MicroRNA164 Affects Plant Responses to UV Radiation in Perennial Ryegrass.

Increasing the ultraviolet radiation (UV) level, particularly UV-B due to damage to the stratospheric ozone layer by human activities, has huge negative effects on plant and animal metabolism. As a widely grown cool-season forage grass and turfgrass in the world, perennial ryegrass (Lolium perenne) is UV-B-sensitive. To study the effects of miR164, a highly conserved microRNA in plants, on perennial ryegrass under UV stress, both OsmiR164a overexpression (OE164) and target mimicry (MIM164) transgenic perennial ryegrass plants were generated using agrobacterium-mediated transformation, and UV-B treatment (~600 µw cm-2) of 7 days was imposed. Morphological and physiological analysis showed that the miR164 gene affected perennial ryegrass UV tolerance negatively, demonstrated by the more scorching leaves, higher leaf electrolyte leakage, and lower relative water content in OE164 than the WT and MIM164 plants after UV stress. The increased UV sensitivity could be partially due to the reduction in antioxidative capacity and the accumulation of anthocyanins. This study indicated the potential of targeting miR164 and/or its targeted genes for the genetic manipulation of UV responses in forage grasses/turfgrasses; further research to reveal the molecular mechanism underlying how miR164 affects plant UV responses is needed.

Effects of antibiotic resistance genes on health risks of rivers in habitat of wild animals under human disturbance - based on analysis of antibiotic resistance genes and virulence factors in microbes of river sediments.

Studying the ecological risk of antibiotic resistance genes (ARGs) to wild animals from human disturbance (HD) is an important aspect of "One Health". The highest risk level of ARGs is reflected in pathogenic antibiotic-resistant bacteria (PARBs). Metagenomics was used to analyze the characteristics of PARBs in river sediments. Then, the total contribution of ARGs and virulence factors (VFs) were assessed to determine the health risk of PARBs to the rivers. Results showed that HD increased the diversity and total relative abundance of ARG groups, as well as increased the kinds of PARBs, their total relative abundance, and their gene numbers of ARGs and VFs. The total health risks of PARBs in wild habitat group (CK group), agriculture group (WA group), grazing group (WG group), and domestic sewage group (WS group) were 0.067 × 10-3, -1.55 × 10-3, 87.93 × 10-3, and 153.53 × 10-3, respectively. Grazing and domestic sewage increased the health risk of PARBs. However, agriculture did not increase the total health risk of the rivers, but agriculture also introduced new pathogenic mechanisms and increased the range of drug resistance. More serious was the increased transfer risk of ARGs in the PARBs from the rivers to wild animals under agriculture and grazing. If the ARGs in the PARBs are transferred from the rivers under HD to wild animals, then wild animals may face severe challenges of acquiring new pathogenic mechanisms and developing resistance to antibiotics. Further analysis showed that the total phosphorus (TP) and dissolved organic nitrogen (DON) were related to the risk of ARGs. Therefore, controlling human emissions of TP and DON could reduce the health risk of rivers.

Blocking miR396 activity by overexpression MIM396 improved switchgrass tiller number and biomass yield.

BACKGROUND: MicroRNA396 (miR396) plays an important role in the regulation of plant growth and development by repressing the expression level of its target growth-regulating factor (GRF) family genes. In our previous study, we found that overexpression of miR396 negatively regulated both tillering and biomass yield in switchgrass (Panicum virgatum L.). We, therefore, speculated that blocking the expression of miR396 could enhance switchgrass tillering and biomass yield. Here, we produced transgenic switchgrass plants overexpressing a target mimicry form of miR396 (MIM396) in wild type (WT) and Os-MIR319b overexpressing switchgrass plant (with higher enzymatic hydrolysis efficiency, but reduced tillering), in which the expression of miR396 was blocked. The phenotype and biological yields of these plants were analyzed. RESULTS: Blocking miR396 to improve its target PvGRFs expression in switchgrass improved the tiller number and dry weight of transgenic plants. Further morphological analysis revealed that MIM396 plants increased the number of aerial branches and basal tillers compared to those of wild-type plants. The enzymatic efficiency of MIM396 plants was reduced; however, the total sugar production per plant was still significantly higher than that of wild-type plants due to the increase in biomass. In addition, blocking miR396 in a transgenic switchgrass plant overexpressing Os-MIR319b (TG21-Ms) significantly increased the PvGRF1/3/5 expression level and tiller number and biomass yield. The miR156-target gene PvSPL4, playing a negative role in aerial and basal buds outgrowth, showed significant downregulated in MIM396 and TG21-Ms. Those results indicate that miR396-PvGRFs, through disrupting the PvSPL4 expression, are involved in miR319-PvPCFs in regulating tiller number, at least partly. CONCLUSIONS: MIM396 could be used as a molecular tool to improving tiller number and biomass yield in switchgrass wild type and miR319b transgenic plants. This finding may be applied to other graminaceous plants to regulate plant biological yield.

The Ser/Thr protein kinase FonKin4-poly(ADP-ribose) polymerase FonPARP1 phosphorylation cascade is required for the pathogenicity of watermelon fusarium wilt fungus Fusarium oxysporum f. sp. niveum.

Poly(ADP-ribosyl)ation (PARylation), catalyzed by poly(ADP-ribose) polymerases (PARPs) and hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG), is a kind of post-translational protein modification that is involved in various cellular processes in fungi, plants, and mammals. However, the function of PARPs in plant pathogenic fungi remains unknown. The present study investigated the roles and mechanisms of FonPARP1 in watermelon Fusarium wilt fungus Fusarium oxysporum f. sp. niveum (Fon). Fon has a single PARP FonPARP1 and one PARG FonPARG1. FonPARP1 is an active PARP and contributes to Fon pathogenicity through regulating its invasive growth within watermelon plants, while FonPARG1 is not required for Fon pathogenicity. A serine/threonine protein kinase, FonKin4, was identified as a FonPARP1-interacting partner by LC-MS/MS. FonKin4 is required for vegetative growth, conidiation, macroconidia morphology, abiotic stress response and pathogenicity of Fon. The S_TKc domain is sufficient for both enzyme activity and pathogenicity function of FonKin4 in Fon. FonKin4 phosphorylates FonPARP1 in vitro to enhance its poly(ADP-ribose) polymerase activity; however, FonPARP1 does not PARylate FonKin4. These results establish the FonKin4-FonPARP1 phosphorylation cascade that positively contributes to Fon pathogenicity. The present study highlights the importance of PARP-catalyzed protein PARylation in regulating the pathogenicity of Fon and other plant pathogenic fungi.

The effects of food provisioning on the gut microbiota community and antibiotic resistance genes of Yunnan snub-nosed monkey.

Yunnan snub-nosed monkeys (Rhinopithecus bieti) are the highest elevation lived non-human primate, and their survival has been threatened for decades. To promote their population growth, a reserve provides a typical monkey population with supplemental food. However, the influences of this food provisioning on their gut microbiota and antibiotic resistance genes (ARGs) were unknown. Therefore, we investigated the gut microbiota and ARGs of the food-provisioned monkey population compared with another wild foraging population. We found that food provisioning significantly increased the gut microbiota diversity and changed the community composition, particularly increased both the Firmicutes abundance and Firmicutes/Bacteroidetes ratio. Meanwhile, the food provisioning decreased the complex and stable gut microbiota network. KEGG functions were also influenced by food provisioning, with wild foraging monkeys showing higher functions of metabolism and genetic information processing, especially the carbohydrate metabolism, while food-provisioned monkeys exhibited increased environmental information processing, cellular processes, and organismal systems, including valine, leucine, and isoleucine degradation. In addition, food provisioning increased the abundance of ARGs in the gut microbiota, with most increasing the abundance of bacA gene and changing the correlations between specific ARGs and bacterial phyla in each population. Our study highlights that even food provisioning could promote wildlife nutrient intake, and it is necessary to pay attention to the increased ARGs and potential effects on gut microbiota stability and functions for this human conservation measure.

Toward a Comprehensive Understanding of the Anomalously Small Contact Angle of Surface Nanobubbles.

Experimental studies have demonstrated that the gas phase contact angle (CA) of a surface nanobubble (SNB) is much smaller than that of a macroscopic gas bubble. This reduced CA plays a crucial role in prolonging the lifetime of SNBs by lowering the bubble pressure and preventing gas molecules from dissolving in the surrounding liquids. Despite extensive efforts to explain the anomalously small CA, a consensus about the underlying reasons is yet to be reached. In this study, we conducted experimental investigations to explore the influence of gas molecules adsorbed at the solid-liquid interface on the CA of SNBs created through the solvent exchange (SE) method and temperature difference (TD). Interestingly, no significant change is observed in the CA of SNBs on highly oriented pyrolytic graphite (HOPG) surfaces. Even for nanobubbles on micro/nano pancakes, the CA only exhibited a slight reduction compared to SNBs on bare HOPG surfaces. These findings suggest that gas adsorption at the immersed solid surface may not be the primary factor contributing to the small CA of the SNBs. Furthermore, the CA of SNBs formed on polystyrene (PS) and octadecyltrichlorosilane (OTS) substrates was also investigated, and a considerable increase in CA was observed. In addition, the effects of other factors including impurity, electric double layer (EDL) line tension, and pinning force upon the CA of SNBs were discussed, and a comprehensive model about multiple factors affecting the CA of SNBs was proposed, which is helpful for understanding the abnormally small CA and the stability of SNBs.

OsATL32 ubiquitinates the reactive oxygen species-producing OsRac5-OsRbohB module to suppress rice immunity.

Ubiquitination-mediated protein degradation is integral to plant immunity, with E3 ubiquitin ligases acting as key factors in this process. Here, we report the functions of OsATL32, a plasma membrane-localized Arabidopsis Tóxicos En Levadura (ATL)-type E3 ubiquitin ligase, in rice (Oryza sativa) immunity and its associated regulatory network. We found that the expression of OsATL32 is downregulated in both compatible and incompatible interactions between rice and the rice blast fungus Magnaporthe oryzae. The OsATL32 protein level declines in response to infection by a compatible M. oryzae strain or to chitin treatment. OsATL32 negatively regulates rice resistance to blast and bacterial leaf blight diseases, as well as chitin-triggered immunity. Biochemical and genetic studies revealed that OsATL32 suppresses pathogen-induced reactive oxygen species (ROS) accumulation by mediating ubiquitination and degradation of the ROS-producing OsRac5-OsRbohB module, which enhances rice immunity against M. oryzae. The protein phosphatase PHOSPHATASE AND TENSIN HOMOLOG enhances rice blast resistance by dephosphorylating OsATL32 and promoting its degradation, preventing its negative effect on rice immunity. This study provides insights into the molecular mechanism by which the E3 ligase OsATL32 targets a ROS-producing module to undermine rice immunity.

Effect of Dl-3-n-Butylphthalide on olfaction in rotenone-induced Parkinson's rats.

Background: Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. Olfactory dysfunction (OD) is an important nonmotor feature of PD. Dl-3-n-Butylphthalide (NBP) is a synthetic compound isolated from Apium graveolens seeds. The present study was conducted to investigate the effect of NBP on olfaction in rotenone-induced Parkinson's rats to explore the mechanism and pathway of OD in PD. Methods: The PD model was established using rotenone-induced SD rats, divided into blank control, model, and treatment groups. A sham group was also established, with 10 rats in each group. The treatment group was given NBP (1 mg/kg, 10 mg/kg, and 100 mg/kg, dissolved in soybean oil) intragastrically for 28 days. Meanwhile, the control group rats were given intra-gastrically soybean oil. After behavioral testing, all rats were executed, and brain tissue was obtained. Proteomics and Proteomic quantification techniques (prm) quantification were used to detect proteomic changes in rat brain tissues. Results: Compared with the control group, the model group showed significant differences in behavioral tests, and this difference was reduced after treatment. Proteomics results showed that after treatment with high-dose NBP, there were 42 differentially expressed proteins compared with the model group. Additionally, the olfactory marker (P08523) showed a significant upregulation difference. We then selected 22 target proteins for PRM quantification and quantified 17 of them. Among them, the olfactory marker protein was at least twofold upregulated in the RTH group compared to the model group.

Effect of Retrogression with Different Cooling Ways on the Microstructure and Properties of T'/η' Strengthened Al-Zn-Mg-Cu Alloys.

Retrogression and re-aging (RRA) treatment has been proven to effectively overcome the trade-off between strength and corrosion resistance. Current research focuses on the heating rate, temperature, and holding time of retrogression treatment while ignoring the retrogression cooling ways. In this paper, the effects of RRA treatment with different retrogression cooling ways on the microstructure and properties of newly developed T'/η' strengthened Al-Zn-Mg-Cu alloys were investigated by performing tests on mechanical properties, intergranular corrosion (IGC) resistance, and electrochemical corrosion behavior. The results show that the mechanical properties of samples subject to RRA treatment with water-quenching retrogression (ultimate tensile strength, yield strength, and elongation of 419.2 MPa, 370.2 MPa, and 15.9, respectively) are better than those of air-cooled and furnace-cooled samples. The corrosion resistance of water-quenching (IGC depth of 162.2 µm, corrosion current density of 0.833 × 10-5 A/cm2) and furnace-cooled samples (IGC depth of 123.7 µm, corrosion current density of 0.712 × 10-5 A/cm2) is better than that of air-cooled samples. Microstructure characterization reveals that the effect of the retrogression cooling rate on mechanical properties is related to the size of T'/η' precipitates with grains as well as the proportion of T' and η', while the difference in corrosion resistance depends on the continuity of grain boundary precipitates (GBPs). With mechanical properties, corrosion resistance, and time cost taken into consideration, it is appropriate to select water quenching for retrogression. These findings offer valuable insights for further design to achieve superior performance in various applications.

Historical changes in the distribution of the Sichuan golden snub-nosed monkey (Rhinopithecus roxellana) in Sichuan Province, China.

The Sichuan golden snub-nosed monkey (Rhinopithecus roxellana) is a rare and endangered primate species endemic to China. Conducting research on the population distribution changes of the Sichuan golden snub-nosed monkey holds paramount importance for its conservation. Our study represented a comprehensive investigation into the population distribution of the Sichuan snub-nosed monkey by integrating data acquired from field surveys, protected areas, and historical records and using Geographic Information Systems (GIS) to explore changes in distribution across various time periods, including the historical (the Mid-to-Late Pleistocene), recent (1980-2000), and current (2001-2023). The research findings demonstrate a significant shift in the distribution range of the Sichuan golden snub-nosed monkey compared to historical time frames. Notably, between 1980 and 2000, there was a sharp decline in distribution area. Analyses revealed that the southernmost distribution county for the Sichuan golden snub-nosed monkey in Sichuan Province has shifted northward from Huili to Kangding. Furthermore, distribution changes in Sichuan Province are not solely characterized by a reduction in habitat area but also by a decrease in vertical distribution zones. Regions in the northeastern part of Sichuan with elevations below 1000 m, such as Guang'an City, Bazhong City, Dazhou City, and Nanchong City, no longer support the presence of the Sichuan golden snub-nosed monkey. At present, the distribution range is confined to elevations between 1000 and 4000 m in the two major mountain ranges of Qionglai and Minshan. A holistic approach is required to safeguard this species. The establishment of movement corridors can play a critical role in enhancing the overall connectivity of current distribution areas. Additionally, we propose implementing a hierarchical approach to protect current habitats. Spatially differentiated conservation measures should be implemented to prioritize the protection of key habitats while simultaneously monitoring anthropogenic activities in non-key habitats to prevent further fragmentation and isolation of the monkey's distribution areas.

OsmiR319-OsPCF5 modulate resistance to brown planthopper in rice through association with MYB proteins.

BACKGROUND: The brown planthopper (BPH) is a kind of piercing-sucking insect specific to rice, with the damage tops the list of pathogens and insects in recent years. microRNAs (miRNAs) are pivotal regulators of plant-environment interactions, while the mechanism underlying their function against insects is largely unknown. RESULTS: Here, we confirmed that OsmiR319, an ancient and conserved miRNA, negatively regulated resistance to BPHs, with overexpression of OsmiR319 susceptible to BPH, while suppression of OsmiR319 resistant to BPH in comparison with wild type. Meanwhile, we identified several targets of OsmiR319 that may mediate BPH resistance. Among them, OsPCF5 was the most obviously induced by BPH feeding, and over expression of OsPCF5 was resistance to BPH. In addition, various biochemical assays verified that OsPCF5 interacted with several MYB proteins, such as OsMYB22, OsMYB30, and OsMYB30C.Genetically, we revealed that both OsMYB22 and OsMYB30C positively regulated BPH resistance. Genetic interaction analyses confirmed that OsMYB22 and OsMYB30C both function in the same genetic pathway with OsmiR319b to mediate BPH resistance. CONCLUSIONS: Altogether, we revealed that OsPCF5 regulates BPH resistance via association with several MYB proteins downstream of OsmiR319, these MYB proteins might function as regulators of BPH resistance through regulating the phenylpropane synthesis.

[Identification and expression analysis of whole gene family of Isatis indigotica 4-coumarate: CoA ligase].

The 4-coumarate: CoA ligase(4CL) is a key enzyme in the upstream pathway of phenylpropanoids such as flavonoids, soluble phenolic esters, lignans, and lignins in plants. In this study, 13 4CL family members of Arabidopsis thaliana were used as reference sequences to identify the 4CL gene family candidate members of Isatis indigotica from the reported I. indigotica genome. Further bioinformatics analysis and analysis of the expression pattern of 4CL genes and the accumulation pattern of flavonoids were carried out. Thirteen 4CL genes were obtained, named Ii4CL1-Ii4CL13, which were distributed on chromosomes 1, 2, 3, 4, and 6. The analysis of the gene structure and conserved structural domains revealed the intron number of I. indigotica 4CL genes was between 1 and 12 and the protein structural domains were highly conserved. Cis-acting element analysis showed that there were multiple response elements in the promoter sequence of I. indigotica 4CL gene family, and jasmonic acid had the largest number of reaction elements. The collinearity analysis showed that there was a close relationship between the 4CL gene family members of I. indigotica and A. thaliana. As revealed by qPCR results, the expression analysis of the 4CL gene family showed that 10 4CL genes had higher expression levels in the aboveground part of I. indigotica. The content assay of flavonoids in different parts of I. indigotica showed that flavonoids were mainly accumulated in the aboveground part of plants. This study provides a basis for further investigating the roles of the 4CL gene family involved in the biosynthesis of flavonoids in I. indigotica.

Post-sepsis psychiatric disorder: Pathophysiology, prevention, and treatment.

Post-sepsis psychiatric disorder, encompassing anxiety, depression, post-traumatic stress disorder and delirium, is a highly prevalent complication secondary to sepsis, resulting in a marked increase in long-term mortality among affected patients. Regrettably, psychiatric impairment associated with sepsis is frequently disregarded by clinicians. This review aims to summarize recent advancements in the understanding of the pathophysiology, prevention, and treatment of post-sepsis mental disorder, including coronavirus disease 2019-related psychiatric impairment. The pathophysiology of post-sepsis psychiatric disorder is complex and is known to involve blood-brain barrier disruption, overactivation of the hypothalamic-pituitary-adrenal axis, neuroinflammation, oxidative stress, neurotransmitter dysfunction, programmed cell death, and impaired neuroplasticity. No unified diagnostic criteria for this disorder are currently available; however, screening scales are often applied in its assessment. Modifiable risk factors for psychiatric impairment post-sepsis include the number of experienced traumatic memories, the length of ICU stay, level of albumin, the use of vasopressors or inotropes, daily activity function after sepsis, and the cumulative dose of dobutamine. To contribute to the prevention of post-sepsis psychiatric disorder, it may be beneficial to implement targeted interventions for these modifiable risk factors. Specific therapies for this condition remain scarce. Nevertheless, non-pharmacological approaches, such as comprehensive nursing care, may provide a promising avenue for treating psychiatric disorder following sepsis. In addition, although several therapeutic drugs have shown preliminary efficacy in animal models, further confirmation of their potential is required through follow-up clinical studies.

The SUMOylation pathway regulates the pathogenicity of Fusarium oxysporum f. sp. niveum in watermelon through stabilizing the pH regulator FonPalC via SUMOylation.

SUMOylation is a key post-translational modification, where small ubiquitin-related modifier (SUMO) proteins regulate crucial biological processes, including pathogenesis, in phytopathogenic fungi. Here, we investigated the function and mechanism of the SUMOylation pathway in the pathogenicity of Fusarium oxysporum f. sp. niveum (Fon), the fungal pathogen that causes watermelon Fusarium wilt. Disruption of key SUMOylation pathway genes, FonSMT3, FonAOS1, FonUBC9, and FonMMS21, significantly reduced pathogenicity, impaired penetration ability, and attenuated invasive growth capacity of Fon. Transcription and proteomic analyses identified a diverse set of SUMOylation-regulated differentially expressed genes and putative FonSMT3-targeted proteins, which are predicted to be involved in infection, DNA damage repair, programmed cell death, reproduction, growth, and development. Among 155 putative FonSMT3-targeted proteins, FonPalC, a Pal/Rim-pH signaling regulator, was confirmed to be SUMOylated. The FonPalC protein accumulation was significantly decreased in SUMOylation-deficient mutant ∆Fonsmt3. Deletion of FonPalC resulted in impaired mycelial growth, decreased pathogenicity, enhanced osmosensitivity, and increased intracellular vacuolation in Fon. Importantly, mutations in conserved SUMOylation sites of FonPalC failed to restore the defects in ∆Fonpalc mutant, indicating the critical function of the SUMOylation in FonPalC stability and Fon pathogenicity. Identifying key SUMOylation-regulated pathogenicity-related proteins provides novel insights into the molecular mechanisms underlying Fon pathogenesis regulated by SUMOylation.

UvHOS3-mediated histone deacetylation is essential for virulence and negatively regulates ustilaginoidin biosynthesis in Ustilaginoidea virens.

Ustilaginoidea virens is the causal agent of rice false smut, which has recently become one of the most important rice diseases worldwide. Ustilaginoidins, a major type of mycotoxins produced in false smut balls, greatly deteriorates grain quality. Histone acetylation and deacetylation are involved in regulating secondary metabolism in fungi. However, little is yet known on the functions of histone deacetylases (HDACs) in virulence and mycotoxin biosynthesis in U. virens. Here, we characterized the functions of the HDAC UvHOS3 in U. virens. The ΔUvhos3 deletion mutant exhibited the phenotypes of retarded growth, increased mycelial branches and reduced conidiation and virulence. The ΔUvhos3 mutants were more sensitive to sorbitol, sodium dodecyl sulphate and oxidative stress/H2 O2 . ΔUvhos3 generated significantly more ustilaginoidins. RNA-Seq and metabolomics analyses also revealed that UvHOS3 is a key negative player in regulating secondary metabolism, especially mycotoxin biosynthesis. Notably, UvHOS3 mediates deacetylation of H3 and H4 at H3K9, H3K18, H3K27 and H4K8 residues. Chromatin immunoprecipitation assays indicated that UvHOS3 regulates mycotoxin biosynthesis, particularly for ustilaginoidin and sorbicillinoid production, by modulating the acetylation level of H3K18. Collectively, this study deepens the understanding of molecular mechanisms of the HDAC UvHOS3 in regulating virulence and mycotoxin biosynthesis in phytopathogenic fungi.

Development of Loop-Mediated Isothermal Amplification Assays for the Rapid and Accurate Diagnosis of Exserohilum turcicum for Field Applications.

Northern corn leaf blight (NCLB), caused by Exserohilum turcicum, is one of the most devastating foliar diseases of maize. Rapid and accurate diagnosis for this disease is urgently needed but still limited. Here, we establish a field-deployable diagnostic method to detect E. turcicum based on loop-mediated isothermal amplification (LAMP) assays. A software application called K-mer Elimination by Cross-reference was used to search for the specific sequences belonging to E. turcicum by comparing the whole genome sequence between E. turcicum and other known maize pathogens. Five LAMP primer sets were designed based on specific and single-copy fragments of E. turcicum. Post-LAMP analyses indicated that only the primer set, Et9468_set1, was the most suitable, producing a ladder-like amplification pattern in the agarose gel electrophoresis and a strong fluorescence signal in the presence of SYBR Green I. The LAMP assay using Et9468_set1 primers demonstrated a high level of specificity in distinguishing E. turcicum from six other common fungal pathogens of maize, as well as 12 more fungal and oomycete strains including the epiphytic fungi from maize leaves and other crop pathogens. Moreover, it exhibited remarkable sensitivity by detecting five copies per reaction, which was approximately 104 times more sensitive compared with conventional PCR. The LAMP assay successfully detected E. turcicum in field maize leaves without DNA extraction, demonstrating its suitability for rapid on-spot detection of NCLB. Our study provides a direct LAMP diagnostic method to detect E. turcicum, which enables on-site pathogen detection in the field and the development of preventive strategies for NCLB management.

Self-Priming Cyclic Amplification Accelerating CRISPR Sensor for Sensitive and Specific MicroRNA Analysis with No Background.

In this work, we demonstrate for the first time the application of the phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) reaction for miRNA assays. A self-priming amplification accelerating CRISPR sensor was well-established for sensitive and specific miRNA detection by integrating the PS-THSP reaction and CRISPR/Cas12a system. The sensor consists of three steps: (1) the formation of a complete PS-THSP template in the presence of target miRNA and ligase; (2) the exponential isothermal amplification of the PS-THSP reaction under the action of DNA polymerase; (3) the activation of the CRISPR/Cas12a fluorescence system to generate signals. We used miR-21 as a model target. The sensor can achieve sensitive detection of miR-21 without the involvement of any primers, and the special design of the CRISPR proto-spacer neighbor motif (PAM) sequence effectively avoids the interference of the background signal. In addition, the sensor can not only identify single-base mutant homologous sequences but also show stable performance in complex biological matrices. We have successfully used this sensor to accurately analyze miR-21 in different cell lines and real clinical samples, demonstrating its great potential in clinical diagnosis.