CRISPR-Cas12a coupled with universal gold nanoparticle strand-displacement probe for rapid and sensitive visual SARS-CoV-2 detection.

Point of care (POC) diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are particularly significant for preventing transmission of coronavirus disease 2019 (COVID-19) by any user at any given time and place. CRISPR/Cas-assisted SARS-CoV-2 assays are viewed as supplemental to RT-PCR due to simple operation, convenient use and low cost. However, most current CRISPR molecular diagnostics based on fluorescence measurement increased the difficulty of POC test with need of the additional light sources. Some instrument-free visual detection with the naked eye has limitations in probe universality. Herein, we developed a universal, rapid, sensitive and specific SARS-CoV-2 POC test that combines the outstanding DNase activity of Cas12a with universal AuNPs strand-displacement probe. The oligo trigger, which is the switch the AuNPs of the strand-displacement probe, is declined as a result of Cas12a recognition and digestion. The amount of released AuNPs produced color change which can be visual with the naked eye and assessed by UV-Vis spectrometer for quantitative detection. Furthermore, a low-cost hand warmer is used as an incubator for the visual assay, enabling an instrument-free, visual SARS-CoV-2 detection within 20 min. A real coronavirus GX/P2V instead of SARS-CoV-2 were chosen for practical application validation. After rapid virus RNA extraction and RT-PCR amplification, a minimum of 2.7 × 102 copies/mL was obtained successfully. The modular design can be applied to many nucleic acid detection applications, such as viruses, bacteria, species, etc., by simply modifying the crRNA, showing great potential in POC diagnosis.

Systematically investigating the fluorescent signal readout of CRISPR-Cas12a for highly sensitive SARS-CoV-2 detection.

The CRISPR/Cas system is widely used for molecular diagnostics after the discovery of trans-cleavage activity, especially now with the COVID-19 outbreak. However, the majority of contemporary trans-cleavage activity-based CRISPR/Cas biosensors exploited standard single-strand DNA (ssDNA) reporters, which were based on the FRET principle from pioneering research. An in-depth comparison and understanding of various fluorescent readout types are essential to facilitate the outstanding analytical performance of CRISPR probes. We investigated various types of fluorescent reporters of Cas12a comprehensively. Results show that trans-cleavage of Cas12a is not limited to ssDNA and dsDNA reporters, but can be extended to molecular beacons (MB). And MB reporters can achieve superior analytical performance compared with ssDNA and ds DNA reporters at the same conditions. Accordingly, we developed a highly-sensitive SARS-CoV-2 detection with the sensitivity as low as 100 fM were successfully achieved without amplification strategy. The model target of ORF1a could robustly identify the current widespread emerging SARS-CoV-2 variants. A real coronavirus GX/P2V instead of SARS-CoV-2 were chosen for practical application validation. And a minimum of 27 copies/mL was achieved successfully. This inspiration can also be applied to other Cas proteins with trans-cleavage activity, which provides new perspectives for simple, highly-sensitive and universal molecular diagnosis in various applications.

Calcium supplementation reducing the risk of hypertensive disorders of pregnancy and related problems: A meta-analysis of multicentre randomized controlled trials.

Hypertensive disorders of pregnancy are closely related to maternal mortality and morbidity. Calcium supplementation during pregnancy seems to reduce the risk of hypertensive disorders. No systematic review on multicentre RCTs of calcium supplementation during pregnancy has been published. The purpose of this study was to report a quantitative systematic review of the effectiveness of calcium supplementation during pregnancy on reducing the risk of hypertensive disorders of pregnancy and related problems. Publications over the years of 1991-2012 were searched through PubMed, Science Direct, EMBASE, CINAHL and Web of Science. The literatures were selected of the multicentre RCTs on calcium supplementation during pregnancy in prevention of hypertensive disorders and related problems. Reference lists from the studies were also examined for additional references. Studies were critically appraised by three independent reviewers, and the Cochrane Handbook was used to assess the quality of those included trials. Four studies were included in this systematic review. All included studies were high quality, with low risk of bias. There was an observed risk reduction in hypertension in calcium group. However, there was no reduction in the risk of severe gestational hypertension, pre-eclampsia, severe pre-eclampsia, preterm birth and low birthweight. Calcium supplementation appears to reduce the risk of hypertension in pregnancy.

Dietary fructose regulates hepatic manganese homeostasis in female mice.

Arginase, an enzyme dependent on manganese (Mn), plays a crucial role in the production of urea and processing of ammonia in the liver. Previous studies have shown that overconsumption of fructose disrupts Mn homeostasis in the liver of male mice. However, the potential sex-specific differences in the impact of fructose on hepatic Mn homeostasis remain uncertain. In this study, we provide evidence that heightened fructose intake disrupts liver Mn homeostasis in female mice. Elevated fructose exposure led to a reduction in liver Mn levels, resulting in decreased arginase and manganese superoxide dismutase (Mn-SOD) activity in the liver of female mice. The underlying mechanism involves the upregulation of carbohydrate-responsive element binding protein (ChREBP) expression and the Mn exporting gene Slc30a10 in the liver in response to fructose consumption. In summary, our findings support the involvement of fructose in liver Mn metabolism via the ChREBP/Slc30a10 pathway in female mice, and indicate that there is no disparity in the impact of fructose on hepatic Mn homeostasis between sexes.

[Evaluation of Heavy Metal Distribution Characteristics and Ecological Risk of Soil of Vegetable Land for Hong Kong in Ningxia].

Heavy metal pollution in farmland soil can affect the growth, development, and yield of vegetable crops, as well as the quality and taste of vegetables, and can be continuously transmitted and enriched through the food chain, which ultimately poses a certain hazard to human health in the long term. Therefore, in order to investigate the distribution characteristics of soil heavy metals after years of multi-crop planting of vegetables supplied to Hong Kong, predict their ecological risks, and analyze the causes of pollution formation, 477 surface soil samples of vegetable fields supplied to Hong Kong in Ningxia were collected for three consecutive years from 2019 to 2021, and the contents and distribution characteristics of eight heavy metals, namely, As, Cd, Cr, Hg, Pb, Cu, Zn, and Ni were analyzed. The soil heavy metal pollution status of vegetable fields supplied to Hong Kong in Ningxia was evaluated using the single-factor pollution index method, Nemero's comprehensive pollution index method, land accumulation index method, and potential ecological risk index method, and the sources of heavy metals in vegetable fields supplied to Hong Kong in Ningxia were analyzed using the Pearson's correlation analysis and the principal component analysis method. The results showed that the mean values of As, Cd, Cr, Hg, Cu, and Zn in the soils of Ningxia's vegetable fields were higher than the background values of Ningxia soils, but the contents of all eight heavy metals were lower than the risk screening values of domestic agricultural soils; in terms of spatial distribution, As, Cr, and Ni showed contiguous high values in the northwestern, central, and southern parts of the study area, whereas Pb, Zn, Cd, Hg, and Cu showed high values in the northwestern and southern parts of the study area. The single-factor index method and the Nemero's comprehensive pollution index method showed that the soil of Ningxia's vegetable farmland for Hong Kong was at the clean level as a whole. The results of the ground accumulation index method showed that the pollution in the study area was mainly Hg and Cd pollution, and the pollution areas were mainly concentrated in the northwest and south of the study area. The potential ecological risk index showed that Hg and Cd were the main risk elements, among which Hg was dominated by moderate, strong, and very strong ecological risks, accounting for 44.65 %, 44.65 %, and 1.26 %, respectively, and Cd was dominated by moderate and strong risks, accounting for 65.83 % and 3.56 %. The comprehensive Pearson correlation analysis and principal component analysis showed that the pollution sources of eight heavy metals could be divided into three categories, namely, natural sources:Cu, Zn, Pb, As, Ni, and Cr; agricultural sources:Cd; and industrial and agricultural sources:Hg. From a comprehensive point of view, the heavy metals of the soil in the fields of vegetables supplied to Hong Kong had not exceeded the standard, and the environmental conditions of the soil were good, such that the production of vegetables supplied to Hong Kong by Ningxia was at a safe level overall. The results of the study can provide a theoretical basis for the safe utilization of soil in vegetable fields and the green production of vegetables supplied to Hong Kong in Ningxia, which were aimed to provide help for the safe production of vegetable fields supplied to Hong Kong, the rational application of fertilizers, agronomic planning, and the adjustment of planting structure.

A novel virus in the family Marnaviridae as a potential pathogen of Penaeus vannamei glass post-larvae disease.

As an aquatic animal of great commercial relevance, Penaeus vannamei is currently the dominant species of cultured shrimp in China and many other countries worldwide. In recent years, the outbreak of glass post-larvae disease (GPD), which accounts for more than 90% of the mortality of shrimp seedlings in serious cases, in many regions of China has caused significant losses and threatened the sustainability of the aquaculture industry and the economy. It is extremely urgent to determine the infectious agent of GPD in P. vannamei. In this work, we performed metagenomic sequencing of glass post-larvae collected from diseased prawns in Tangshan Hebei, where GPD broke out recently. An evolutionary tree was constructed by MEGA 7 to understand the evolutionary history and relationship of the pathogen genome. A novel virus in the family Marnaviridae was first identified in P. vannamei suffering from GPD, and we tentatively named this virus Baishivirus (GenBank: ON550424). The identified pathogen was validated according to Koch's rule with a pathogenic challenge assay and reverse transcription-polymerase chain reaction. There was only 8% query coverage with 64.96% identity in the Baishivirus genome when compared with its most closely related genome sequence of Wenzhou picorna-like virus 21 reported in 2016. Baishivirus genomic RNA is 9.895 kb in length and encodes three potential open reading frames (ORFs). The identification of Baishivirus in P. vannamei enriches the family Marnaviridae and potentially provides a new candidate to study and prevent GPD in the aquaculture industry.

Importance of sample input volume for accurate SARS-CoV-2 qPCR testing.

Nucleic acid testing is the most widely used detection method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Currently, a number of COVID-19 real-time quantitative reverse transcription PCR (qPCR) kits with high sensitivity and specificity are available for SARS-CoV-2 testing. However, these qPCR assays are not always reliable in detecting low viral load samples (Ct-value ≥ 35), resulting in inconclusive or false-negative results. Here, we used a Poisson distribution to illustrate the inconsistent performance of qPCR tests in detecting low viral load samples. From this, we concluded that the false-negative outcomes resulted from the random occurrences of sampling zero target molecules in a single test, and the probability to sample zero target molecules in one test decreased significantly with increasing purified RNA or initial sample input volume. At a given RNA concentration of 0.5 copy/µL, the probability of sampling zero RNA molecules decreased from 36.79% to close to 0.67% after increasing the RNA input volume from 2 to 10 µL. A SARS-CoV-2 qPCR assay with an LOD of 300 copies/mL was used to validate the improved consistency of the qPCR tests. We found that the false-negative qPCR results of clinical COVID-19 samples with a Ct ≥ 35 decreased by 50% after increasing the input of purified RNA from 2 to 10 µL. The consistency, accuracy, and robustness of nucleic acid testing for SARS-CoV-2 samples with low viral loads can be improved by increasing the sample input volume.

Rapid and highly sensitive one-tube colorimetric RT-LAMP assay for visual detection of SARS-CoV-2 RNA.

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a highly contagious disease. To tame the continuously raging outbreak of COVID-19, developing a cheap, rapid and sensitive testing assay is absolutely imperative. Herein, we developed a one-tube colorimetric RT-LAMP assay for the visual detection of SARS-CoV-2 RNA. The assay integrates Si-OH magnetic beads (MBs)-based fast RNA extraction and rapid isothermal amplification in a single tube, thus bypassing the RNA elution step and directly amplifying on-beads RNA molecules with the visualized results. This one-tube assay has a limit of detection (LOD) as low as 200 copies/mL for sample input volumes of up to 600 µL, and can be performed in less than 1 h from sample collection to result readout. This assay demonstrated a 100% concordance with the gold standard test RT-qPCR test by using 29 clinical specimens and showed high specificity. This one-tube colorimetric RT-LAMP assay can serve as an alternative platform for a rapid and sensitive diagnostic test for COVID-19 and is particularly suitable for use at community clinics or township hospitals.

Circular RNA hsa_circ_0000700 promotes cell proliferation and migration in Esophageal Squamous Cell Carcinoma by sponging miR-1229.

Accumulating evidence has demonstrated that circular RNAs (circRNAs) are involved in the pathogenesis of cancer, including that of esophageal squamous cell carcinoma (ESCC). The current study aimed to investigate the role of hsa_circ_0000700 in ESCC. hsa_circ_0000700, miR-1229, and related functional gene expression was measured by RT-qPCR. To characterize the functions of hsa_circ_0000700 and miR-1229, ESCC cells were infected with hsa_circ_0000700-specific siRNA, miR-1229 mimics, and an inhibitor alone or in combination. Cell Counting Kit-8 (CCK8), colony formation, EdU, flow cytometry, and Transwell assays were employed to evaluate cell proliferation, apoptosis, and migration. Luciferase reporter and RNA immunoprecipitation assays were used to confirm the targeting relationship between hsa_circ_0000700 and miR-1229. Finally, a competing endogenous RNAs (ceRNA) network was built for hsa_circ_0000700, and miR-1229 targets were analyzed by bioinformatics. circ_0000700 expression was significantly upregulated in ESCC cell lines. Actinomycin D and RNase R treatment confirmed that circ_0000700 was more stable than its linear CDH9 mRNA form. Moreover, a cytoplasmic and nuclear fractionation assay suggested that circ_0000700 was mainly distributed in the cytoplasm of ECA-109 and TE-1 cells. In vitro, the proliferative and migratory capacities of ECA-109 and TE-1 cells were inhibited by knocking down circ_0000700 expression. Additionally, miR-1229 silencing reversed the circ_0000700-specific siRNA-induced attenuation of malignant phenotypes. Mechanistically, circ_0000700 was identified as a sponge of miR-1229 and could activate PRRG4, REEP5, and PSMB5 indirectly to promote ESCC progression. In summary, our results suggest that hsa_circ_0000700 functions as an oncogenic factor by sponging miR-1229 in ESCC.

Radiation-induced gray matter atrophy in patients with nasopharyngeal carcinoma after intensity modulated radiotherapy: a MRI magnetic resonance imaging voxel-based morphometry study.

BACKGROUND: Gray matter (GM) damage after radiotherapy (RT) in nasopharyngeal carcinoma (NPC) patients can result in cognitive impairment, while there may be no visible brain tissue change according to the conventional magnetic resonance imaging (MRI). This study investigated radiation-induced GM volume differences between NPC patients who received RT and those who did not. METHODS: High-resolution brain structural MRI data from two groups of patients were acquired. The pre-RT group was composed of 56 newly diagnosed but not yet medically treated NPC patients, while the after-RT group consisted of 40 NPC patients who had completed RT more than 1 year ago. Voxel-based morphometry (VBM) was applied to assess GM volumes. Two sample t-test was used to analyze GM volumes voxel-by-voxel using the VBM8 toolbox built in the SPM software. Radiation-induced cortical volume alteration in all NPC patients after RT and dosimetry of 36 patients were analyzed. RESULTS: Compared to pre-treatment group, cortical volumes of GM were significantly smaller in the left hippocampus, the right pulvinar and the right middle temporal gyrus (MTG, P<0.001, AlphaSim correction, cluster size ≥157). The mean dose (Dmean) for bilateral hippocampal heads were significantly higher than other different parts of the brain (P<0.001). No significant correlations between the GM volume in any brain regions and the mean dose of corresponding position of these brain regions were observed (P>0.05). CONCLUSIONS: Radiation to the NPC patients can not only induce damage of the hippocampus, but also other secondary damages of GM.