Visual isothermal amplification detection of ASFV based on trimeric G-quadruplex cis-cleavage activity of Cas-12a.

African swine fever virus (ASFV) is a kind of DNA virus and can infect both domestic pigs and wild boars with fatality up to 100%. The contaminated meat products mainly led to the worldwide transmission of ASFV. The outbreak of ASF greatly affects the supply stability of meat products as well as the development of the global pig industry. In this study, a visual isothermal amplification detection assay for ASFV based on trimeric G-quadruplex cis-cleavage activity of Cas12a was developed. The introduction of Cas12a could discriminate the specific amplification from the non-specific amplification and improve the sensitivity. The detection limit was as low as 0.23 copies/µL. This assay had good potential in the detection of ASFV and would be helpful for the stability of meat production and supply.

A visual denaturation bubble-mediated strand exchange amplification and RGB visual analysis-based assay for quantitative detection of Helicobacter pylori in saliva.

Helicobacter pylori (H. pylori) is a class I carcinogen causing gastric cancer. Almost 50% of people on earth have been infected and it is worse in developing countries. Early diagnosis of H. pylori infection is the most important strategy for preventing the spread and worse consequences. H. pylori can be isolated from human saliva, and the sampling of saliva is easy and convenient. Therefore, we developed a visual denaturation bubble-mediated strand exchange amplification and RGB visual analysis-based assay for quantitative detection of H. pylori in saliva in this study. Under the optimized reaction temperature and time, the SEA reaction could be finished in 30 min with a simple reaction system and low dependency on equipment. The detection results could be qualitatively identified by the naked eye and quantitatively analyzed by a developed RGB visual analysis method. The limit of detection (LOD) of RGB visual analysis was 10.8 CFU/mL. This assay had good specificity and anti-interference capacity. In the artificial contamination test, the recovery rate of our assay was between 99.3% and 111.5%, with RSD values ranging from 1.7% to 3.5%. These indicated our assay also had good reliability in the detection of saliva. We believe this assay showed good potential for better non-invasive diagnosis of H. pylori infection.

A time-resolved fluorescent microsphere-lateral flow immunoassay strip assay with image visual analysis for quantitative detection of Helicobacter pylori in saliva.

Helicobacter pylori (H. pylori) is a kind of microaerobic and food-borne pathogen. More than 4.4 billion individuals have been infected by H. pylori and H. pylori was listed as Group I carcinogen by WHO in 1994. Considering the high infection rate of H. pylori and the limited medical resources, self-testing is helpful for diagnosis and timely treatment. Although the amount of H. pylori in human saliva is low, the sampling of saliva is simple and convenient compared with stomach, blood and stool samples. Therefore, H. pylori in human saliva can be an indicator for self-testing, and a sensitive and easy-to-use assay is necessary. In this study, we developed a time-resolved fluorescent microsphere-lateral flow immunoassay (TRFM-LFIA) strip assay with image visual analysis for detection of H. pylori in saliva. The detection of the TRFM-LFIA strip was easy to use and had a low dependency on equipment. With optimized preparation and detection parameters, the whole detection process could be finished in 8 min and the LOD by naked eyes was 102 CFU/mL. For quantitative analysis by image visual analysis, the LOD was as low as 1.05 CFU/mL in the linear range of 101-105 CFU/mL. Besides, the TRFM-LFIA strip also had good stability, reliability, repeatability and accuracy. All these advantages indicated that the TRFM-LFIA strips developed in this study had a good potential for self-testing for H. pylori infection.

Impact of Germinated Brown Rice and Brown Rice on Metabolism, Inflammation, and Gut Microbiome in High Fat Diet-Induced Insulin Resistant Mice.

The constituents of germinated brown rice (GBR), brown rice (BRR), and white rice (WHR) and their impact on metabolism, inflammation, and gut microbiota in high fat (HF) diet-fed mice were examined. The contents of total fiber and γ-aminobutyric acid in BRR and GBR were higher than that in WHR (p < 0.05). Male C57 BL/6J mice received HF diet+26 g% of WHR, BRR, or GBR for 12 weeks. BRR and GBR comparably reduced HF diet-induced increases in fasting plasma glucose, lipids, insulin resistance, and inflammatory markers compared to WHR (p < 0.01). The abundance of fecal Bacteroidetes in mice fed HF+GBR or HF+BRR was higher than in HF+WHR-fed mice (p < 0.05). The abundance of fecal Lactobacillus gasseri in GBR-fed mice was greater than that in WHR- or BRR-fed mice (p < 0.05). The results indicated that GBR or BRR attenuated hyperglycemia, insulin resistance, and inflammation in mice. HF+GBR, but not HF+BRR, increased a probiotic bacteria in the gut.

A visual on-site biosensor for low-cost detection of chloramphenicol based on aptamer and split DNAzyme.

Chloramphenicol (CAP) is a kind of broad-spectrum antibiotic, which has been forbidden in food by most countries because of its side effects. In this study, a simple and low-cost biosensor for CAP detection in food was developed. The biosensor consisted of an aptamer specific to CAP and a pair of split probes that could self-assemble as DNAzyme. The detection result could be identified by the naked eye and the visual limit was 10 nM CAP. The absorbance of final reaction products at 417 nm had a linear relationship with the logarithm of the CAP concentration in a range from 10 to 200 nM, and the limit of detection was 87.3 pM. The visual analysis by imageJ also showed a linear detection range between 25 and 200 nM. The entire detection procedure could be completed in about 1.5 h at a cost of about 0.16 dollars per reaction. We believe that the biosensor shows great potential in the rapid and sensitive detection of CAP in food.

Signal-enhanced visual strand exchange amplification detection of African swine fever virus by the introduction of multimeric G-quadruplex/hemin DNAzyme.

African swine fever virus (ASFV) causes hemorrhagic infectious disease in pigs with a fatality rate of nearly 100%. In this study, we developed a visual strand exchange amplification detection assay for ASFV. In the presence of ASFV, DNA amplification products containing multimeric G-quadruplex sequences were amplified by strand exchange amplification. These G-quadruplexes, assembled with hemin to form DNAzyme, displayed enhanced significant "turned-on" colorimetric signals to indicate detection results. The results showed that dimeric DNAzyme had the best visualization effect. Under the optimal reaction parameters, there was a linear relationship between the absorbance of the reaction solution at 417 nm and the logarithm of ASFV concentration ranged from 1 × 101 to 1 × 103 copies/µL, and the detection limit was 2.7 copies/µL. We hoped this visual assay could be helpful in the rapid and sensitive detection of ASFV, and the results of multimeric G-quadruplex/hemin DNAzyme could be helpful for the development of better visual detection assays.

Fe-N-C single-atom nanozymes based sensor array for dual signal selective determination of antioxidants.

Machine learning algorithms as a powerful tool can efficiently utilize and process large quantities of data generated by high-throughput experiments in various fields. In this work, we used a general ionic salt-assisted synthesis method to prepare oxidase-like Fe-N-C SANs. The possible reason for the excellent enzyme-mimicking activity and affinity of Fe-N-C SANs was further verified by density functional theory calculations. Due to the remarkable oxidase-mimicking activity, the prepared Fe-N-C SANs were used to detect ascorbic acid (AA) with a detection limit of 0.5 µM. Based on the machine learning algorithms, we successfully distinguished six antioxidants (ascorbic acid, glutathione, L-cysteine, dithiothreitol, uric acid, and dopamine) with the same concentration by either one kind of Fe-N-C SANs or three kinds of different Fe-N-C SANs. The usefulness of the Fe-N-C SANs sensor arrays was further validated by the hierarchal cluster analysis, where they also can be correctly identified. More importantly, a SANs-based digital-image colorimetric sensor array has also been successfully constructed and thereby achieved visual and informative colorimetric analysis for practical samples out of the lab. This work not only provides a design synthesis method to prepare SANs but also combines machine learning algorithms with SANs sensors to identify analytes with similar properties, which can further expand to the detection of proteins and cells related to diseases in the future.

The Chinese pine genome and methylome unveil key features of conifer evolution.

Conifers dominate the world's forest ecosystems and are the most widely planted tree species. Their giant and complex genomes present great challenges for assembling a complete reference genome for evolutionary and genomic studies. We present a 25.4-Gb chromosome-level assembly of Chinese pine (Pinus tabuliformis) and revealed that its genome size is mostly attributable to huge intergenic regions and long introns with high transposable element (TE) content. Large genes with long introns exhibited higher expressions levels. Despite a lack of recent whole-genome duplication, 91.2% of genes were duplicated through dispersed duplication, and expanded gene families are mainly related to stress responses, which may underpin conifers' adaptation, particularly in cold and/or arid conditions. The reproductive regulation network is distinct compared with angiosperms. Slow removal of TEs with high-level methylation may have contributed to genomic expansion. This study provides insights into conifer evolution and resources for advancing research on conifer adaptation and development.

Diagnostic techniques for COVID-19: A mini-review.

COVID-19, a new respiratory infectious disease, was first reported at the end of 2019, in Wuhan, China. Now, COVID-19 is still causing major loss of human life and economic productivity in almost all countries around the world. Early detection, early isolation, and early diagnosis of COVID-19 patients and asymptomatic carriers are essential to blocking the spread of the pandemic. This paper briefly reviewed COVID-19 diagnostic assays for clinical application, including nucleic acid tests, immunological methods, and Computed Tomography (CT) imaging. Nucleic acid tests (NAT) target the virus genome and indicates the existence of the SARS-CoV-2 virus. Currently, real-time quantitative PCR (qPCR) is the most widely used NAT and, basically, is the most used diagnostic assay for COVID-19. Besides qPCR, many novel rapid and sensitive NAT assays were also developed. Serological testing (detection of serum antibodies specific to SARS-CoV-2), which belongs to the immunological methods, is also used in the diagnosis of COVID-19. The positive results of serological testing indicate the presence of antibodies specific to SARS-CoV-2 resulting from being infected with the virus. Viral antigen detection assays are also important immunological methods used mainly for rapid virus detection. However, only a few of these assays had been reported. CT imaging is still an important auxiliary diagnosis tool for COVID-19 patients, especially for symptomatic patients in the early stage, whose viral load is low and different to be identified by NAT. These diagnostic techniques are all good in some way and applying a combination of them will greatly improve the accuracy of COVID-19 diagnostics.

Development of an in-situ signal amplified electrochemical assay for detection of Listeria monocytogenes with label-free strategy.

Listeria monocytogenes is an important foodborne pathogen, which imposes great burdens on public health. The current methods for detecting L. monocytogene are limited in several ways such as time consuming and lab equipment dependent. In this study, we developed a new electrochemical assay to improve the efficacy. This assay allows us to generate numerous G-quadruplex sequences while loop-mediated isothermal amplification happens. Then, these G-quadruplex sequences form DNAzyme to produce a color change and an electrochemical signal by oxidizing tetramethylbenzidine. This assay could be finished in 2 h, which significantly reduced the detection time. Also, we confirmed the limit of detection of this assay at 6.8 CFU/mL according to 3σ criterion. Our assay shows good sensitivity to detect bacteria range from 52.5 to 5.25 × 104 CFU/mL. This assay's reliability was also confirmed by detecting artificially contaminated pork samples. Thus, we propose this electrochemical assay for rapid and sensitive detection of L. monocytogenes in food.

A signal cascade amplification strategy based on RT-PCR triggering of a G-quadruplex DNAzyme for a novel electrochemical detection of viable Cronobacter sakazakii.

Cronobacter sakazakii is an important opportunistic food-borne pathogen, and it can cause severe diseases with main symptoms including neonatal meningitis, necrotizing enterocolitis, and sepsis. For the achievement of practical and convenient detection of viable C. sakazakii, a simple and robust strategy based on the cascade signal amplification of RT-PCR triggered G-quadruplex DNAzyme catalyzed reaction was firstly used to develop an effective and sensitive DNAzyme electrochemical assay. Without viable C. sakazakii in the samples there are no RT-PCR and DNAzyme products, which can cause a weak electrochemical response. Once viable C. sakazakii exists in the samples, an obvious enhancement of the electrochemical response can be achieved after the target signal is amplified by RT-PCR and the resulting DNAzyme, which catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 with the assistance of the cofactor hemin. Our novel assay can be performed in a range of 2.4 × 107 CFU mL-1 to 3.84 × 104 CFU mL-1 (R2 = 0.9863), with a detection limit of 5.01 × 102 CFU mL-1. Through the assay of 15 real samples, electrochemical detection assay provided the same results as conventional detection methods. Therefore, detection of viable C. sakazakii based on G-quadruplex DNAzyme electrochemical assay with RT-PCR demonstrates the significant advantages of high sensitivity, low cost and simple manipulation over existing approaches and offers an opportunity for potential application in pathogen detection.

A label-free fluorescent enhancement nanosensor for ultrasensitive and highly selective detection of miRNA-378 through signal synergy amplification.

For early detection and diagnosis of gastric cancer, a novel, highly sensitive detection of microRNA-378 was developed through rolling circle amplification and DNA-templated silver nanoclusters as a lable-free fluorescent probe. DNA-templated fluorescent silver nanoclusters (DNA/AgNCs) to make target signal cascade amplification were prepared and identified through their fluorescent spectrum. MiRNA-378 trigged rolling circle amplification to obtain the complementary sequence (cDNA) to combine with two DNA/AgNCs in the middle of a sandwich structure to induce the new fluorescent signal at a new wavelength. In the presence of microRNA-378, a large amount of RCA product cDNAs were hybridized with DNA/AgNCs as the fluorescence nanocluster beacon, resulting in fluorescence enhanced "turn-on" phenomenon. This study indicated that amplified fluorescence detection of microRNA-378 is a stable, low-cost, highly specific, and ultra-low as 1.07 fM detectability. The proposed approach of signal synergetic amplification facilitating the fluorescence detection microRNA shows great potential for potentially clinically diagnosis.

Development of DNAzyme-based PCR signal cascade amplification for visual detection of Listeria monocytogenes in food.

Listeria monocytogenes is an important foodborne pathogen, and it can cause severe diseases. Rapid detection of L. monocytogenes is crucial to control this pathogen. A simple and robust strategy based on the cascade of PCR and G-quadruplex DNAzyme catalyzed reaction was used to detect L. monocytogenes. In the presence of hemin and the aptamer formed during PCR, the catalytic horseradish peroxidase-mimicking G-quadruplex DNAzymes allow the colorimetric responses of target DNA from L. monocytogenes. This assay can detect genomic DNA of L. monocytogenes specifically with as low as 50 pg/reaction with the naked eye. Through 20 pork samples assay, visual detection assay had the same results as conventional detection methods, and had a good performance. This is a powerful demonstration of the ability of G-quadruplex DNAzyme to be used for PCR-based assay with significant advantages of high sensitivity, low cost and simple manipulation over existing approaches and offers the opportunity for application in pathogen detection.

Visual diagnostic of Helicobacter pylori based on a cascade amplification of PCR and G-quadruplex DNAzyme as a color label.

Helicobacter pylori is a spiral-shaped, Gram-negative, microaerophilic and fastidious bacterium. It is the main cause of chronic gastritis as well as gastric and duodenal ulcers. The diagnosis of H. pylori infection is significant for the selection of therapy and for the follow up of eradication success. A simple and robust strategy based on the cascade of PCR and DNAzyme catalyzed reaction was utilized to detect H. pylori. The design of the primer pair would enable PCR to synthesize aptamer of DNAzyme at the 3' end of PCR products. G-quadruplex DNAzyme as a color label can exhibit peroxidase-like activity to amplify the specific signal and demonstrate a colorimetric signal to indicate the diagnostic result. This assay can detect genomic DNA of H. pylori specifically with as low as 100 pg/reaction by the naked eye. This is a powerful demonstration of G-quadruplex DNAzyme to be used for PCR-based assay with significant advantages of high sensitivity, low cost and simple manipulation over existing approaches and offers the potential opportunity for clinical application.

Colorimetric detection of Cucumber green mottle mosaic virus using unmodified gold nanoparticles as colorimetric probes.

Cucumber green mottle mosaic virus (CGMMV)causes a severe mosaic symptom of watermelon and cucumber, and can be transmitted via infected cucumber seeds, leaves and soil. It remains a challenge to detect this virus to prevent its introduction and infection and spread in fields. For this purpose, a simple and sensitive label-free colorimetric detection method for CGMMV has been developed with unmodified gold nanoparticles (AuNPs) as colorimetric probes. The method is based on the finding that the presence of RT-PCR target products of CGMMV and species-specific probes results in color change of AuNPs from red to blue after NaCl induction. Normally, species-specific probes attach to the surface of AuNPs and thereby increasing their resistance to NaCl-induced aggregation. The concentration of sodium, probes in the reaction system and evaluation of specificity and sensitivity of a novel assay, visual detection of Cucumber green mottle mosaic virus using unmodified AuNPs has been carried out with simple preparation of samples in our study. Through this assay, as low as 30pg/µL of CGMMV RNA was thus detected visually, by the naked eye, without the need for any sophisticated, expensive instrumentation and biochemical reagents. The specificity was 100% and exhibited good reproducibility in our assays. The results note that this assay is highly species-specific, simple, low-cost, and visual for easy detection of CGMMV in plant tissues. Therefore, visual assay is a potentially useful tool for middle or small-scales corporations and entry-exit inspection and quarantine bureau to detect CGMMV in cucumber seeds or plant tissues.

Influence of Ship Emissions on Urban Air Quality: A Comprehensive Study Using Highly Time-Resolved Online Measurements and Numerical Simulation in Shanghai.

Shanghai has become an international shipping center in the world. In this study, the multiyear measurements and the high resolution air quality model with hourly ship emission inventory were combined to determine the influence of ship emissions on urban Shanghai. The aerosol time-of-flight mass spectrometer (ATOFMS) measurements were carried out at an urban site from April 2009 to January 2013. During the entire sampling time, most of the half-hourly averaged number fractions of primary ship emitted particles varied between 1.0-10.0%. However, the number fraction could reach up to 50% during the ship plume cases. Ship-plume-influenced periods usually occurred in spring and summer. The simulation of Weather Research and Forecasting/Community Multiscale Air Quality model (WRF/CMAQ) with hourly ship emission inventory provided the highly time-resolved concentrations of ship-related air pollutants during a ship plume case. It showed ships could contribute 20-30% (2-7 µg/m3) of the total PM2.5 within tens of kilometers of coastal and riverside Shanghai during ship-plume-influenced periods. Our results showed that ship emissions have substantial contribution to the air pollution in urban Shanghai. The control measures of ship emission should be taken considering its negative environment and human health effects.

Optimization of fermentation conditions of pectin production from Aspergillus terreus and its partial characterization.

Figures of persimmons for the world's top ten persimmon producing countries are about 4000,000 tons in 2011 and are increasing every year according to FAO statistics. However, there is not any report on pectin production by microbial with persimmon peel as the source. Optimization of fermentation conditions of pectin production from Aspergillus terreus in submerged culture and partial characterization of pectin were carried out in the work. An optimum fermentation condition for pectin production was obtained through a central composite rotatable design in response surface methodology as follows: fermentation time, 30.09 h, temperature, 25.00 °C and the initial pH in the fermentation medium, 6.90, respectively and the pectin yield reached the maximal value 0.449 g/g. Persimmon peel pectin had highly methoxylated (62.51%), high galacturonic acid content (82.28%) than citrus pectin, and was classified as the highly methoxylated pectin, the results indicated that persimmon peel had potential good resources for pectin production. The investigation can make it available to utilize persimmon peel to produce high methoxyl pectin for food industry, pharmacy and cosmetic manufacture.

Genome-wide identification, characterization and expression analysis of the auxin response factor gene family in Vitis vinifera.

KEY MESSAGE: Our study has identified and analyzed the VvARF gene family that may be associated with the development of grape berry and other tissues. Auxin response factors (ARFs) are transcription factors that regulate the expression of auxin responsive genes through specific binding to auxin response elements (AuxREs). The ARF genes are represented by a large multigene family in plants. Until now, many ARF families have been characterized based on genome resources. However, there is no specialized research about ARF genes in grapevine (Vitis vinifera). In this study, a comprehensive bioinformatics analysis of the grapevine ARF gene family is presented, including chromosomal locations, phylogenetic relationships, gene structures, conserved domains and expression profiles. Nineteen VvARF genes were identified and categorized into four groups (Classes 1, 2, 3 and 4). Most of VvARF proteins contain B3, AUX_RESP and AUX_IAA domains. The VvARF genes were widely expressed in a range of grape tissues, and fruit had higher transcript levels for most VvARFs detected in the EST sources. Furthermore, analysis of expression profiles indicated some VvARF genes may play important roles in the regulation of grape berry maturation processes. This study which provided basic genomic information for the grapevine ARF gene family will be useful in selecting candidate genes related to tissue development in grapevine and pave the way for further functional verification of these VvARF genes.

Protective effects of baicalin against bromocriptine induced abortion in mice.

The Chinese herbal medicine Huang Qin (Radix Scutellariae) had been used for restless fetus for hundreds of years in China, however, little attention had been given to the components of the herb, specifically its ability to exert abortion-preventing effects at the maternal fatal interface. The present study was carried out to investigate the protective effects of baicalin and the possible mechanisms on pregnancies. Baicalin (at 10, 20, and 50 mg/kg BW respectively) was gavaged to bromocriptine-treated mice from gestation day (GD) 1 through GD 7. Abortion rates were calculated and the changes of interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and progesterone were assayed on different gestation days. Results showed that the embryonic death rates were significantly decreased in groups supplemented with 20 or 50 mg/kg BW of baicalin, accompanied with reduced IFN-gamma and enhanced progesterone contents. Moreover, the highest levels of IFN-gamma appeared on GD 5 both in the control and in baicalin treated groups. It is concluded that baicalin can exert an anti-abortive effect by cutting down the production of IFN-gamma and elevating the levels of progesterone in a dose dependent manner and IFN-gamma is involved in an inflammatory reaction which is beneficial for a successful implantation.

Species-specific diagnostic marker for rapid identification of Staphylococcus aureus.

Staphylococcus aureus is a common bacterial pathogen that has emerged as an increasingly important health concern. Following the recent publication of the genome sequences of 9 S. aureus strains (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi), a gene of S. aureus that relates signal transduction as a target for rapid detection and identification of the pathogen has been investigated. By sequence analysis of S. aureus signal transduction genes from the complete genome of S. aureus ATCC N315 and their comparison with other DNA sequences using GenBank BLAST searches, we identified a unique gene, vicK. Polymerase chain reaction primers (vicK1 and vicK2) derived from this gene allowed amplification of a 289-bp DNA fragment only from S. aureus and not from other Staphylococcus species and other common bacteria tested. Besides offering an additional target for specific confirmation of S. aureus, further analysis of the signal transduction gene vicK and its related protein product may lead to new insights into the molecular mechanisms of S. aureus maintenance and pathogenicity.