The CRISPR-Cas13a Gemini System for noncontiguous target RNA activation.

Simultaneous multi-target detection and multi-site gene editing are two key factors restricting the development of disease diagnostic and treatment technologies. Despite numerous explorations on the source, classification, functional features, crystal structure, applications and engineering of CRISPR-Cas13a, all reports use the contiguous target RNA activation paradigm that only enables single-target detection in vitro and one-site gene editing in vivo. Here we propose a noncontiguous target RNA activation paradigm of Cas13a and establish a CRISPR-Cas13a Gemini System composed of two Cas13a:crRNA binary complexes, which can provide rapid, simultaneous, highly specific and sensitive detection of two RNAs in a single readout, as well as parallel dual transgene knockdown. CRISPR-Cas13a Gemini System are demonstrated in the detection of two miRNAs (miR-155 and miR-375) for breast cancer diagnosis and two small RNAs (EBER-1 and EBER-2) for Epstein-Barr virus diagnosis using multiple diagnostic platforms, including fluorescence and colorimetric-based lateral flow systems. We also show that CRISPR-Cas13a Gemini System can knockdown two foreign genes (EGFP and mCherry transcripts) in mammalian cells simultaneously. These findings suggest the potential of highly effective and simultaneous detection of multiple biomarkers and gene editing of multiple sites.

The short- and long-term outcomes of laparoscopic D2 lymphadenectomy plus complete mesogastrium excision for lymph node-negative gastric cancer.

BACKGROUND: Patients with T1-3N0M0 gastric cancer (GC) who undergo radical gastrectomy maintain a high recurrence rate. The free cancer cells in the mesogastric adipose connective tissue (Metastasis V) maybe the reason for recurrence in these individuals. We aimed to evaluate whether D2 lymphadenectomy plus complete mesogastrium excision (D2 + CME) was superior to D2 lymphadenectomy with regard to safety and oncological efficacy for T1-3N0M0 GC. METHODS: Patients with T1-3N0M0 GC who underwent radical resection from January 2014 to July 2018 were retrospectively analyzed; there were 323 patients, of whom 185 were in the D2 + CME group and 138 in the D2 group. The primary endpoint was 5-year disease-free survival (DFS). Secondary endpoints include the 5-year overall survival (OS), recurrence pattern, morbidity, mortality, and surgical outcomes. RESULTS: D2 + CME was associated with less intraoperative bleeding loss, a greater number of lymph nodes harvested, and less time to first postoperative flatus, but the postoperative morbidity was similar. The 5-year DFS was 95.6% (95% CI 92.7-98.5%) and 90.4% (95% CI 85.5-95.3%) in the D2 + CME group and the D2 group, respectively, with a hazard ratio (HR) of 0.455 (95% CI 0.188-1.097; p = 0.071). In terms of recurrence patterns, local recurrence was more prone to occur in the D2 group (p = 0.031). Subgroup analysis indicated that for patients with T1b-3N0M0 GC, the 5-year DFS in the D2 + CME group was considerably greater than that in the D2 group (95.3% [95% CI 91.6-99.0%] vs. 87.6% [95% CI 80.7-94.5%], HR 0.369, 95% CI 0.138-0.983; log-rank p = 0.043). CONCLUSION: Laparoscopic D2 + CME for T1-3N0M0 GC is safe and feasible. Furthermore, it not only reduces the local recurrence rate but also improves the 5-year DFS in cases of T1b-3N0M0 GC.

Organic Pollutants Associated with Plastic Debris in Marine Environment: A Systematic Review of Analytical Methods, Occurrence, and Characteristics.

Plastic pollution has become one of the most serious environmental problems, and microplastics (MPs, particles < 5 mm size) may behave as a vehicle of organic pollutants, causing detrimental effects to the environment. Studies on MP-sorbed organic pollutants lack methodological standardization, resulting in a low comparability and replicability. In this work, we reviewed 40 field studies of MP-sorbed organic contaminants using PRISMA guidelines for acquiring information on sampling and analytical protocols. The papers were also scored for their reliability on the basis of 7 criteria, from 0 (minimum) to 21 (maximum). Our results showed a great heterogeneity of the methods used for the sample collection, MPs extraction, and instruments for chemicals' identification. Measures for cross-contamination control during MPs analysis were strictly applied only in 13% of the studies, indicating a need for quality control in MPs-related research. The most frequently detected MP-sorbed chemicals were polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and organochlorine pesticides (OCPs). Most of the studies showed a good reliability (>75% of the total score), with 32 papers scoring 16 or higher. On the basis of the collected information, a standardizable protocol for the detection of MPs and MP-sorbed chemicals has been suggested for improving the reliability of MPs monitoring studies.

Genetic dissection of the impact of lncRNA AI662270 during the development of atherosclerosis.

BACKGROUND: Atherosclerosis is driven by synergistic interactions between pathological biomechanical and lipid metabolic factors. Long noncoding RNAs (LncRNAs) have been implicated in atherogenesis. The purpose of this study was to investigate the potential mechanism of lncRNA AI662270 on macrophage cholesterol transport in atherosclerosis. METHODS: Apolipoprotein E deficiency (ApoE-/-) mice were fed a high fat diet for 16 weeks to construct atherosclerotic model, and the mice were injected with recombinant lentivirus carrying AI662270 gene to overexpress AI662270. Macrophages were cleared by liposomal clondronate in vivo. Fundamental experiments and functional assays, hematoxylin and eosin staining, oil red O staining and others, were performed to evaluate the function of AI662270 on atherogenesis. Peritoneal macrophages were treated with oxidized low density lipoprotein (ox-LDL) to simulate in vitro model. Mechanism assays, RNA-interacting protein immunoprecipitation, RNA-protein pulldown and others, were performed to study the regulatory mechanism of AI662270 in macrophages. RESULTS: The novel AI662270 was mainly enriched in macrophages, but not in endothelial cells, smooth muscle cells and fibroblasts of mouse atherosclerotic lesions and was upregulated by ox-LDL. Overexpression of AI662270 resulted in lipid accumulation, larger atherosclerotic plaques and cardiac dysfunction in vivo. After macrophages were removed, the pro-atherogenic effect of AI662270 disappeared. Downregulation of AI662270 in macrophages protected against foam cell formation by potentiating cholesterol efflux and reducing intracellular total cholesterol. The opposite effect was observed in macrophage-specific AI662270-overexpressed cells in vitro. AI662270 bound to adenosine triphosphate-binding cassette transporter A1 (Abca1) responsible for regulating cholesterol efflux in macrophages. Forced expression of AI662270 in macrophages decreased Abca1 expression. The reverse occurred when expression of AI662270 was repressed. CONCLUSION: These findings reveal an essential role for AI662270 in atherosclerosis progression by regulating cholesterol efflux from macrophages.

lncR-GAS5 upregulates the splicing factor SRSF10 to impair endothelial autophagy, leading to atherogenesis.

Long noncoding RNAs (lncRNAs) play a critical role in the regulation of atherosclerosis. Here, we investigated the role of the lncRNA growth arrest-specific 5 (lncR-GAS5) in atherogenesis. We found that the enforced expression of lncR-GAS5 contributed to the development of atherosclerosis, which presented as increased plaque size and reduced collagen content. Moreover, impaired autophagy was observed, as shown by a decreased LC3II/LC3I protein ratio and an elevated P62 level in lncR-GAS5-overexpressing human aortic endothelial cells. By contrast, lncR-GAS5 knockdown promoted autophagy. Moreover, serine/arginine-rich splicing factor 10 (SRSF10) knockdown increased the LC3II/LC3I ratio and decreased the P62 level, thus enhancing the formation of autophagic vacuoles, autolysosomes, and autophagosomes. Mechanistically, lncR-GAS5 regulated the downstream splicing factor SRSF10 to impair autophagy in the endothelium, which was reversed by the knockdown of SRSF10. Further results revealed that overexpression of the lncR-GAS5-targeted gene miR-193-5p promoted autophagy and autophagic vacuole accumulation by repressing its direct target gene, SRSF10. Notably, miR-193-5p overexpression decreased plaque size and increased collagen content. Altogether, these findings demonstrate that lncR-GAS5 partially contributes to atherogenesis and plaque instability by impairing endothelial autophagy. In conclusion, lncR-GAS5 overexpression arrested endothelial autophagy through the miR-193-5p/SRSF10 signaling pathway. Thus, miR-193-5p/SRSF10 may serve as a novel treatment target for atherosclerosis.

Virus contamination and infectivity in beach environment: Focus on sand and stranded material.

To assess the exposure of beachgoers to viruses, a study on seawater, sand, and beach-stranded material was carried out, searching for human viruses, fecal indicator organisms, and total fungi. Moreover, for the first time, the genome persistence and infectivity of two model viruses was studied in laboratory-spiked sand and seawater samples during a one-week experiment. Viral genome was detected in 13.6 % of the environmental samples, but it was not infectious (Human Adenovirus - HAdV, and enterovirus). Norovirus and SARS-CoV-2 were not detected. The most contaminated samples were from sand and close to riverine discharges. In lab-scale experiments, the infectivity of HAdV5 decreased by ~1.5-Log10 in a week, the one of Human Coronavirus-229E disappeared in <3 h in sand. The genome of both viruses persisted throughout the experiment. Our results confirm viral contamination of the beach and suggest HAdV as an index pathogen for beach monitoring and quantitative risk assessment.

Multiscale Analysis of Runoff Complexity in the Yanhe Watershed.

Runoff complexity is an important indicator reflecting the sustainability of a watershed ecosystem. In order to explore the multiscale characteristics of runoff complexity and analyze its variation and influencing factors in the Yanhe watershed in China during the period 1991-2020, we established a new analysis method for watershed runoff complexity based on the complete ensemble empirical mode decomposition with adaptive noise (CEEMDAN) method for the decomposition of multiscale characteristics and the refined composite multiscale entropy (RCMSE) method for the quantification of the system complexity. The results show that runoff and its components all present multiscale complexity characteristics that are different from random signals, and the intermediate frequency modes contribute the most to runoff complexity. The runoff complexity of the Yanhe watershed has decreased gradually since 1991, and 2010 was a turning point of runoff complexity, when it changed from a decline to an increase, indicating that the ecological sustainability of this basin has improved since 2010, which was mainly related to the ecological restoration measures of the Grain for Green Project. This study expands the research perspective for analyzing the variation characteristics of runoff at the multiscale, and provides a reference for the study of watershed ecological sustainability and ecological management.

Aloe-emodin derivative produces anti-atherosclerosis effect by reinforcing AMBRA1-mediated endothelial autophagy.

Atherosclerosis is an inflammatory disease of high lethality associated with endothelial dysfunction. Due to the pathophysiological complexity and our incomplete understanding of the mechanisms for the development and progression of atherosclerosis, effective means for the prevention and treatment of atherosclerosis still need further exploration. This study was designed to investigate the potential effects and underlying mechanisms of aloe-emodin derivative (AED) on atherosclerosis. High fat diet (HFD) treated ApoE-/- mice were used as an animal model of atherosclerosis. Intragastric administration of aloe-emodin (AE) or AED for 12 weeks markedly reduced the atherosclerotic plaque in aorta with decreased plaque area, lipid accumulation, macrophage infiltration, collagen content and metabolic abnormalities. By comparison, AED produced more potent anti-atherosclerosis effects than AE at the same dose. AED enhanced production of autophagy flux in cultured human aortic endothelial cells (HAECs). Moreover, AED increased the expression of activating molecule in Beclin1-regulated autophagy 1 (AMBRA1), a key protein involved in autophagosome formation. Furthermore, knockdown of AMBRA1 blocked the promotion effect of AED on autophagy in HAECs. Taken together, AED facilitates endothelial autophagy via AMBRA1 during the progression of atherosclerosis, suggesting the potential application of this compound for atherosclerosis treatment.

CYP4G8 is responsible for the synthesis of methyl-branched hydrocarbons in the polyphagous caterpillar of Helicoverpa armigera.

Insect cuticular hydrocarbons (CHCs) have dual functions as physical barrier and chemical signals. The last step of CHC biosynthesis is known to be catalyzed by cytochrome P450 CYP4G in a number of insects. Until recently, studies on CYP4Gs in the context of functional evolution are rare. In this study, we analyzed sequence similarity and temporal-spatial expression patterns of the five CYP4G genes in the cotton bollworm Helicoverpa armigera, an important agricultural pest and also typical representative of lepidopteran insects. Moreover, the CRISPR/Cas9-induced knockout was used to clarify the roles of the five CYP4Gs in CHC biosynthesis. Temporal-spatial expression patterns revealed that CYP4G8 was highly expressed at all developmental stages and in most tissues examined. Larvae with CYP4G8 knocked out could not produce methyl-branched CHCs and failed to pupate, while larvae with the other four CYP4G genes knocked out (4G1-type-KO) showed no significant changes in their CHC profiles, weight gain and survival. Comparative transcriptomics revealed that knocking out CYP4G8 affected the global gene expression in larvae, especially down-regulated the expression of genes in the fatty acid biosynthetic pathway, while no significant change in 4G1-type-KO transcriptome was observed. These findings indicate that the five members of the CYP4G subfamily have undergone functional divergence: CYP4G8 maintains the essential function in CHC biosynthesis, while the function of the other four CYP4G genes remains unclear. Intriguingly, CYP4G8 has evolved to be a P450 enzyme responsible for the synthesis of larval methyl-branched hydrocarbons. The observation that CYP4G8 knockout is lethal strongly suggest that CYP4G8 may serve as a candidate target for the development of insecticidal agents for the control of cotton bollworms.

An Urban Scaling Estimation Method in a Heterogeneity Variance Perspective.

Urban scaling laws describe powerful universalities of the scaling relationships between urban attributes and the city size across different countries and times. There are still challenges in precise statistical estimation of the scaling exponent; the properties of variance require further study. In this paper, a statistical regression method based on the maximum likelihood estimation considering the lower bound constraints and the heterogeneous variance of error structure, termed as CHVR, is proposed for urban scaling estimation. In the CHVR method, the heterogeneous properties of variance are explored and modeled in the form of a power-of-the-mean variance model. The maximum likelihood fitting method is supplemented to satisfy the lower bound constraints in empirical data. The CHVR method has been applied to estimating the scaling exponents of six urban attributes covering three scaling regimes in China and compared with two traditional methods. Method evaluations based on three different criteria validate that compared with both classical methods, the CHVR method is more effective and robust. Moreover, a statistical test and long-term variations of the parameter in the variance function demonstrate that the proposed heterogeneous variance function can not only describe the heterogeneity in empirical data adequately but also provide more meaningful urban information. Therefore, the CHVR method shows great potential to provide a valuable tool for effective urban scaling studies across the world and be applied to scaling law estimation in other complex systems in the future.

Spatial Heterogeneity Analysis: Introducing a New Form of Spatial Entropy.

Distinguishing and characterizing different landscape patterns have long been the primary concerns of quantitative landscape ecology. Information theory and entropy-related metrics have provided the deepest insights in complex system analysis, and have high relevance in landscape ecology. However, ideal methods to compare different landscape patterns from an entropy view are still lacking. The overall aim of this research is to propose a new form of spatial entropy (Hs) in order to distinguish and characterize different landscape patterns. Hs is an entropy-related index based on information theory, and integrates proximity as a key spatial component into the measurement of spatial diversity. Proximity contains two aspects, i.e., total edge length and distance, and by including both aspects gives richer information about spatial pattern than metrics that only consider one aspect. Thus, Hs provides a novel way to study the spatial structures of landscape patterns where both the edge length and distance relationships are relevant. We compare the performances of Hs and other similar approaches through both simulated and real-life landscape patterns. Results show that Hs is more flexible and objective in distinguishing and characterizing different landscape patterns. We believe that this metric will facilitate the exploration of relationships between landscape patterns and ecological processes.

Using a Mixed Model to Explore Evaluation Criteria for Bank Supervision: A Banking Supervision Law Perspective.

Financial supervision means that monetary authorities have the power to supervise and manage financial institutions according to laws. Monetary authorities have this power because of the requirements of improving financial services, protecting the rights of depositors, adapting to industrial development, ensuring financial fair trade, and maintaining stable financial order. To establish evaluation criteria for bank supervision in China, this study integrated fuzzy theory and the decision making trial and evaluation laboratory (DEMATEL) and proposes a fuzzy-DEMATEL model. First, fuzzy theory was applied to examine bank supervision criteria and analyze fuzzy semantics. Second, the fuzzy-DEMATEL model was used to calculate the degree to which financial supervision criteria mutually influenced one another and their causal relationship. Finally, an evaluation criteria model for evaluating bank and financial supervision was established.

Activation of Autophagy Contributes to the Angiotensin II-Triggered Apoptosis in a Dopaminergic Neuronal Cell Line.

Our recent study indicated that angiotensin II (Ang II), the main component of renin-angiotensin system, participated in the pathogenesis of Parkinson's disease (PD) by triggering the apoptosis of dopaminergic neuronal cells. However, the underlying mechanisms are still not fully understood. In this study, by using CATH.a cells, a dopaminergic neuronal cell line stably expressing angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R), we tested the hypothesis that activation of autophagy contributed to the apoptosis triggered by Ang II. We showed that Ang II activated autophagy and triggered apoptosis in CATH.a cells in a dose-dependent manner. More importantly, inhibition of autophagy by 3-methyladenine markedly attenuated the apoptosis caused by Ang II in CATH.a cells. In addition, the Ang II-induced autophagy and subsequent cell apoptosis could be fully abolished by an AT1R antagonist losartan rather than PD1223319, an antagonist for AT2R. Taken together, our study provides the first evidence that Ang II triggers apoptosis via activation of autophagy in a dopaminergic neuronal cell line through an AT1R-mediated manner. These findings have deepened our understanding on the role of Ang II in the pathogenesis of PD and support the use of AT1R antagonists for the treatment of this devastating neurodegenerative disease.

Angiotensin II triggers apoptosis via enhancement of NADPH oxidase-dependent oxidative stress in a dopaminergic neuronal cell line.

Numerous studies reveal that Angiotensin II (Ang II), the main effector of renin-angiotensin system, contributes to the pathogenesis of Parkinson's disease (PD) via triggering dopaminergic cell loss. However, the underlying mechanisms remain largely unclear. In the current study, by using CATH.a cell, a dopaminergic neuronal cell line stably expressing Angiotensin II type 1 receptor (AT1R) and Angiotensin II type 2 receptor (AT2R), we showed that Ang II treatment triggered cell apoptosis in a dose-dependent manner, providing the first evidence that apoptotic cell death contributed to the dopaminergic cell loss induced by Ang II. Ang II treatment also led to a significant increment in intracellular reactive oxygen species generation, which could be fully abolished by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors apocynin or diphenylene iodonium, indicating that Ang II enhanced oxidative stress via a NADPH oxidase-dependent manner. More importantly, inhibition of oxidative stress by NADPH oxidase inhibitors partially attenuated cell apoptosis caused by Ang II, implying that the enhancement of NADPH oxidase-dependent oxidative stress contributed to the cell apoptosis triggered by Ang II. Furthermore, the Ang II-induced oxidative stress and subsequent apoptosis could be completely abolished by AT1R blocker losartan rather than AT2R blocker PD1223319, suggesting that the aforementioned detrimental effects of Ang II are mediated by AT1R. In summary, these findings have deepened our understanding on the role of Ang II in PD pathogenesis, and support the use of AT1R blockers in the treatment of this devastating disease.

Angiotensin AT2 receptor stimulation inhibits activation of NADPH oxidase and ameliorates oxidative stress in rotenone model of Parkinson's disease in CATH.a cells.

Oxidative stress has long been considered as a major contributing factor in the pathogenesis of Parkinson's disease (PD). The brain has an independent local renin-angiotensin system (RAS). Angiotensin II (Ang II) activates NADPH-dependent oxidases, which are a major source of superoxide and are upregulated in major aging-related diseases such as hypertension and neurodegenerative disease. In this study, we firstly examined whether CGP42112, an AT2 receptor (AT2R) agonist, may exert direct protective effects on the rotenone-induced CATH.a cell injury in vitro. We used CATH.a cell line to evaluate changes in cultured dopaminergic neuron levels of superoxide dismutase (SOD), glutathione (GSH) and reactive oxygen species (ROS). We also evaluated expression of NADPH oxidase, AT1 and AT2 receptors in treated with phosphate buffer saline (PBS), rotenone, Ang II, AT2R agonist CGP42112, or AT2R antagonist PD123319, alone and combined (n=6, each group). Quantitative reverse transcriptase PCR (qRT-PCR) and western blot were used to determine messenger RNA (mRNA) and protein levels of the AT1, AT2 receptors and NADPH oxidase. ROS generation was determined by the dichlorodihydrofluorescein diacetate fluorescent probe assay. The levels of SOD and GSH were measured by using available kits. In our study, CGP42112 (100nM) significantly reduced rotenone-induced oxidative stress and elevated the total SOD activity and GSH level. In addition, CGP42112 significantly increased AT2R expression and attenuated Ang II-induced NADPH oxidase activation, and these effects were completely abolished by the AT2R antagonist, PD123319 (1µM). Our results suggest that CGP42112 attenuates rotenone-induced oxidative stress in CATH.a neuron via activating AT2R and suppressing NADPH oxidase expression.

Salubrinal protects against rotenone-induced SH-SY5Y cell death via ATF4-parkin pathway.

Parkinson's disease (PD) is a progressive neurodegenerative disorder, for which there are no effective disease-modifying therapies. Growing evidence from studies in human PD brain, in addition to genetic and toxicological models, indicates that endoplasmic reticulum (ER) stress is a common feature of the disease and contributes to neurodegeneration. We examine whether salubrinal, a ER stress inhibitor, can protect the rotenone-induced SH-SY5Y cell death and explore the mechanisms underlying this protection. Our results demonstrated that rotenone induced a significant ER stress response and caused cell apoptosis, which was inhibited by salubrinal. Activating transcription factor 4 (ATF4), a member of the ATF/CREB family of basic leucine zipper transcription factors, has been implicated in the pathogenesis of neurodegeneration. We showed that salubrinal increased the up-regulation of ATF4 expression. An ATF4 siRNA significantly increased the rotenone cytotoxicity and decreased the salubrinal's protection. Further, we showed that ATF4 siRNA inhibited the expression of parkin, and parkin knockdown similarly aggravated the rotenone cytotoxicity and reduced the salubrinal's protection. Additionally, the protein level of parkin was declined after treatment with rotenone, whereas this reduction was rescued by salubrinal. These findings indicate ATF4-parkin pathway plays an important role in the salubrinal-mediated neuroprotection of rotenone-induced dopaminergic cell death.