The use of ratiometric fluorescence can eradicate environmental effects by self-calibration. Consequently, the proposed approach had a detection limit of 2.02 fM. This approach could distinguish between colorectal cancer and precancerous tissue, as well as between colorectal patients and healthy people. Therefore, the developed method can serve as an excellent site-specific methylation detection tool, which is promising for biological and disease studies.

MicroRNA Sensors Based on CRISPR/Cas12a Technologies: Evolution From Indirect to Direct Detection.

MicroRNA (miRNA) has emerged as a promising biomarker for disease diagnosis and a potential therapeutic targets for drug development. The detection of miRNA can serve as a noninvasive tool in diseases diagnosis and predicting diseases prognosis. CRISPR/Cas12a system has great potential in nucleic acid detection due to its high sensitivity and specificity, which has been developed to be a versatile tool for nucleic acid-based detection of targets in various fields. However, conversion from RNA to DNA with or without amplification operation is necessary for miRNA detection based on CRISPR/Cas12a system, because dsDNA containing PAM sequence or ssDNA is traditionally considered as the activator of Cas12a. Until recently, direct detection of miRNA by CRISPR/Cas12a system has been reported. In this review, we provide an overview of the evolution of biosensors based on CRISPR/Cas12a for miRNA detection from indirect to direct, which would be beneficial to the development of CRISPR/Cas12a-based sensors with better performance for direct detection of miRNA.

Non-canonical CRISPR/Cas12a-based technology: A novel horizon for biosensing in nucleic acid detection.

Nucleic acids are essential biomarkers in molecular diagnostics. The CRISPR/Cas system has been widely used for nucleic acid detection. Moreover, canonical CRISPR/Cas12a based biosensors can specifically recognize and cleave target DNA, as well as single-strand DNA serving as reporter probe, which have become a super star in recent years in the field of nucleic acid detection due to its high specificity, universal programmability and simple operation. However, canonical CRISPR/Cas12a based biosensors are hard to meet the requirements of higher sensitivity, higher specificity, higher efficiency, larger target scope, easier operation, multiplexing, low cost and diversified signal reading. Then, advanced non-canonical CRISPR/Cas12a based biosensors emerge. In this review, applications of non-canonical CRISPR/Cas12a-based biosensors in nucleic acid detection are summarized. And the principles, peculiarities, performances and perspectives of these non-canonical CRISPR/Cas12a based biosensors are also discussed.

4-Vinylbenzodioxinones as a new type of precursor for palladium-catalyzed (4+3) cycloaddition of azomethine imines.

In this paper, benzo-fused cyclic carbonates were designed and synthesized as a new type of precursor of π-allylpalladium zwitterionic intermediates, and were applied in Pd-catalyzed diastereo- and enantioselective (4+3) cycloaddition with C,N-cyclic azomethine imines, leading to various biologically important 1,3,4-benzoxadiazepine derivatives in 43-99% yields with 6 : 1 to >20 : 1 dr and up to 95% ee.

Phosphine-Catalyzed (3 + 2) Annulation of γ-Substituted Cinnamic Aldehyde-Derived Morita-Baylis-Hillman Carbonates through Remote Activation.

A series of γ-substituted Morita-Baylis-Hillman (MBH) carbonates were synthesized and subjected to phosphine-catalyzed annulations with electrophilic exocyclic alkenes, giving various valuable spirocyclopentenes in moderate to excellent yields with moderate to excellent diastereoselectivities. A large scope of MBH carbonates bearing γ-hydrogen, alkenyl, and alkynyl substituents was well tolerated. The annulation unprecedentedly involves ß-, γ-, and δ-carbons of MBH carbonates.

Palladium-Catalyzed Asymmetric (3 + 2) Cycloaddition of 5-Allenyloxazolidine-2,4-Diones with Barbiturate-Derived Alkenes: Synthesis of Spirobarbiturate-γ-Lactams.

The 5-allenyloxazolidine-2,4-diones had been synthesized as novel precursors of π-allyl palladium zwitterion and were applied in a palladium-catalyzed enantioselective (3 + 2) annulation by using barbiturate-derived alkenes as the reaction partner in the presence of an axially chiral phosphoramidite ligand. This reaction proceeded smoothly under mild reaction conditions, affording highly functionalized spirobarbiturate-γ-lactam derivatives in excellent yields along with high diastereo- and enantioselectivities. The scale-up reaction and further transformation of the product were also successful.

Association between dinner timing and glucose metabolism in rural China: A large-scale cross-sectional study.

OBJECTIVES: Meal timing is a major risk factor for metabolic disease. The aim of this study was to assess the relationship between dinner timing and glucose metabolism in the rural Chinese population. METHODS: This cross-sectional study included 7701 participants from a Henan rural cohort study. Basic information was collected by in-person questionnaires. Multiple linear regression analysis was used to evaluate the relationship between dinner timing and fasting insulin (FINS), fasting plasma glucose (FPG), and homeostatic model assessment for insulin resistance (HOMA-IR). Restricted cubic spline was employed to investigate the dose-response relationship between dinner timing and FINS, FPG, and HOMA-IR. A generalized linear model was used to explore the interaction effect of age and dinner timing on FINS, FPG, and HOMA-IR. RESULTS: After adjusting for confounding factors, FINS concentration was reduced by 0.482 mmol/L (P < 0.001) for each hour delay in dinner timing. Furthermore, the HOMA-IR index decreased by 0.122 mmol/L for each hour delay. The results indicated a noticeable trend of decreasing values associated with later dinner timing (FINS: Poverall association < 0.001, Pnonlinear association = 0.144; HOMA-IR: Poverall association = 0.001, Pnonlinear association = 0.186). The interaction between age and dinner time significantly correlated with FINS and HOMA-IR (P < 0.05). This relationship was statistically significant before 69 y (P < 0.05). CONCLUSION: A significant association between dinner timing and glucose metabolism was observed in the rural Chinese population. Delayed dinner timing may be associated with lower fasting insulin. The negative effect of dinner timing on FINS and HOMA-IR was diminished with age.

Phosphine-Catalyzed Tandem Annulation of Allenylic Alcohols with 1,1-Dicyanoalkenes.

The phosphine-catalyzed tandem annulation of allenylic alcohols with 1,1-dicyanoalkenes has been developed, giving various bicyclic tetrahydrocyclopentafuran derivatives in 40-89% yields with moderate to excellent diastereoselectivities. The fused ring was furnished through a sequential (3 + 2) annulation/nucleophilic addition reaction. The unusual nucleophilic addition of an alkoxide ion to a cyano group led to a formation of tetrahydrofuran ring having an imino substituent.

Fluorescence quenching determination of tetracyclines based on the synergistic oxidation effect between Fe3O4nanoparticles and tetracyclines.

In recent years, tetracyclines (TCs) is a hot research topic. Herein, we report an interesting discovery using the complexation of oxytetracycline and metal ions. In this study, according to the properties of Fe3O4nanoparticles (Fe3O4NPs) as a nanoenzyme, it can be used to catalyze the oxidation of KI by H2O2to produceI3-,while at the same timeI3-binds to rhodamine 6G (Rh6G) to form a conjoined particle (Rh6G ∼ I3)n, leading to a decrease in the fluorescence intensity of Rh6G. However, in the presence of TCs, Fe3O4NPs have a synergistic effect with TCs, leading to enhanced catalytic activity, as well as better selectivity compared to the activity of other reducing enzymes. Consequently,the fluorescent signal based on a resonance scattering effect between Rh6G andI3-is dependent on the concentration of TCs, thus achieving highly facile and robust detection of TCs. The limits of detection (LOD) of the method were 20 nM, 10 nM and 40 nM for oxytetracycline(OTC), tetracycline(TC) and chlortetracycline(CTC), respectively. Most importantly, the method can be successfully applied to the detection of TCs in milk, eggs, and honey. The recoveries of spiked samples ranged from 83.11 to 118.95%. Thus, a stable, hands-on strategy for the detection of TCs is proposed, which has potential applications in the field of food safety and environmental protection.

Fungicide-inspired precursors of π-allylpalladium intermediates for palladium-catalyzed decarboxylative cycloadditions.

Inspired by a fungicide, we designed 5-vinyloxazolidine-2,4-diones as new precursors of π-allylpalladium zwitterionic intermediates and developed palladium-catalyzed asymmetric (5 + 3) cycloaddition with azomethine imines and (3 + 2) cycloaddition with 1,1-dicyanoalkenes. Both reactions proceeded smoothly under mild reaction conditions to produce various chiral heterocyclic compounds in high yields with excellent enantioselectivities. These results revealed that 5-vinyloxazolidine-2,4-diones were a type of suitable precursor for palladium catalysis and will find extensive applications in Pd-catalyzed reactions such as cycloaddition and allylic alkylation.

Association between Meal Frequency and Type 2 Diabetes Mellitus in Rural Adults: A Large-Scale Cross-Sectional Study.

Diet frequency may potentially influence metabolic health. However, general population-based evidence on the association between meal frequency and type 2 diabetes mellitus (T2DM) remains limited and inconclusive. Thus, this study aimed to investigate the association between meal frequency and T2DM in resource limited area. A total of 29,405 qualified participants were enrolled from the Henan rural cohort study. Data on meal frequency were collected through a validated face-to-face questionnaire survey. Logistic regression models were utilized to explore the association between meal frequency and T2DM. Compared with 21 times per week meal frequency group, the adjusted odds ratios (ORs) and 95% confidence intervals (95%CIs) were 0.75 (0.58, 0.95) and 0.70 (0.54, 0.90) for 16-20 times/week group and 14-15 times/week group, respectively. For the analysis of the three meals, significant associations were only found between dinner frequency and T2DM. Compared with seven times per week dinner group, the ORs (95%CIs) were 0.66 (0.42, 0.99) and 0.51 (0.29, 0.82) for the group with three to six times/week and zero to two times/week. Reduced meal frequency, especially dinner frequency, was associated with lower prevalence of T2DM, which suggests that an appropriate reduction in meal frequency per week may have a role in decreasing the risk of T2DM.

Polymeric Memristor Based Artificial Synapses with Ultra-Wide Operating Temperature.

Neuromorphic electronics, being inspired by how the brain works, hold great promise to the successful implementation of smart artificial systems. Among several neuromorphic hardware issues, a robust device functionality under extreme temperature is of particular importance for practical applications. Given that the organic memristors for artificial synapse applications are demonstrated under room temperature, achieving a robust device performance at extremely low or high temperature is still utterly challenging. In this work, the temperature issue is addressed by tuning the functionality of the solution-based organic polymeric memristor. The optimized memristor demonstrates a reliable performance under both the cryogenic and high-temperature environments. The unencapsulated organic polymeric memristor shows a robust memristive response under test temperature ranging from 77 to 573 K. Utilizing X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary-ion mass spectrometry (ToF-SIMS) depth profiling, the device working mechanism is unveiled by comparing the compositional profiles of the fresh and written organic polymeric memristors. A reversible ion migration induced by an applied voltage contributes to the characteristic switching behavior of the memristor. Herein, both the robust memristive response achieved at extreme temperatures and the verified device working mechanism will remarkably accelerate the development of memristors in neuromorphic systems.

Both early and late maternal age at childbirth is associated with increasing odds of central obesity in offspring.

OBJECTIVE: Despite studies on offspring obesity and delayed parenthood, little attention has been paid to the central obesity of offspring. The purpose of this study was to test the hypothesis that maternal age at childbirth (MAC) was associated with central obesity in offspring among the adult population, and fasting insulin may play a role in this association as a mediating factor. METHODS: A total of 423 adults (mean age 37.9 years, 37.1% female) were included. Information about maternal variables and other confounders was collected by face-to-face interview. Waist circumference and insulin were determined through physical measurements and biochemical examinations. Logistic regression model and restricted cubic spline model were used to analyze the relationship between MAC and central obesity of offspring. The mediating effect of fasting insulin levels on association between MAC and offspring waist circumference was also analyzed. RESULTS: There was a nonlinear relationship between MAC and central obesity in offspring. Compared with subjects with MAC 27-32 years, those with MAC 21-26 years (OR = 1.814, 95% CI: 1.129-2.915) and MAC ≥33 years (OR = 3.337, 95% CI: 1.638-6.798) had higher odds to develop central obesity. Offspring fasting insulin was also higher in MAC 21-26 years and MAC ≥33 years compared with those with MAC 27-32 years. Taking the group MAC 27-32 years as reference, the mediating effect of fasting insulin levels on the waist circumference was 20.6% and 12.4% for MAC 21-26 years and ≥ 33 years, respectively. CONCLUSION: MAC 27-32 years has the lowest odds of central obesity in offspring. Fasting insulin levels may have a partial mediating effect on the association between MAC and central obesity.

Locus-Specific Detection of DNA Methylation: The Advance, Challenge, and Perspective of CRISPR-Cas Assisted Biosensors.

Deoxyribonucleic acid (DNA) methylation is one of the epigenetic characteristics that result in heritable and revisable phenotype changes but without sequence changes in DNA. Aberrant methylation occurring at a specific locus was reported to be associated with cancers, insulin resistance, obesity, Alzheimer's disease, Parkinson's disease, etc. Therefore, locus-specific DNA methylation can serve as a valuable biomarker for disease diagnosis and therapy. Recently, Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are applied to develop biosensors for DNA, ribonucleic acid, proteins, and small molecules detection. Because of their highly specific binding ability and signal amplification capacity, CRISPR-Cas assisted biosensor also serve as a potential tool for locus-specific detection of DNA methylation. In this perspective, based on the detection principle, a detailed classification and comprehensive discussion of recent works about the latest advances in locus-specific detection of DNA methylation using CRISPR-Cas systems are provided. Furthermore, current challenges and future perspectives of CRISPR-based locus-specific detection of DNA methylation are outlined.

Universal DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a bioassay for the synchronous detection of two exosomal proteins and its application in intelligent diagnosis of cancer.

Exosomal proteins are considered to be promising indicators of cancer. Herein, a novel DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a strategy was proposed for the synchronous determination of exosomal proteins: serum amyloid A-1 protein (SAA1) and coagulation factor V (FV). In this design, the paired antibodies were used to recognize targets, thereby ensuring the specificity. DNAzyme walkers were employed to convert the contents of SAA1 and FV into activators (P1 and P2), and one target can produce abundant activators, thus achieving an initial amplification of signal. Furthermore, the P1 and P2 can activate CRISPR-Cas12a/Cas13a system, which in turn trans-cleaves the reporters, enabling a second amplification and generating two fluorescent signals. The assay is highly sensitive (limits of detection as low as 30.00 pg/mL for SAA1 and 200.00 pg/mL for FV), highly specific and ideally accurate. More importantly, it is universal and can be used to detect both non-membrane and membrane proteins in exosome. Besides, the method can be successfully applied to detect SAA1 and FV in plasma exosomes to differentiate between lung cancer patients and healthy individuals. To explore the application of the developed method in tumor diagnosis, a deep learning model based on the expressions of SAA1 and FV was developed. The accuracy of this model can achieve 86.96%, which proves that it has a promising practical application capacity. Thus, this study does not only provide a new tool for the detection of exosomal proteins and cancer diagnosis, but also propose a new strategy to detect non-nucleic acid analytes for CRISPR-Cas system.

Evidence of a casual relationship between vitamin D deficiency and hypertension: a family-based study.

An association between vitamin D deficiency and hypertension has been observed in numerous studies. However, blood pressure improvements resulting from supplementation with vitamin D have been inconsistent. The causal relationship between vitamin D deficiency and hypertension is still unclear and was investigated in this family-based study. A total of 1370 individuals from both vitamin D deficiency and hypertension families were included. First, the heritability of vitamin D deficiency was estimated by the Falconer method. Second, SNPs (single nucleotide polymorphisms) of vitamin D metabolic and functional pathway genes associated with vitamin D deficiency were screened by a family-based association test, and the findings were further verified in nuclear families with vitamin D deficiency. Finally, a family-based association test was applied to investigate the association between selected SNPs associated with vitamin D deficiency and hypertension. The heritability of vitamin D deficiency was 50.4% in this family-based study. Allele C of rs3847987 was a risk factor for vitamin D deficiency (OR: 1.639, 95% CI: 1.170-2.297, P = 0.004). Furthermore, a family-based association of rs3847987 with hypertension was found in both additive and recessive models (P < 0.05). In addition, vitamin D deficiency was associated with hypertension (OR: 1.317, 95% CI: 1.022-1.698, P = 0.033). In conclusion, rs3847987 in the VDR gene was associated with both vitamin D deficiency and hypertension. Therefore, vitamin D deficiency may be a causal factor for hypertension.

Mutation of D201G near the receptor binding site significantly drives antigenic drift of circulating H9N2 subtype avian influenza virus.

The H9N2 subtype of avian influenza virus (H9N2 AIV) has caused significant losses in chicken flocks throughout China. Our previous research has shown that field isolates of H9N2 underwent antigenic drift to evolve into distinct groups with significant antigenic divergence from the commercially available vaccines. The present study sought to identify which single mutations that have naturally appeared in isolates from the past 5 years have driven antigenic drift. Six high-frequency mutation sites in/near the receptor binding site region were screened by comparing amino acid alignments of the H9N2 AIVs isolated from China between 2014 and 2019. Two substitutions (A168N and D201G) were demonstrated to have a significant impact on the antigenicity but did not change the growth kinetics of the virus. It is worth noting that the D201G substitution not only significantly changed the antigenicity but also caused immune escape against the parental virus. In conclusion, A168N and D201G substitution are newly discovered determinants that can significantly change the antigenicity of H9N2 AIV, which should be tracked during outbreaks.

Sex differences in the association between dinner-bedtime interval and abdominal obesity: a large-scale cross-sectional study.

PURPOSE: Dinner-bedtime interval was reported to be associated with general obesity. However, the association between dinner-bedtime interval and abdominal obesity is still unclear. This study was conducted to investigate the association of dinner-bedtime interval and abdominal obesity. METHODS: A total of 7600 participants from Henan rural cohort study were included in this study. A standard questionnaire was used to obtain the time of dinner and sleep by the way of face-to-face interview. Sleep quality of each participant was evaluated by Pittsburgh Sleep Quality Index. Logistic regression and restricted cubic spline were used to assess the association between dinner-bedtime interval and abdominal obesity. Line regression was used to estimate the association between dinner-bedtime interval and lipid metabolism indexes. The mediation effect of sleep quality on the relationship between dinner-bedtime interval and abdominal obesity was evaluated. RESULTS: In male, increased dinner-bedtime interval was associated with abdominal obesity risk (Adjusted OR: 1.084, 95% CI 1.009-1.164). Compared with participants with dinner-bedtime interval ≤ 2 h, those dinner-bedtime interval > 2 h had an elevated risk of abdominal obesity (Adjusted OR: 1.199, 95% CI 1.009-1.425). In addition, a positive linear dose-response relationship was detected between dinner-bedtime interval and abdominal obesity. Moreover, total cholesterol concentration increased by 0.047 mmol/L for each 1-h increase in dinner-bedtime interval (P = 0.019). In addition, sleep quality mediated 11.45% of the relationship between dinner-bedtime interval and abdominal obesity (adjusted mediation effect: - 0.010, 95% CI - 0.019 to - 0.003). But in female, these associations were not significant. CONCLUSION: It is suggested that increased dinner-bedtime interval was related to a higher risk of abdominal obesity in rural China and this association was differed by sex. LEVEL OF EVIDENCE: Level V: cross-sectional descriptive study.

The correlation between testosterone and stroke and the mediating role of blood pressure: The Henan rural cohort study.

AIMS: We aimed to investigate the association of serum testosterone with stroke and calculate the proportion explained by blood pressure on this association. MATERIALS AND METHODS: A total of 6175 subjects were included in this study. Serum testosterone was quantified by liquid chromatography-tandem mass spectrometry. The logistic regression model was used to evaluate the association between serum testosterone and stroke. Linear regression analysis was used to assess the associations of serum testosterone with blood pressure. In addition, mediation analysis was performed to identify the mediation effects of blood pressure on the association of serum testosterone with stroke. Sex-stratified analysis was employed throughout the research. RESULTS: After adjusting for multiple variables, serum testosterone levels were negatively associated with stroke in males (per 1 unit natural log-transformed, odds ratio (OR) = 0.81, 95% confidence interval (CI): 0.69-0.94; Tertile 3 vs Tertile 1, OR = 0.65, 95% CI:0.44-0.96). Furthermore, blood pressure played a partial mediating role in the relationship between testosterone and stroke in males. The indirect effect/total effect of systolic blood pressure, diastolic blood pressure and mean arterial pressure were 7.37%, 9.54% and 9.22%, respectively. Notably, the relationship between testosterone and stroke and the role of blood pressure in regulating them was not observed in females. CONCLUSION: This study describes that in rural Chinese males, testosterone can reduce the risk of stroke by affecting blood pressure. To some extent, we provide a new epidemiological evidence for the relationship between testosterone and stroke.

New horizons in the identification of circulating tumor cells (CTCs): An emerging paradigm shift in cytosensors.

Circulating tumor cells (CTCs) are cancer cells that are shed from a primary tumor into the bloodstream and function as seeds for cancer metastasis at distant locations. Enrichment and identification methods of CTCs in the blood of patients plays an important role in diagnostic assessments and personalized treatments of cancer. However, the current traditional identification methods not only impact the viability of cells, but also cannot determine the type of cancer cells when the disease is unknown. Hence, new methods to identify CTCs are urgently needed. In this context, many advanced and safe technologies have emerged to distinguish between cancer cells and blood cells, and to distinguish specific types of cancer cells. In this review, at first we have briefly discussed recent advances in technologies related to the enrichment of CTCs, which lay a good foundation for the identification of CTCs. Next, we have summarized state-of-the-art technologies to confirm whether a given cell is indeed a tumor cell and determine the type of tumor cell. Finally, the challenges for application and potential directions of the current identification methods in clinical analysis of CTCs have been discussed.