A comprehensive workflow for optimizing RNA-seq data analysis.

BACKGROUND: Current RNA-seq analysis software for RNA-seq data tends to use similar parameters across different species without considering species-specific differences. However, the suitability and accuracy of these tools may vary when analyzing data from different species, such as humans, animals, plants, fungi, and bacteria. For most laboratory researchers lacking a background in information science, determining how to construct an analysis workflow that meets their specific needs from the array of complex analytical tools available poses a significant challenge. RESULTS: By utilizing RNA-seq data from plants, animals, and fungi, it was observed that different analytical tools demonstrate some variations in performance when applied to different species. A comprehensive experiment was conducted specifically for analyzing plant pathogenic fungal data, focusing on differential gene analysis as the ultimate goal. In this study, 288 pipelines using different tools were applied to analyze five fungal RNA-seq datasets, and the performance of their results was evaluated based on simulation. This led to the establishment of a relatively universal and superior fungal RNA-seq analysis pipeline that can serve as a reference, and certain standards for selecting analysis tools were derived for reference. Additionally, we compared various tools for alternative splicing analysis. The results based on simulated data indicated that rMATS remained the optimal choice, although consideration could be given to supplementing with tools such as SpliceWiz. CONCLUSION: The experimental results demonstrate that, in comparison to the default software parameter configurations, the analysis combination results after tuning can provide more accurate biological insights. It is beneficial to carefully select suitable analysis software based on the data, rather than indiscriminately choosing tools, in order to achieve high-quality analysis results more efficiently.

PTI-ETI synergistic signal mechanisms in plant immunity.

Plants face a relentless onslaught from a diverse array of pathogens in their natural environment, to which they have evolved a myriad of strategies that unfold across various temporal scales. Cell surface pattern recognition receptors (PRRs) detect conserved elicitors from pathogens or endogenous molecules released during pathogen invasion, initiating the first line of defence in plants, known as pattern-triggered immunity (PTI), which imparts a baseline level of disease resistance. Inside host cells, pathogen effectors are sensed by the nucleotide-binding/leucine-rich repeat (NLR) receptors, which then activate the second line of defence: effector-triggered immunity (ETI), offering a more potent and enduring defence mechanism. Moreover, PTI and ETI collaborate synergistically to bolster disease resistance and collectively trigger a cascade of downstream defence responses. This article provides a comprehensive review of plant defence responses, offering an overview of the stepwise activation of plant immunity and the interactions between PTI-ETI synergistic signal transduction.

The miR6445-NAC029 module regulates drought tolerance by regulating the expression of glutathione S-transferase U23 and reactive oxygen species scavenging in Populus.

MicroRNAs are essential in plant development and stress resistance, but their specific roles in drought stress require further investigation. Here, we have uncovered that a Populus-specific microRNAs (miRNA), miR6445, targeting NAC (NAM, ATAF, and CUC) family genes, is involved in regulating drought tolerance of poplar. The expression level of miR6445 was significantly upregulated under drought stress; concomitantly, seven targeted NAC genes showed significant downregulation. Silencing the expression of miR6445 by short tandem target mimic technology significantly decreased the drought tolerance in poplar. Furthermore, 5' RACE experiments confirmed that miR6445 directly targeted NAC029. The overexpression lines of PtrNAC029 (OE-NAC029) showed increased sensitivity to drought compared with knockout lines (Crispr-NAC029), consistent with the drought-sensitive phenotype observed in miR6445-silenced strains. PtrNAC029 was further verified to directly bind to the promoters of glutathione S-transferase U23 (GSTU23) and inhibit its expression. Both Crispr-NAC029 and PtrGSTU23 overexpressing plants showed higher levels of PtrGSTU23 transcript and GST activity while accumulating less reactive oxygen species (ROS). Moreover, poplars overexpressing GSTU23 demonstrated enhanced drought tolerance. Taken together, our research reveals the crucial role of the miR6445-NAC029-GSTU23 module in enhancing poplar drought tolerance by regulating ROS homeostasis. This finding provides new molecular targets for improving the drought resistance of trees.

An alternative splicing variant of PtRD26 delays leaf senescence by regulating multiple NAC transcription factors in Populus.

During leaf senescence, the final stage of leaf development, nutrients are recycled from leaves to other organs, and therefore proper control of senescence is thus critical for plant fitness. Although substantial progress has been achieved in understanding leaf senescence in annual plants, the molecular factors that control leaf senescence in perennial woody plants are largely unknown. Using RNA sequencing, we obtained a high-resolution temporal profile of gene expression during autumn leaf senescence in poplar (Populus tomentosa). Identification of hub transcription factors (TFs) by co-expression network analysis of genes revealed that senescence-associated NAC family TFs (Sen-NAC TFs) regulate autumn leaf senescence. Age-dependent alternative splicing (AS) caused an intron retention (IR) event in the pre-mRNA encoding PtRD26, a NAC-TF. This produced a truncated protein PtRD26IR, which functions as a dominant-negative regulator of senescence by interacting with multiple hub Sen-NAC TFs, thereby repressing their DNA-binding activities. Functional analysis of senescence-associated splicing factors identified two U2 auxiliary factors that are involved in AS of PtRD26IR. Correspondingly, silencing of these factors decreased PtRD26IR transcript abundance and induced early senescence. We propose that an age-dependent increase of IR splice variants derived from Sen-NAC TFs is a regulatory program to fine tune the molecular mechanisms that regulate leaf senescence in trees.

Rational design of waste anode graphite-derived carbon catalyst to activate peroxymonosulfate for atrazine degradation.

As the rapidly growing number of waste lithium-ion batteries (LIBs), the recycling and reutilization of anode graphite is of increasing interest. Converting waste anode graphite into functional materials may be a sensible option. Herein, a series of carbonaceous catalysts (TG) were successfully prepared using spent anode graphite calcined at various temperatures and applied for activating peroxymonosulfate (PMS) to degrade atrazine (ATZ). The catalyst obtained at 800 °C (TG-800) showed the optimum performance for ATZ removal (99.2% in 6 min). Various experimental conditions were explored to achieve the optimum efficiency of the system. In the TG-800/PMS system, free radicals (e.g., SO4·-, HO·), singlet oxygen (1O2), together with a direct electron transfer pathway all participated in ATZ degradation, and the ketonic (CO) group was proved as the leading catalytic site for PMS activation. The potential degradation routes of ATZ have also been presented. According to the toxicity assessment experiments, the toxicity of the intermediate products decreased. The reusability and universal applicability of the TG-800 were also confirmed. This research not only provides an efficient PMS activator for pollutant degradation, but also offers a meaningful reference for the recovery of waste anode graphite to develop environmentally functional materials.

Yield stress Measurement of municipal sludge: A comprehensive evaluation of testing methods and concentration effects using a rotational rheometer.

Accurate prediction and measurement of yield stress are crucial for optimizing sludge treatment and disposal. However, the differences and applicability of various methods for measuring yield stress are subjects of ongoing debate. Meanwhile, literature on measuring sludge yield stress is limited to low solid concentrations (TS <10%), understanding and studying the yield stress of medium to high solid concentration sludge is crucial due to increasingly stringent standards for sludge treatment and disposal. So, this study employed a rotational rheometer to measure sludge yield stress across a wide range of TS (4-50%) using steady shear, dynamic oscillatory shear, and transient shear. The study derived significant conclusions by comparing and summarizing the applicability and limitations of each testing method: Dynamic oscillatory shear methods, including G'-σ curve method, γ-σ curve method, and G**γc method can measure sludge yield stress ranging from 4% to 40% TS, while other methods are restricted to low or limited solid concentrations; The G' = G″ method, utilizing the intersection of G' and G″ curves, consistently yields the highest value for yield stress when 4%≤ TS ≤ 12%; The rotational rheometer cannot measure sludge yield stress when the solid concentration exceeds 40% TS; The relationship between sludge yield stress and solid concentration is stronger as a power-law for TS ≤ 25%, transitioning to linear for higher concentrations (28%≤ TS <40%). This study systematically explores the applicability and limitations of various measurement methods for characterizing sludge yield stress across a wide range of solid concentrations, providing valuable guidance for scientific measurement and highlighting challenging research issues.

Identification of WUSCHEL-related homeobox gene and truncated small peptides in transformation efficiency improvement in Eucalyptus.

BACKGROUND: The WUSCHEL-related Homeobox (WOX) genes, which encode plant-specific homeobox (HB) transcription factors, play crucial roles in regulating plant growth and development. However, the functions of WOX genes are little known in Eucalyptus, one of the fastest-growing tree resources with considerable widespread cultivation worldwide. RESULTS: A total of nine WOX genes named EgWOX1-EgWOX9 were retrieved and designated from Eucalyptus grandis. From the three divided clades marked as Modern/WUS, Intermediate and Ancient, the largest group Modern/WUS (6 EgWOXs) contains a specific domain with 8 amino acids: TLQLFPLR. The collinearity, cis-regulatory elements, protein-protein interaction network and gene expression analysis reveal that the WUS proteins in E. grandis involve in regulating meristems development and regeneration. Furthermore, by externally adding of truncated peptides isolated from WUS specific domain, the transformation efficiency in E. urophylla × E. grandis DH32-29 was significant enhanced. The transcriptomics data further reveals that the use of small peptides activates metabolism pathways such as starch and sucrose metabolism, phenylpropanoid biosynthesis and flavonoid biosynthesis. CONCLUSIONS: Peptides isolated from WUS protein can be utilized to enhance the transformation efficiency in Eucalyptus, thereby contributing to the high-efficiency breeding of Eucalyptus.

Spliceosomal protein U2B″ delays leaf senescence by enhancing splicing variant JAZ9ß expression to attenuate jasmonate signaling in Arabidopsis.

The regulatory framework of leaf senescence is gradually becoming clearer; however, the fine regulation of this process remains largely unknown. Here, genetic analysis revealed that U2 small nuclear ribonucleoprotein B (U2B″), a component of the spliceosome, is a negative regulator of leaf senescence. Mutation of U2B″ led to precocious leaf senescence, whereas overexpression of U2B″ extended leaf longevity. Transcriptome analysis revealed that the jasmonic acid (JA) signaling pathway was activated in the u2b″ mutant. U2B″ enhances the generation of splicing variant JASMONATE ZIM-DOMAIN 9ß (JAZ9ß) with an intron retention in the Jas motif, which compromises its interaction with CORONATINE INSENSITIVE1 and thus enhances the stability of JAZ9ß protein. Moreover, JAZ9ß could interact with MYC2 and obstruct its activity, thereby attenuating JA signaling. Correspondingly, overexpression of JAZ9ß rescued the early senescence phenotype of the u2b″ mutant. Furthermore, JA treatment promoted expression of U2B″ that was found to be a direct target of MYC2. Overexpression of MYC2 in the u2b″ mutant resulted in a more pronounced premature senescence than that in wild-type plants. Collectively, our findings reveal that the spliceosomal protein U2B″ fine-tunes leaf senescence by enhancing the expression of JAZ9ß and thereby attenuating JA signaling.

Histone variant HTB4 delays leaf senescence by epigenetic control of Ib bHLH transcription factor-mediated iron homeostasis.

Leaf senescence is an orderly process regulated by multiple internal factors and diverse environmental stresses including nutrient deficiency. Histone variants are involved in regulating plant growth and development. However, their functions and underlying regulatory mechanisms in leaf senescence remain largely unclear. Here, we found that H2B histone variant HTB4 functions as a negative regulator of leaf senescence. Loss of function of HTB4 led to early leaf senescence phenotypes that were rescued by functional complementation. RNA-seq analysis revealed that several Ib subgroup basic helix-loop-helix (bHLH) transcription factors (TFs) involved in iron (Fe) homeostasis, including bHLH038, bHLH039, bHLH100, and bHLH101, were suppressed in the htb4 mutant, thereby compromising the expressions of FERRIC REDUCTION OXIDASE 2 (FRO2) and IRON-REGULATED TRANSPORTER (IRT1), two important components of the Fe uptake machinery. Chromatin immunoprecipitation-quantitative polymerase chain reaction analysis revealed that HTB4 could bind to the promoter regions of Ib bHLH TFs and enhance their expression by promoting the enrichment of the active mark H3K4me3 near their transcriptional start sites. Moreover, overexpression of Ib bHLH TFs or IRT1 suppressed the premature senescence phenotype of the htb4 mutant. Our work established a signaling pathway, HTB4-bHLH TFs-FRO2/IRT1-Fe homeostasis, which regulates the onset and progression of leaf senescence.

Metabolic control of arginine and ornithine levels paces the progression of leaf senescence.

Leaf senescence can be induced by stress or aging, sometimes in a synergistic manner. It is generally acknowledged that the ability to withstand senescence-inducing conditions can provide plants with stress resilience. Although the signaling and transcriptional networks responsible for a delayed senescence phenotype, often referred to as a functional stay-green trait, have been actively investigated, very little is known about the subsequent metabolic adjustments conferring this aptitude to survival. First, using the individually darkened leaf (IDL) experimental setup, we compared IDLs of wild-type (WT) Arabidopsis (Arabidopsis thaliana) to several stay-green contexts, that is IDLs of two functional stay-green mutant lines, oresara1-2 (ore1-2) and an allele of phytochrome-interacting factor 5 (pif5), as well as to leaves from a WT plant entirely darkened (DP). We provide compelling evidence that arginine and ornithine, which accumulate in all stay-green contexts-likely due to the lack of induction of amino acids (AAs) transport-can delay the progression of senescence by fueling the Krebs cycle or the production of polyamines (PAs). Secondly, we show that the conversion of putrescine to spermidine (SPD) is controlled in an age-dependent manner. Thirdly, we demonstrate that SPD represses senescence via interference with ethylene signaling by stabilizing the ETHYLENE BINDING FACTOR1 and 2 (EBF1/2) complex. Taken together, our results identify arginine and ornithine as central metabolites influencing the stress- and age-dependent progression of leaf senescence. We propose that the regulatory loop between the pace of the AA export and the progression of leaf senescence provides the plant with a mechanism to fine-tune the induction of cell death in leaves, which, if triggered unnecessarily, can impede nutrient remobilization and thus plant growth and survival.

What affects the accuracy and applicability of determining wastewater sludge water content via low-field nuclear magnetic resonance?

The accurate determination of waster sludge water content is crucial to sludge dewatering treatment and its disposal management. Though previous studies highlight the great advantages of low-field nuclear magnetic resonance (LF-NMR) in the determination of sludge water content, its accuracy and applicability are not well studied. Herein, this study investigated the settling of operating parameters and the properties of sludge samples on the accuracy and applicability of LF-NMR method in measuring sludge water content. The results showed that the setting of basic parameters such as standard curve, number of scanning times (NS) and sample weight affected the accuracy of sludge water content by LF-NMR. The standard calibration curve constructed by 3 g/L CuSO4, NS = 8 and the sample weight of about 5 g, were suitable for the accurate determination of sludge water content. Furthermore, the existence of magnetic substances in sludge can affect the distribution gradient of main magnetic field, and thus restricted the applicability of LF-NMR. The saturation magnetization of chemical reagents strongly correlated with the measured relative errors of sludge water content (r = 0.995, p < 0.01), the greater the saturation magnetization of the magnetic material, the greater the error of the test results. On the whole, it is necessary to fully consider the influence of process parameters and sludge properties to evaluate the accuracy and applicability of the LF-NMR method, rather than simply copying the parameters in literatures.

Enantioseparation of amino acid and mandelic acid enantiomers using Josiphos-metal complexes as chiral extractants.

The development of new and efficient chiral extractants has always been the research hotspot and difficulty in the field of chiral extraction. Josiphos, a famous ferrocene derivative catalyst, is employed as a chiral extractant in enantioseparation of amino acid and mandelic acid enantiomers. The influences of metal ions, organic solvents, pH of the aqueous solution, extractant concentrations, and extraction temperature on enantioselectivities are systematically studied. The result reveals that Josiphos-Pd has good capabilities to enantioseparate 4-nitro-phenylalanine (Nphe), 3-chloro-phenylglycine (Cpheg), and mandelic acid (MA) with separation factors (α) of 3.30, 2.65, and 2.18, respectively. The pH of the aqueous phase and Josiphos-Pd concentration affect the extraction significantly, whereas extraction temperature shows little influence. After optimizing by response surface method, the mathematical models for extractions are established. And the highest experimental performance factors (pf) for Nphe, Cpheg, and MA are 0.1843, 0.1335, and 0.08884, respectively.

Cuprous-mediated peroxymonosulfate activation for Fenton-like removal of micropollutants: The function of co-catalyst and the accelerated degradation mechanism.

Introducing co-catalysts to enhance the activation of cuprous-mediated peroxymonosulfate (PMS) and induce the continuous generation of highly reactive oxygen species is promising. The function, effectiveness, and acceleration mechanism of co-catalysts in the cuprous-mediated PMS activation process were fully explored in this work, which focused on rhodamine B as the target contaminants. The results demonstrated that molybdenum (Mo) powder was a superb co-catalyst, and that the reaction of cuprous-mediated PMS system was carried out by surface Mo species as opposed to Mo ions in the solution. The Cu (II)/Cu(I) cycle was primarily encouraged by the Mo0, which also caused abundant ·HO and 1O2 and minimal SO4·- and ·O2- to be produced from PMS. The Mo/Cu2+/PMS system exhibited high removal efficiency towards typical pollutants, especially ciprofloxacin, methyl orange, malachite green, and crystal violet, with removal rates up to 93%, 99%, 97%, and 92%, respectively. Additionally, this system showed excellent adaptability to complex water environments. After four cycles, the Mo powder retained its properties and morphology, and the target pollutants could still maintain an 82% degradation efficiency. This study provides a basis for enhancing cuprous-mediated PMS activation for wastewater treatment.

Genome-Wide Analysis of the FBA Subfamily of the Poplar F-Box Gene Family and Its Role under Drought Stress.

F-box proteins are important components of eukaryotic SCF E3 ubiquitin ligase complexes, which specifically determine protein substrate proteasomal degradation during plant growth and development, as well as biotic and abiotic stress. It has been found that the FBA (F-box associated) protein family is one of the largest subgroups of the widely prevalent F-box family and plays significant roles in plant development and stress response. However, the FBA gene family in poplar has not been systematically studied to date. In this study, a total of 337 F-box candidate genes were discovered based on the fourth-generation genome resequencing of P. trichocarpa. The domain analysis and classification of candidate genes revealed that 74 of these candidate genes belong to the FBA protein family. The poplar F-box genes have undergone multiple gene replication events, particularly in the FBA subfamily, and their evolution can be attributed to genome-wide duplication (WGD) and tandem duplication (TD). In addition, we investigated the P. trichocarpa FBA subfamily using the PlantGenIE database and quantitative real-time PCR (qRT-PCR); the results showed that they are expressed in the cambium, phloem and mature tissues, but rarely expressed in young leaves and flowers. Moreover, they are also widely involved in the drought stress response. At last, we selected and cloned PtrFBA60 for physiological function analysis and found that it played an important role in coping with drought stress. Taken together, the family analysis of FBA genes in P. trichocarpa provides a new opportunity for the identification of P. trichocarpa candidate FBA genes and elucidation of their functions in growth, development and stress response, thus demonstrating their utility in the improvement of P. trichocarpa.

Prediction of arsenic adsorption onto metal organic frameworks and adsorption mechanisms interpretation by machine learning.

Metal-organic frameworks (MOFs) are promising adsorbents for the removal of arsenic (As) from wastewater. The As removal efficiency is influenced by several factors, such as the textural properties of MOFs, adsorption conditions, and As species. Examining all of the relevant factors through traditional experiments is challenging. To predict the As adsorption capacities of MOFs toward organic, inorganic, and total As and reveal the adsorption mechanisms, four machine learning-based models were developed, with the adsorption conditions, MOF properties, and characteristics of different As species as inputs. The results demonstrated that the extreme gradient boosting (XGBoost) model exhibited the best predictive performance (test R2 = 0.93-0.96). The validation experiments demonstrated the high accuracy of the inorganic As-based XGBoost model. The feature importance analysis showed that the concentration of As, the surface area of MOFs, and the pH of the solution were the three key factors governing inorganic-As adsorption, while those governing organic-As adsorption were the concentration of As, the pHpzc value of MOFs, and the oxidation state of the metal clusters. The formation of coordination complexes between As and MOFs is possibly the major adsorption mechanism for both inorganic and organic As. However, electrostatic interaction may have a greater effect on organic-As adsorption than on inorganic-As adsorption. Overall, this study provides a new strategy for evaluating As adsorption on MOFs and discovering the underlying decisive factors and adsorption mechanisms, thereby facilitating the investigation of As wastewater treatment.

Enhancing data reliability in quantitative characterization of moisture distribution in sludge using DSC: Impact of sample attributes and test parameters.

Exploring moisture distribution, especially bound water content, is vital for studying and applying sludge dewatering. The differential scanning calorimetry (DSC) method has been extensively utilized for the quantitative characterization of moisture distribution in sludge. However, this method has certain limitations, such as low reproducibility of results, leading to controversial parameter values in different papers and hindering result comparison. In this study, we investigated the influence of key sample attributes on measuring sludge bound water using the DSC method.The findings demonstrated that the moisture content and mass of sludge samples substantially influenced the reproducibility and stability of DSC test results. To ensure data reliability, the moisture content of the sludge sample should be minimized and kept below 84%, with the mass not exceeding 10 mg. Compared to the influence of sludge moisture content and sample mass, the heating rate (1⁓5 °C/min) minimally affected DSC test results. This study offers a comprehensive insight into how sample attributes and test parameters affect the quantitative characterization of bound water in sludge using the DSC method. Furthermore, practical strategies are presented to enhance the method's applicability in sludge bound water characterization.

Linkage Microenvironment of Azoles-Related Covalent Organic Frameworks Precisely Regulates Photocatalytic Generation of Hydrogen Peroxide.

Artificial H2 O2 photosynthesis by covalent organic frameworks (COFs) photocatalysts is promising for wastewater treatment. The effect of linkage chemistry of COFs as functional basis to photoelectrochemical properties and photocatalysis remains a significant challenge. In this study, three kinds of azoles-linked COFs including thiazole-linked TZ-COF, oxazole-linked OZ-COF and imidazole-linked IZ-COF were successfully synthesized. More accessible channels of charge transfer were constructed in TZ-COF via the donor-π-acceptor structure between thiazole linkage and pyrene linker, leading to efficient suppression of photoexcited charge recombination. Density functional theory calculations support the experimental studies, demonstrating that the thiazole linkage is more favorable for the formation of *O2 intermediate in H2 O2 production than that of the oxazole and imidazole linkages. The real active sites in COFs located at the benzene ring fragment between pyrene unit and azole linkage.

Directing the Architecture of Surface-Clean Cu2O for CO Electroreduction.

Tailoring the morphology of nanocrystals is a promising way to enhance their catalytic performance. In most previous shape-controlled synthesis strategies, surfactants are inevitable due to their capability to stabilize different facets. However, the adsorbed surfactants block the intrinsic active sites of the nanocrystals, reducing their catalytic performance. For now, strategies to control the morphology without surfactants are still limited but necessary. Herein, a facile surfactant-free synthesis method is developed to regulate the morphology of Cu2O nanocrystals (e.g., solid nanocube, concave nanocube, cubic framework, branching nanocube, branching concave nanocube, and branching cubic framework) to enhance the electrocatalytic performance for the conversion of CO to n-propanol. Specifically, the Cu2O branching cubic framework (BCF-Cu2O), which is difficult to fabricate using previous surfactant-free methods, is fabricated by combining the concentration depletion effect and the oxidation etching process. More significantly, the BCF-Cu2O-derived catalyst (BCF) presents the highest n-propanol current density (-0.85 mA cm-2) at -0.45 V versus the reversible hydrogen electrode (VRHE), which is fivefold higher than that of the surfactant-coated Cu2O nanocube-derived catalyst (SFC, -0.17 mA cm-2). In terms of the n-propanol Faradaic efficiency in CO electroreduction, that of the BCF exhibits a 41% increase at -0.45 VRHE as compared with SFC. The high catalytic activity of the BCF that results from the clean surface and the coexistence of Cu(100) and Cu(110) in the lattice is well-supported by density functional theory calculations. Thus, this work presents an important paradigm for the facile fabrication of surface-clean nanocrystals with an enhanced application performance.

Structure-Switchable Hairpin-Powered Exponential Replications for Sensing Attomolar microRNA-Related Single Nucleotide Polymorphisms in Human Cancer Tissues with Zero Background.

MicroRNA (miRNA)-related single-nucleotide polymorphisms (miR-SNPs) are a novel class of genetic variations involved in multiple cellular functions, and they have emerged as promising biomarkers for cancer diagnostics and prognostics. Herein, we demonstrate for the first time the structure-switchable hairpin-powered exponential replications for sensing attomolar miR-SNPs in human cancer tissues with zero background. In the presence of target miR-196a2T, hairpin probes (i.e., HP1 and HP2) are splinted together to construct the dumbbell-shaped probe (DSP) with SplintR ligase catalysis. Once the DSP is formed, the self-primed polymerization extension and linear FIP/BIP-primed strand displacement DNA synthesis (SDS) are automatically repeated to activate self-circulated exponential amplification, producing large amounts of double-stranded DNAs (dsDNAs) which can be real-time monitored using SYBR Green I. This nanodevice can detect miR-196a2T with an ultralow detection limit of 2.46 aM; distinguish rare miR-196a2 SNP with a selectivity factor of 0.001%; and even profile miR-196a2T in human tissues for nonsmall cell lung cancer (NSCLC) diagnosis, risk assessment, and cancer type prediction. Notably, this nanodevice can be rapidly and homogeneously used in one tube in a real-time and label-free manner, providing a powerful point-of-care platform for noninvasive diagnostics and prognostics of miR-SNPs-related human cancers.

CLE42 delays leaf senescence by antagonizing ethylene pathway in Arabidopsis.

Leaf senescence is the final stage of leaf development and is influenced by numerous internal and environmental factors. CLE family peptides are plant-specific peptide hormones that regulate various developmental processes. However, the role of CLE in regulating Arabidopsis leaf senescence remains unclear. Here, we found that CLE42 is a negative regulator of leaf senescence by using a CRISPR/Cas9-produced CLE mutant collection. The cle42 mutant displayed earlier senescence phenotypes, while overexpression of CLE42 delayed age-dependent and dark-induced leaf senescence. Moreover, application of the synthesized 12-amino-acid peptide (CLE42p) also delayed leaf senescence under natural and dark conditions. CLE42 and CLE41/44 displayed functional redundancy in leaf senescence, and the cle41 cle42 cle44 triple mutant displayed more pronounced earlier senescence phenotypes than any single mutant. Analysis of differentially expressed genes obtained by RNA-Seq methodology revealed that the ethylene pathway was suppressed by overexpressing CLE42. Moreover, CLE42 suppressed ethylene biosynthesis and thus promoted the protein accumulation of EBF, which in turn decreased the function of EIN3. Accordingly, mutation of EIN3/EIL1 or overexpression of EBF1 suppressed the earlier senescence phenotypes of the cle42 mutant. Together, our results reveal that the CLE peptide hormone regulates leaf senescence by communicating with the ethylene pathway.