Four thermostatic steps: A novel CRISPR-Cas12-based system for the rapid at-home detection of respiratory pathogens.

The outbreak of coronavirus disease 2019 (COVID-19) in 2019 has severely damaged the world's economy and public health and made people pay more attention to respiratory infectious diseases. However, traditional quantitative real-time polymerase chain reaction (qRT-PCR) nucleic acid detection kits require RNA extraction, reverse transcription, and amplification, as well as the support of large-scale equipment to enrich and purify nucleic acids and precise temperature control. Therefore, novel, fast, convenient, sensitive and specific detection methods are urgently being developed and moving to proof of concept test. In this study, we developed a new nucleic acid detection system, referred to as 4 Thermostatic steps (4TS), which innovatively allows all the detection processes to be completed in a constant temperature device, which performs extraction, amplification, cutting of targets, and detection within 40 min. The assay can specifically and sensitively detect five respiratory pathogens, namely SARS-CoV-2, Mycoplasma felis (MF), Chlamydia felis (CF), Feline calicivirus (FCV), and Feline herpes virus (FHV). In addition, a cost-effective and practical small-scale reaction device was designed and developed to maintain stable reaction conditions. The results of the detection of the five viruses show that the sensitivity of the system is greater than 94%, and specificity is 100%. The 4TS system does not require complex equipment, which makes it convenient and fast to operate, and allows immediate testing for suspected infectious agents at home or in small clinics. Therefore, the assay system has diagnostic value and significant potential for further reducing the cost of early screening of infectious diseases and expanding its application. KEY POINTS: • The 4TS system enables the accurate and specific detection of nucleic acid of pathogens at 37 °C in four simple steps, and the whole process only takes 40 min. •A simple alkali solution can be used to extract nucleic acid. • A small portable device simple to operate is developed for home diagnosis and detection of respiratory pathogens.

Task-Wise Sampling Convolutions for Arbitrary-Oriented Object Detection in Aerial Images.

Arbitrary-oriented object detection (AOOD) has been widely applied to locate and classify objects with diverse orientations in remote sensing images. However, the inconsistent features for the localization and classification tasks in AOOD models may lead to ambiguity and low-quality object predictions, which constrains the detection performance. In this article, an AOOD method called task-wise sampling convolutions (TS-Conv) is proposed. TS-Conv adaptively samples task-wise features from respective sensitive regions and maps these features together in alignment to guide a dynamic label assignment for better predictions. Specifically, sampling positions of the localization convolution in TS-Conv are supervised by the oriented bounding box (OBB) prediction associated with spatial coordinates, while sampling positions and convolutional kernel of the classification convolution are designed to be adaptively adjusted according to different orientations for improving the orientation robustness of features. Furthermore, a dynamic task-consistent-aware label assignment (DTLA) strategy is developed to select optimal candidate positions and assign labels dynamically according to ranked task-aware scores obtained from TS-Conv. Extensive experiments on several public datasets covering multiple scenes, multimodal images, and multiple categories of objects demonstrate the effectiveness, scalability, and superior performance of the proposed TS-Conv.

A General Gaussian Heatmap Label Assignment for Arbitrary-Oriented Object Detection.

Recently, many arbitrary-oriented object detection (AOOD) methods have been proposed and attracted widespread attention in many fields. However, most of them are based on anchor-boxes or standard Gaussian heatmaps. Such label assignment strategy may not only fail to reflect the shape and direction characteristics of arbitrary-oriented objects, but also have high parameter-tuning efforts. In this paper, a novel AOOD method called General Gaussian Heatmap Label Assignment (GGHL) is proposed. Specifically, an anchor-free object-adaptation label assignment (OLA) strategy is presented to define the positive candidates based on two-dimensional (2D) oriented Gaussian heatmaps, which reflect the shape and direction features of arbitrary-oriented objects. Based on OLA, an oriented-bounding-box (OBB) representation component (ORC) is developed to indicate OBBs and adjust the Gaussian center prior weights to fit the characteristics of different objects adaptively through neural network learning. Moreover, a joint-optimization loss (JOL) with area normalization and dynamic confidence weighting is designed to refine the misalign optimal results of different subtasks. Extensive experiments on public datasets demonstrate that the proposed GGHL improves the AOOD performance with low parameter-tuning and time costs. Furthermore, it is generally applicable to most AOOD methods to improve their performance including lightweight models on embedded platforms.

miR-146b-5p activation of hepatic stellate cells contributes to the progression of fibrosis by directly targeting HIPK1.

The present study aimed to explore the biological functions of microRNA (miR)-146b-5p and homeodomain interacting protein kinase 1 (HIPK1) in the progression of hepatic fibrosis (HF) and to identify the underlying mechanism. A rat HF model was established by administering a subcutaneous injection of carbon tetrachloride (CCl4). Relative levels of miR-146b-5p and HIPK1 in fibrotic rat liver tissues and the rat hepatic stellate cell (HSC) line HSC-T6 were measured by quantitative reverse transcription PCR, western blotting and immunohistochemistry. Following activation of HSC-T6 cells by lipopolysaccharide (LPS) induction, cell viability was examined by MTT assay. Transfection of miR-146b-5p mimic or inhibitor into HSC-T6 cells was performed, with the aim to identify the influence of miR-146b-5p on HSC-T6 cell behavior. The targeting relationship between miR-146b-5p and HIPK1 was predicted by TargetScan 7.2 and StarBase 3.0 and it was later verified by a dual-luciferase reporter assay. Through lentivirus transfection, the biological function of HIPK1 in regulating the progression of HF and the underlying mechanism were investigated. The results showed that miR-146b-5p was upregulated in liver tissues of rats with HF and activated HSC-T6 cells, while HIPK1 was downregulated in liver tissues of rats with HF and activated HSC-T6 cells. miR-146b-5p was able to upregulate the activation markers of LPS-induced HSC-T6 cells, upregulate COL1A1 and TGF-ß, increase cell viability and contribute to fibrosis progression. HIPK1 was validated as the direct target of miR-146b-5p and its overexpression could effectively reduce the effect of miR-146b-5p in contribution to the progression of HF. In conclusion, miR-146b-5p was significantly upregulated during the progression of HF. By targeting and downregulating HIPK1, miR-146b-5p could significantly activate HSCs, upregulate COL1A1 and TGF-ß and contribute to fibrosis progression. miR-146b-5p is a potential biomarker and therapeutic target for HF.

CRISPR-Cas13: A new technology for the rapid detection of pathogenic microorganisms.

Pathogenic microorganisms have major impacts on human lives. Rapid and sensitive diagnostic tools are urgently needed to facilitate the early treatment of microbial infections and the effective control of microbial transmission. CRISPR-Cas13 employs programmable RNA to produce a sensitive and specific method with high base resolution and thus to provide a novel tool for the rapid detection of microorganisms. The review aims to provide insights to spur further development by summarizing the characteristics of effectors of the CRISPR-Cas13 system and by describing the latest research into its application in the rapid detection of pathogenic microorganisms in combination with nucleic acid extraction, isothermal amplification, and product detection.

[Studies on performance standard for hemolytic toxins in harmful bloom algae].

OBJECTIVE: To explore a performance standard for hemolytic toxins in harmful bloom algae. METHODS: Using Chattonella marina as hemolytic substances producing organism, methods and conditions were compared and optimized including cell breakage, distillation temperature, blood origin and storage of algal pellets in extraction and activity determination of hemolytic toxins. RESULTS: The hemolytic activity of C. marina broken by supersonic method was 288.23 HU/L, higher than that by freezing--thawing method (94.89 HU/L), suggesting that supersonic method could be more optimal to break microalgal cells. When the supersonic treatment times were 5, 10, 20 and 30 min, the hemolytic activities were 80.57, 157.45, 288.23 and 279.17 HU/L, respectively, indicating that 20 min of supersonic treatment was suitable. When the distillation temperature were 40, 60 and 80 degrees C, the hemolytic activities were 288.23, 124.97 and 120.68 HU/L, respectively, meaning that high distillation temperature in extraction of hemolytic substances lowed the hemolytic activities of samples. Bloods from various animals such as human, fish, rat and rabbit exhibited different sensitivity to the hemolytic toxins, of which rabbit erythrocyte was the most sensitive. The hemolytic activities to human, fish, rat and rabbit were 244.98, 288.23, 266.35 and 195.47HU/L, respectively. The storage of algal pellets for 3 days at the temperature of 0 degrees C did not reveal a significant loss in hemolytic activity, while significant losses were observed at the temperature of 20 degrees C or -20 degrees C only after one day. CONCLUSION: Supersonic method could be more optimal to break cell in comparison with freeze-thaw method. Optimal conditions for broken algal cells by supersonic method were 200 W for 20 min at the temperature of 4 degrees C. The distillation temperature in extraction of hemolytic substances should be maintained under the temperature of 40 degrees C. The rabbit erythrocyte could be the most optimal blood to detect hemolytic activity due to its high sensitivity. The algal pellets could be kept at the temperature of 0 degrees C for 3 days before determination of activity.