A rapid and high-throughput system for the detection of transgenic products based on LAMP-CRISPR-Cas12a.

With the increasing acreage of genetically modified crops worldwide, rapid and efficient detection technologies have become very important for the regulation and screening of GM organisms. We constructed a method based on loop-mediated isothermal amplification (LAMP), CRISPR-Cas12a and lateral flow assay (LAMP-CRISPR-Cas12a-LFA). It is an intuitive, sensitive and specific fluorescence detection and test strip system to detect CP4-EPSPS and Cry1Ab/Ac genes in field screening. The LAMP-CRISPR-Cas12a-LFA method has a limit of detection (LOD) of 100 copies based on lateral flow test strips after optimization of the conditions with screened specific primers, and the entire detection process can be completed within 1 h at 61 °C. The system was used to evaluate field test samples and showed high reproducibility after testing products containing CP4-EPSPS and Cry1Ab/Ac genes, and both were detectable. The LAMP-CRISPR-Cas12a-LFA method established in this paper functions as a rapid field detection method. It requires only one portable thermostatic instrument, which renders it compatible with the rapid detection of field samples and useable at experimental workstations, in law enforcement field work, and in local inspection and quarantine departments.

A Study on Vascular Regulators as Early Biomarkers of Hand Arm Vibration Syndrome.

OBJECTIVES: Purposes of this study are to study the changes in expression of vascular regulators after vibration exposure and during the onset of vibration-induced white finger (VWF) and to screen for vascular regulatory factors that could be used as early biomarkers of HAVS. METHODS: Using judgmental sampling from a Chinese factory, workers with VWF and hand-transmitted vibration exposure but without VWF were selected for research. Blood samples were taken from all subjects, and the levels of nine of the vascular regulators were measured using ELISA. RESULTS: Receiver operating characteristic curve analysis was performed on nine vascular regulators to assess their diagnostic sensitivity for VWF with the following area under the curve results: PGI 2 = 0.861, ANP = 0.840. CONCLUSIONS: Collectively, PGI 2 and ANP exhibited the most potential for the early diagnosis of HAVS.

Sulfate-reducing bacteria loaded in hydrogel as a long-lasting H2S factory for tumor therapy.

The continuous supply of hydrogen sulfide (H2S) gas at high concentrations to tumors is considered a promising and safe strategy for tumor therapy. However, the absence of a durable and cost-effective H2S-producing donor hampers its extensive application. Sulfate-reducing bacteria (SRB) can serve as an excellent H2S factory due to their ability to metabolize sulfate into H2S. Herein, a novel injectable chondroitin sulfate (ChS) hydrogel loaded with SRB (SRB@ChS Gel) is proposed to sustainably produce H2S in tumor tissues to overcome the limitations of current H2S gas therapy. In vitro, the ChS Gel not only supports the growth of encapsulated SRB, but also supplies a sulfate source to the SRB to produce high concentrations of H2S for at least 7 days, resulting in mitochondrial damage and immunogenic cell death. Once injected into tumor tissue, the SRB@ChS Gel can constantly produce H2S for >5 days, significantly inhibiting tumor growth. Furthermore, such treatment activates systemic anti-tumor immune responses, suppresses the growth of distant and recurrent tumors, as well as lung metastases, meanwhile with negligible side effects. Therefore, the injectable SRB@ChS Gel, as a safe and long-term, self-sustained H2S-generating factory, provides a promising strategy for anti-tumor therapy.

A rapid and high-throughput Helicobacter pylori RPA-CRISPR/Cas12a-based nucleic acid detection system.

BACKGROUND: Helicobacter pylori lives in the human stomach and causes gastric cancer and other gastric diseases. The development of molecular technology has facilitated low-cost, rapid, and high-throughput detection of H. pylori. MATERIALS AND METHODS: The combination of isothermal recombinase polymerase amplification (RPA) and CRISPR-Cas12a was used for early diagnosis and monitoring of H. pylori in clinical settings. The UreB genes from 242 H. pylori strains were subjected to cluster analysis, and we designed corresponding RPA primers and screened 2 sets of CRISPR-derived RNAs (crRNAs) for accurate H. pylori recognition. We then performed specificity and sensitivity validation of seven strains using this RPA-CRISPR/Cas12a method. In addition, the cut-off values of this RPA-CRISPR/Cas12a method based on fluorescence values (i.e., RPA-CRISPR/Cas12a-FT) were determined by comparison with quantitative PCR (qPCR), and further experiments comparing different methods were performed using clinical samples. RESULTS: We developed a rapid detection system based on the combination of RPA and CRISPR-Cas12a, which was applied to the early diagnosis and monitoring of H. pylori in clinical settings. The RPA-CRISPR/Cas12a system was used to detect the UreB gene. We found that the limit of detection (LOD) for the CRISPR/Cas12a method based on the lateral flow dipstick result (i.e., CRISPR/Cas12a-LFD) was 100 copies, the cut-off value was 1.4; and for CRISPR/Cas12a-FT the LOD was 50 copies. This system was used to assess clinical samples and showed high reproducibility with proof-of-concept sensitivity, and the whole detection process was completed within 40 min. CONCLUSION: As a diagnostic method that can detect the UreB gene of H. pylori in gastric tissue samples rapidly, sensitively, visually, and in a high throughput manner, our method provides a new diagnostic option for clinicians. This system is ideal for hospitals or testing sites with limited medical resources.

Near-Infrared Nano-Optogenetic Activation of Cancer Immunotherapy via Engineered Bacteria.

Certain anaerobic microbes with the capability to colonize the tumor microenvironment tend to express the heterologous gene in a sustainable manner, which will inevitably compromise the therapeutic efficacy and induce off-tumor toxicity in vivo. To improve the therapeutic precision and controllability of bacteria-based therapeutics, Escherichia coli Nissle 1917 (EcN), engineered to sense blue light and release the encoded flagellin B (flaB), is conjugated with lanthanide upconversion nanoparticles (UCNPs) for near-infrared (NIR) nano-optogenetic cancer immunotherapy. Upon 808 nm photoirradiation, UCNPs emit at the blue region to photoactivate the EcN for secretion of flaB, which subsequently binds to Toll-like receptor 5 expressed on the membrane of macrophages for activating immune response via MyD88-dependent signal pathway. Such synergism leads to significant tumor regression in different tumor models and metastatic tumors with negligible side effects. These studies based on the NIR nano-optogenetic platform highlight the rational of leveraging the optogenetic tools combined with natural propensity of certain bacteria for cancer immunotherapy.

Rapid detection of genetically modified products based on CRISPR-Cas12a combined with recombinase polymerase amplification.

With the large-scale planting of genetically modified (GM) crops, consumers were more aware of biosafety. Onsite rapid diagnostic methods were advantageous to the regulation of GM products. In this study, a rapid, sensitive and portable detection method based on recombinase polymerase amplification were proposed based on RPA reaction and Cas12a cleavage reaction for GM ingredients, named RPA-Cas12a-GM. The results would be displayed by fluorescence signal (FS) and visual bands of lateral flow strip (LFS). RPA-Cas12a-GM method could be completed within 45 min, and the detection limit was as low as 45 copies/µL of the standard plasmid containing CP4-EPSPS gene and Cry1Ab/Ac gene. Furthermore, the detection coincidence rate of RPA-Cas12a-GM method was 100%. In conclusion, the proposed RPA-Cas12a-GM method based FS and LFS were sensitive, specific, rapid and visible for diagnosis of CP4-EPSPS gene and Cry1Ab/Ac gene without complex equipment, which provides technical support for the regulation of GM products in the field.

Electroencephalograph-Based Emotion Recognition Using Brain Connectivity Feature and Domain Adaptive Residual Convolution Model.

In electroencephalograph (EEG) emotion recognition research, obtaining high-level emotional features with more discriminative information has become the key to improving the classification performance. This study proposes a new end-to-end emotion recognition method based on brain connectivity (BC) features and domain adaptive residual convolutional network (short for BC-DA-RCNN), which could effectively extract the spatial connectivity information related to the emotional state of the human brain and introduce domain adaptation to achieve accurate emotion recognition within and across the subject's EEG signals. The BC information is represented by the global brain network connectivity matrix. The DA-RCNN is used to extract high-level emotional features between different dimensions of EEG signals, reduce the domain offset between different subjects, and strengthen the common features between different subjects. The experimental results on the large public DEAP data set show that the accuracy of the subject-dependent and subject-independent binary emotion classification in valence reaches 95.15 and 88.28%, respectively, which outperforms all the benchmark methods. The proposed method is proven to have lower complexity, better generalization ability, and domain robustness that help to lay a solid foundation for the development of high-performance affective brain-computer interface applications.

Dietary titanium dioxide particles (E171) promote diet-induced atherosclerosis through reprogramming gut microbiota-mediated choline metabolism in APOE-/- mice.

Food-grade titanium dioxide (E171) has been reported to induce changes in some intestinal metabolites related to development of atherosclerosis (AS). However, little is known about the effects of chronic dietary intake of E171 on AS development, particularly in AS-prone populations with high-choline western diet (HCD). Herein, we disclosed that E171 obviously exacerbated HCD-induced AS through increasing production of trimethylamine (TMA) and pro-atherogenic trimethylamine-N-oxide (TMAO) via remodeling gut microbiota structure in APOE-/- mice. Oral administration of 40 mg/kg E171 daily for 4 months significantly increased the atherosclerotic lesion area, especially in the HCD group. Mechanistic studies revealed that E171 induced much more TMAO production by increasing the gut microbial expression of choline TMA lyases (CutC/D), which converted dietary choline to TMA by a glycyl radical reaction. The 16S rDNA sequencing analysis demonstrated that bacterial strains expressing CutC/D were enriched by E171 in HCD-fed mice. In contrast, gut microbiota depletion eliminated the impact of E171 on choline/TMA/TMAO pathway and AS progression, indicating gut flora shifts were responsible for the exacerbation effects of E171 ingestion on HCD-induced AS. All the results emphasized the alarming role of E171 on AS progression and stated the importance of reevaluating the impact of food additives on the development of chronic diseases.