Amino acid mutation of succinate dehydrogenase complex induced resistance to benzovindiflupyr in Magnaporthe oryzae.

Magnaporthe oryzae is a rice blast pathogen that seriously threatens rice yield. Benzovindiflupyr is a succinate dehydrogenase inhibitor (SDHI) fungicide that effectively controls many crop diseases. Benzovindiflupyr has a strong inhibitory effect on M. oryzae; however, control of rice blast by benzovindiflupyr and risk of resistance to benzovindiflupyr are not well studied in this pathogen. In this study, six benzovindiflupyr-resistant strains were obtained by domestication induced in the laboratory. The MoSdhBH245D mutation was the cause of M. oryzae resistance to benzovindiflupyr, which was verified through succinate dehydrogenase (SDH) activity assays, molecular docking, and site-specific mutations. Survival fitness analysis showed no significant difference between the benzovindiflupyr-resistant and parent strains. Positive cross-resistance to benzovindiflupyr and other SDHIs and negative cross-resistance to azoxystrobin were observed. Therefore, the risk of benzovindiflupyr resistance in M. oryzae might be medium to high. It should be combined with other classes of fungicides (tebuconazole and azoxystrobin) to slow the development of resistance.

CRISPR/Cas12a-coupled multiplexed strand displacement amplification for miRNA155 one-tube detection: via a dual-cavity PCR tube.

A CRISPR/Cas12a-coupled multiplexed strand displacement amplification (CMSDA) for the detection of miR155 has been developed. Non-specific amplification was avoided by designing a single-stranded DNA template with a hairpin structure. The detection target miR155 was used as a primer to initiate a multiple-strand displacement reaction to produce abundant ssDNA. ssDNA was recognized by the Cas12a/CrRNA binary complex, activating the trans-cleaving activity of Cas12a. The multiple-strand displacement reaction is more efficiently detected compared with a single-strand displacement reaction. The detection range is from 250 pM to 1 nM, and the limit of the detection is 6.5 pM. The proposed method showed a good applicability in complex serum environments, indicating that the method has a broad prospect for disease detection and clinical application. In addition, we designed a dual-cavity PCR tube, which realized one-tube detection of miRNA155 and avoided open-cap contamination.

Hypoxia-activated XBP1s recruits HDAC2-EZH2 to engage epigenetic suppression of ΔNp63α expression and promote breast cancer metastasis independent of HIF1α.

Hypoxia is a hallmark of cancer development. However, the molecular mechanisms by which hypoxia promotes tumor metastasis are not fully understood. In this study, we demonstrate that hypoxia promotes breast cancer metastasis through suppression of ΔNp63α in a HIF1α-independent manner. We show that hypoxia-activated XBP1s forms a stable repressor protein complex with HDAC2 and EZH2 to suppress ΔNp63α transcription. Notably, H3K27ac is predominantly occupied on the ΔNp63 promoter under normoxia, while H3K27me3 on the promoter under hypoxia. We show that XBP1s binds to the ΔNp63 promoter to recruit HDAC2 and EZH2 in facilitating the switch of H3K27ac to H3K27me3. Pharmacological inhibition or the knockdown of either HDAC2 or EZH2 leads to increased H3K27ac, accompanied by the reduced H3K27me3 and restoration of ΔNp63α expression suppressed by hypoxia, resulting in inhibition of cell migration. Furthermore, the pharmacological inhibition of IRE1α, but not HIF1α, upregulates ΔNp63α expression in vitro and inhibits tumor metastasis in vivo. Clinical analyses reveal that reduced p63 expression is correlated with the elevated expression of XBP1, HDAC2, or EZH2, and is associated with poor overall survival in human breast cancer patients. Together, these results indicate that hypoxia-activated XBP1s modulates the epigenetic program in suppression of ΔNp63α to promote breast cancer metastasis independent of HIF1α and provides a molecular basis for targeting the XBP1s/HDAC2/EZH2-ΔNp63α axis as a putative strategy in the treatment of breast cancer metastasis.

Real time imaging of cell-permeable nanoreactor with SERS for insight into cellular processes.

Intracellular glucose detection is crucial due to its pivotal role in metabolism and various physiological processes. Precise glucose monitoring holds significance in diabetes management, metabolic studies, and biotechnological applications. In this study, we developed an innovative and expedient cell-permeable nanoreactor for intracellular glucose based on surface-enhanced Raman scattering (SERS). The nanoreactor was designed with gold nanoparticles (AuNPs), which were engineered with glucose oxide (GOx) and a H2O2-responsive Raman reporter 2-mercaptohydroquinone (2-MHQ). The interaction between 2-MHQ and H2O2 generated by glucose and GOx could simultaneously induce the appearance in the peak at 985 cm-1. Our results showed excellent performance in detecting glucose within the concentration range from 0.1 µM to 10 mM, with a low detection limitation of 14.72 nM. In addition, the glucose distribution in single HeLa cells was evaluated by real time SERS mapping. By combining noble metal particles and natural oxidases, the nanoreactor possesses both Raman activity and enzymatic functionality, thus enables sensitive glucose detection and facilitates imaging at a single cell level, which offers an insightful monitoring of cellular processes.

Rapid, portable, and sensitive detection of CaMV35S by RPA-CRISPR/Cas12a-G4 colorimetric assays with high accuracy deep learning object recognition and classification.

Fast, sensitive, and portable detection of genetic modification contributes to agricultural security and food safety. Here, we developed RPA-CRISPR/Cas12a-G-quadruplex colorimetric assays that can combine with intelligent recognition by deep learning algorithms to achieve sensitive, rapid, and portable detection of the CaMV35S promoter. When the crRNA-Cas12a complex recognizes the RPA amplification product, Cas12 cleaves the G-quadruplex, causing the G4-Hemin complex to lose its peroxide mimetic enzyme function and be unable to catalyze the conversion of ABTS2- to ABTS, allowing CaMV35S concentration to be determined based on ABTS absorbance. By utilizing the RPA-CRISPR/Cas12a-G4 assay, we achieved a CaMV35S limit of detection down to 10 aM and a 0.01 % genetic modification sample in 45 min. Deep learning algorithms are designed for highly accurate classification of color results. Yolov5 objective finding and Resnet classification algorithms have been trained to identify trace (0.01 %) CaMV35S more accurately than naked eye colorimetry. We also coupled deep learning algorithms with a smartphone app to achieve portable and rapid photo identification. Overall, our findings enable low cost ($0.43), high accuracy, and intelligent detection of the CaMV35S promoter.