A facile fluorescence method for the effective detection of ampicillin using antioxidant carbon dots with specific fluorescent response to ˙OH.

Monitoring methods for beta-lactam (ß-lactam) antibiotics, especially for ampicillin (AMP), with simple operation and sensitivity for realtime applications are highly required. To address this need, antioxidant carbon dots (E-CDs) with excellent fluorescence properties were synthesized using citric acid and ethylenediamine as raw materials. With a quantum yield of 81.97%, E-CDs exhibited a specific and sensitive response to ˙OH. The quenched fluorescence of E-CDs by the formed ˙OH could be restored through a competition reaction with AMP. Leveraging the signal-quenching strategy of E-CDs, H2O2, and Fe2+, a fluorescence signal-on strategy was developed using AMP as the fluorescence recovery agent for the sensitive detection of AMP. The mechanism of the quenching of E-CDs by ˙OH was attributed to the damaging effect of ˙OH on E-CDs. Under optimal conditions, the detection limit of this method for AMP was determined to be 0.38 µg mL-1. This method was successful in drug quality control and the spiked detection of AMP in lake water, milk, and sea cucumber, presenting a viable option for convenient and rapid antibiotic monitoring methods.

Stefin B alleviates the gouty arthritis in mice by inducing the M2 polarization of macrophages.

The present study aims to explore the therapeutic effect of Stefin B on gouty arthritis (GA) and the polarization of macrophages in mice. Stefin B-overexpressed or knockdown M0 macrophages were constructed. The GA model was established in mice by injecting 25 mg/mL MSU, followed by a single injecting of Stefin B-overexpressing adenovirus vector (GA model + Stefin B OE) or an empty vector (GA model + Stefin B OE NC). Stefin B was found lowly expressed in M1 macrophages. CD206 was markedly upregulated and IL-10 release was signally increased in Stefin B-overexpressed macrophages. In gouty arthritis mice, marked redness and swelling were observed in the ankle joint. Dramatical infiltration of inflammatory cells was observed in the GA model and GA model + Stefin B OE NC groups, which was suppressed in the Stefin B OE group. Increased proportion of F4/80+CD86+ cells observed in GA mice was markedly repressed by Stefin B overexpression, accompanied by the declined level of Caspase-1 and IL-17. Collectively, Stefin B alleviated the GA in mice by inducing the M2 polarization of macrophages.

Risk of deep vein thrombosis (DVT) in lower extremity after total knee arthroplasty (TKA) in patients over 60 years old.

PURPOSE: There is a significant risk of DVT after TKA. We aim to evaluate the potential risk factors for postoperative DVT in the lower extremities in TKA patients over 60 years of age and provide a reference for the effective prevention of DVT. METHODS: This retrospective study included patients older than 60 who underwent TKA surgery in our hospital from May 2015 to May 2022 and compared and analyzed patients' personal characteristics and clinical data with or without postoperative DVT. Logistic regression analysis was performed to determine the potential risk factors for DVT after TKA. The sensitivity and specificity of each risk factor in the diagnosis of DVT were compared by the ROC curve, and the value of this model in the diagnosis of DVT was further investigated using a multivariable combined diagnosis ROC curve model. RESULTS: A total of 661 patients over 60 who underwent TKA were included. Preoperative Hematocrit (HCT), platelet count, anesthesia mode, postoperative D-dimer, ESR, diabetes mellitus, and other aspects of the DVT group and non-DVT group were statistically significant after TKA (P < 0.05). Multivariate logistics regression analysis showed that preoperative HCT, anesthesia mode, and diabetes were independent risk factors for DVT in patients over 60 years old after TKA. Compared with the univariate ROC model, the multivariable combined ROC curve analysis model has a higher diagnostic value for the diagnosis of DVT. CONCLUSION: DVT is common in patients over 60 years of age after TKA, and there is a multivariable influence on its pathogenesis. For patients over 60 with diabetes, neuraxial anesthesia is recommended for patients with high preoperative HCT levels, which may reduce the incidence of postoperative DVT.

Effectively synthesized functional Si-doped carbon dots with the applications in tyrosinase detection and lysosomal imaging.

There has been significant interest in the preparation and versatile applications of carbon dots (CDs) due to their immense potential value in sensors and imaging. In this work, silicon-doped green carbon dots (Si-CDs) with high quantum yield and rich epoxypropyl were effectively synthesized. Given the clinical diagnostic importance of abnormal levels of tyrosinase (TYR), sensitive detection of TYR is significant for clinical research. A fluorescence signal-off strategy with Si-CDs as probe was constructed to determine TYR based on the oxidation of dopamine by TYR. The detection ranges of this method were 0.01-1.5 and 10-30 U/mL with the detection limit of 0.0046 U/mL, the lower limit of quantification (LLOQ) was 0.01 U/mL, and TYR was successfully and accurately monitored in human serum. Additionally, due to the role of lysosomes in cellular regulatory processes, including TYR levels and fluorescence stability characteristics of Si-CDs in acidic conditions, it was envisaged to use Si-CDs as probe to establish real-time monitoring of lysosomes. According to fluorescence colocation analysis, Si-CDs had intrinsic lysosomal targeting ability to HepG2 and L-02 (with Pearson correlation coefficients were 0.90 and 0.91, respectively). The targeting of Si-CDs to lysosomes was due to the acidophilic effect of the epoxypropyl on its surface.

Detection of Doxycycline Using Carbon Quantum dots as Probe Based on Internal Filtering Effect.

The establishment of a convenient and effective detection method for doxycycline (DC) holds significant importance in drug monitoring and drug residue assessment. In this work, carbon quantum dots (CQDs) with excellent and stable luminescence performance (the quantum yield of CQDs was 21.8%) were synthesized by the melting method and employed as probes to monitor the fluorescence intensity variations before and after the introduction of DC. A fluorescence analytical method based on the internal filtration effect (IFE) was developed for DC determination. The mechanism of DC quenching CQDs was verified using fluorescence lifetime tests, absorption spectroscopy, and evaluation of internal filtration parameters. After optimizing experimental conditions, it was found that the DC concentration (CDC) exhibited a good linear relationship with the fluorescence quenching efficiency ((F0-F)/F0) of CQDs in the range of 5-30 µM. The fitted linear equation was Y = 0.01249*CDC+0.03625, R2 = 0.9987, and the detection limit was 2.343 µM (n = 8). This developed method has been successfully applied to accurately determine DC concentrations in both doxycycline hydrochloride tablets and human serum samples. It stands out for its simplicity, rapidity, and acceptable detection performance. Due to its advantages, this method holds great promise for application in the biomedical field for monitoring DC drug concentrations and ensuring quality control.

Isolation of Meiocytes and Cytological Analyses of Male Meiotic Chromosomes in Soybean, Lettuce, and Maize.

Meiosis is a specialized cell division that halves the number of chromosomes following a single round of DNA replication, thus leading to the generation of haploid gametes. It is essential for sexual reproduction in eukaryotes. Over the past several decades, with the well-developed molecular and cytogenetic methods, there have been great advances in understanding meiosis in plants such as Arabidopsis thaliana and maize, providing excellent references to study meiosis in other plants. A chapter in the previous edition described molecular cytological methods for studying Arabidopsis meiosis in detail. In this chapter, we focus on methods for studying meiosis in soybean (Glycine max), lettuce (Lactuca sativa), and maize (Zea mays). Moreover, we include the method that was recently developed for examination of epigenetic modifications, such as DNA methylation and histone modifications on meiotic chromosomes in plants.

Evaluation of Crossover Number, Distribution, and Interference Using Cytological Assays in Arabidopsis.

Meiosis involves the replication of nuclear chromosomes in a parent cell, followed by two successive nuclear divisions to produce haploid spores, which differentiate into the gametophyte generations that produce the egg and sperm in plants. Meiotic recombination or crossover (CO) is a hallmark of meiosis that allows shuffling of genetic information between homologous chromosomes (homologs), thereby giving rise to genetically diverse progeny cells and, ultimately, individuals in the progeny; this opens vast opportunities for genetic differentiation and hence speciation. Meiotic COs also ensure the formation of bivalents and fidelity of their equal segregation. Therefore, mechanisms that regulate meiotic recombination have been extensively studied in multiple species. Several approaches have been developed to observe or estimate the frequency of CO, in which CO can be visualized and analyzed cytologically by estimating the number of chiasma (plural chiasmata), a position where non-sister chromatids exchange genetic material between homologs. Furthermore, one CO event might influence the occurrence of another one nearby, along a chromosome; this is known as CO interference. Over the past decades, visualizing CO events and measuring CO interference have contributed greatly to our understanding of the regulatory mechanisms of meiotic recombination. Here, we describe protocols to estimate the number of chiasmata and CO interference in Arabidopsis using cytological methods involving chromosome spreads and immunostaining. Specifically, we describe how chromosome spreads can be used to estimate the number of chiasmata based on the conformations of metaphase I bivalents and provide a revised acid-based quick immunostaining assay that permits high-throughput and quantitative digital estimation of the relative distance between adjacent interference-sensitive CO foci at diakinesis. These methods can be easily established or modified, if necessary, for studying meiotic recombination in other plants and crops. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Estimation of the number of chiasmata per nucleus based on metaphase I bivalent conformations Basic Protocol 2: A chromosome spread-based immunostaining method for relative distance analysis of adjacent interference-sensitive CO foci at diakinesis in Arabidopsis meiocytes.

Decorated Polyetheretherketone Implants with Antibacterial and Antioxidative Effects through Layer-by-Layer Nanoarchitectonics Facilitate Diabetic Bone Integration with Infection.

Patients suffering diabetic bone defects still need some new and effective strategies to achieve enhanced prognostic effects. Although medical implants are the common treatment of bone defects, the excessive oxidative stress and high risk of bacterial infection in diabetes mellitus lead to a higher risk of implant failure. To improve the healing ability of diabetic bone defects, herein, polyetheretherketone (PEEK) was modified through a developed layer-by-layer (LBL) construction strategy to obtain multifunctional PEEK (SP@(TA-GS/PF)*3) by the assembly of tannic acid (TA), gentamicin sulfate (GS) and Pluronic F127 (PF127) on the basis of prepared porous PEEK through sulfonation (SPEEK). The prepared SP@(TA-GS/PF)*3 exhibited sustained antimicrobial activity and enhanced the differentiation of osteoblast (MC3T3-E1) for needed osteogenesis. Moreover, SP@(TA-GS/PF)*3 scavenged excessive oxidative stress to promote the growth of H2O2 damaged HUVEC with enhanced secretion of VEGF for neovascularization. In addition, the remarkable in vivo outcomes of angiogenesis and osseointegration were revealed by the subcutaneous implant model and bone tissue implant model in diabetic rats, respectively. The in vitro and in vivo results demonstrated that modified PEEK with multifunction can be an attractive tool for enhancing bone integration under diabetic conditions, underpinning the clinical application potential of modified implants for diabetic osseointegration.

DNA polymerase epsilon binds histone H3.1-H4 and recruits MORC1 to mediate meiotic heterochromatin condensation.

Heterochromatin is essential for genomic integrity and stability in eukaryotes. The mechanisms that regulate meiotic heterochromatin formation remain largely undefined. Here, we show that the catalytic subunit (POL2A) of Arabidopsis DNA polymerase epsilon (POL ε) is required for proper formation of meiotic heterochromatin. The POL2A N terminus interacts with the GHKL adenosine triphosphatase (ATPase) MORC1 (Microrchidia 1), and POL2A is required for MORC1's localization on meiotic heterochromatin. Mutations affecting the POL2A N terminus cause aberrant morphology of meiotic heterochromatin, which is also observed in morc1. Moreover, the POL2A C-terminal zinc finger domain (ZF1) specifically binds to histone H3.1-H4 dimer or tetramer and is important for meiotic heterochromatin condensation. Interestingly, we also found similar H3.1-binding specificity for the mouse counterpart. Together, our results show that two distinct domains of POL2A, ZF1 and N terminus bind H3.1-H4 and recruit MORC1, respectively, to induce a continuous process of meiotic heterochromatin organization. These activities expand the functional repertoire of POL ε beyond its classic role in DNA replication and appear to be conserved in animals and plants.

DNA polymerase epsilon interacts with SUVH2/9 to repress the expression of genes associated with meiotic DSB hotspot in Arabidopsis.

Meiotic recombination is initiated by the SPORULATION 11 (SPO11)-triggered formation of double-strand breaks (DSBs) that usually occur in open chromatin with active transcriptional features in many eukaryotes. However, gene transcription at DSB sites appears to be detrimental for repair, but the regulatory mechanisms governing transcription at meiotic DSB sites are largely undefined in plants. Here, we demonstrate that the largest DNA polymerase epsilon subunit POL2A interacts with SU(VAR)3 to 9 homologs SUVH2 and SUVH9. N-SIM (structured illumination microscopy) observation shows that the colocalization of SUVH2 with the meiotic DSB marker γ-H2AX is dependent on POL2A. RNA-seq of male meiocytes demonstrates that POL2A and SUVH2 jointly repress the expression of 865 genes, which have several known characteristics associated with meiotic DSB sites. Bisulfite-seq and small RNA-seq of male meiocytes support the idea that the silencing of these genes by POL2A and SUVH2/9 is likely independent of CHH methylation or 24-nt siRNA accumulation. Moreover, pol2a suvh2 suvh9 triple mutants have more severe defects in meiotic recombination and fertility compared with either pol2a or suvh2 suvh9. Our results not only identify a epigenetic regulatory mechanism for gene silencing in male meiocytes but also reveal roles for DNA polymerase and SUVH2/9 beyond their classic functions in mitosis.

Identifying small RNAs and Analyzing Their Association with Gene Expression Using Isolated Arabidopsis Male Meiocytes.

Meiosis is a specialized cell division that generates gametes and is essential for sexual reproduction. Studying meiosis in plants, like the model flowering plant Arabidopsis thaliana, contributes to our understanding of the fundamental biology of reproductive biology and has practical implications for improving economically important crop species. In this chapter, we provide a detailed protocol for capillary collection of Arabidopsis male meiocytes followed by total RNA extraction, RNA-Seq, and bioinformatics analysis of small-RNAs (sRNAs) including analysis of sRNA cluster that correlate with genomic features.

Risk factors and new diagnostic index for deep venous thrombosis of lower extremities in elderly patients with traumatic femoral neck fracture.

Purpose: To determine the incidence and risk factors of deep vein thrombosis (DVT) of lower extremities at admission in elderly Chinese patients with femoral neck fracture, and to establish and evaluate a new DVT predictor based on these risk factors. Methods: Patients who were hospitalized from January 2018 to December 2020 at three independent centers were reviewed. According to the results of lower extremities vascular ultrasound at admission, they were divided into DVT group and non-DVT group. Single and multivariate logistic regression analysis were applied to identify independent risk factors for DVT occurrence, and then a prediction formula for DVT based on the risk factors was developed. The new predictive index of DVT was calculated by the formula. Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of different factors and the new predictive index. Results: There were 203 elder patients were included in the final analysis after application of the exclusion criteria. Thirty seven patients (18.2%) were diagnosed as DVT by ultrasound, including 33 patients (89.2%) of peripheral type, 1 patient (2.7%) of central type and 3 patients (8.1%) of mixed type.Multivariate logistic regression analysis showed that four factors including injured side, hemoglobin, fibrinogen, d-dimer were the independent risk factors for the incidence of DVT. On this basis, a new formula for DVT predictive factor was constructed: New predictive index = 0.895 * injured side (right = 1, left = 0) + 0.899 * hemoglobin (<109.5 g/L = 1, > 109.5 g/L = 0) + 1.19 * fibrinogen (>4.24 g/L = 1, < 4.24 g/L = 0) + 1.221* d-dimer (>2.4 mg/L = 1, < 2.4 mg/L = 0). The AUC value of this new developed index was 0.735. Conclusions: This work showed that the incidence of DVT in elderly patients with femoral neck fracture in China was high at admission. New DVT predictive value can be used as an effective diagnosis strategy for evaluation of thrombosis at admission.

Regulation of interference-sensitive crossover distribution ensures crossover assurance in Arabidopsis.

During meiosis, crossovers (COs) are typically required to ensure faithful chromosomal segregation. Despite the requirement for at least one CO between each pair of chromosomes, closely spaced double COs are usually underrepresented due to a phenomenon called CO interference. Like Mus musculus and Saccharomyces cerevisiae, Arabidopsis thaliana has both interference-sensitive (Class I) and interference-insensitive (Class II) COs. However, the underlying mechanism controlling CO distribution remains largely elusive. Both AtMUS81 and AtFANCD2 promote the formation of Class II CO. Using both AtHEI10 and AtMLH1 immunostaining, two markers of Class I COs, we show that AtFANCD2 but not AtMUS81 is required for normal Class I CO distribution among chromosomes. Depleting AtFANCD2 leads to a CO distribution pattern that is intermediate between that of wild-type and a Poisson distribution. Moreover, in Atfancm, Atfigl1, and Atrmi1 mutants where increased Class II CO frequency has been reported previously, we observe Class I CO distribution patterns that are strikingly similar to Atfancd2. Surprisingly, we found that AtFANCD2 plays opposite roles in regulating CO frequency in Atfancm compared with either in Atfigl1 or Atrmi1. Together, these results reveal that although AtFANCD2, AtFANCM, AtFIGL1, and AtRMI1 regulate Class II CO frequency by distinct mechanisms, they have similar roles in controlling the distribution of Class I COs among chromosomes.

Facile surface functional polyetheretherketone with antibacterial and immunoregulatory activities for enhanced regeneration toward bacterium-infected bone destruction.

Existing biologically inert or unmodified implants to treat infectious bone defects or osteomyelitis still cannot effectively solve bacterial infection and osseointegration. In this work, a simple co-deposition strategy was developed to modify porous polyetheretherketone (PEEK) with improved antibacterial activity and controllable immunoregulatory ability. After PEEK was treated by H2SO4 to obtain porous PEEK (SPEEK), the self-polymerization of dopamine was operated on SPEEK in the solution of dopamine and gentamicin sulfate (GS) to prepare polydopamine (pDA) and GS layer-modified SPEEK (labeled as SPEEK-pDA-GS). The morphology, surface property, and molecular structure of SPEEK-pDA-GS were investigated. Besides the antibacterial property of SPEEK-pDA-GS ascribed to the successful immobilization of GS, SPEEK-pDA-GS exhibited promoted osseointegration through the results of mineralization, alkaline phosphatase (ALP) levels and osteogenic gene expression. Furthermore, the evaluation of the cell proliferation suggested that SPEEK-pDA-GS possessed the biocompatibility and the immunoregulatory ability that induced macrophages to anti-inflammatory M2 phenotype. Using rat as model, in vivo results containing X-ray, µ-CT, immunohistochemistry, and pathological analysis showed the excellent healing effect of SPEEK-pDA-GS on bone defect with infection with biological safety. This work illustrates a new insight into the simple and effective modification of PEEK and other implants with antibacterial, immunoregulatory, and osseointegration abilities for clinical requirement.

Comparative transcriptomic analysis of thermally stressed Arabidopsis thaliana meiotic recombination mutants.

BACKGROUND: Meiosis is a specialized cell division that underpins sexual reproduction in most eukaryotes. During meiosis, interhomolog meiotic recombination facilitates accurate chromosome segregation and generates genetic diversity by shuffling parental alleles in the gametes. The frequency of meiotic recombination in Arabidopsis has a U-shaped curve in response to environmental temperature, and is dependent on the Type I, crossover (CO) interference-sensitive pathway. The mechanisms that modulate recombination frequency in response to temperature are not yet known. RESULTS: In this study, we compare the transcriptomes of thermally-stressed meiotic-stage anthers from msh4 and mus81 mutants that mediate the Type I and Type II meiotic recombination pathways, respectively. We show that heat stress reduces the number of expressed genes regardless of genotype. In addition, msh4 mutants have a distinct gene expression pattern compared to mus81 and wild type controls. Interestingly, ASY1, which encodes a HORMA domain protein that is a component of meiotic chromosome axes, is up-regulated in wild type and mus81 but not in msh4. In addition, SDS the meiosis-specific cyclin-like gene, DMC1 the meiosis-specific recombinase, SYN1/REC8 the meiosis-specific cohesion complex component, and SWI1 which functions in meiotic sister chromatid cohesion are up-regulated in all three genotypes. We also characterize 51 novel, previously unannotated transcripts, and show that their promoter regions are associated with A-rich meiotic recombination hotspot motifs. CONCLUSIONS: Our transcriptomic analysis of msh4 and mus81 mutants enhances our understanding of how the Type I and Type II meiotic CO pathway respond to environmental temperature stress and might provide a strategy to manipulate recombination levels in plants.

Author Correction: Cell-type-dependent histone demethylase specificity promotes meiotic chromosome condensation in Arabidopsis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cell-type-dependent histone demethylase specificity promotes meiotic chromosome condensation in Arabidopsis.

Histone demethylation is crucial for proper chromatin structure and to ensure normal development, and requires the large family of Jumonji C (JmjC)-containing demethylases; however, the molecular mechanisms that regulate the substrate specificity of these JmjC-containing demethylases remain largely unknown. Here, we show that the substrate specificity of the Arabidopsis histone demethylase JMJ16 is broadened from Lys 4 of histone H3 (H3K4) alone in somatic cells to both H3K4 and H3K9 when it binds to the meiocyte-specific histone reader MMD1. Consistent with this, the JMJ16 catalytic domain exhibits both H3K4 and H3K9 demethylation activities. Moreover, the JMJ16 C-terminal FYR domain interacts with the JMJ16 catalytic domain and probably restricts its substrate specificity. By contrast, MMD1 can compete with the N-terminal catalytic domain of JMJ16 for binding to the FYR-C domain, thereby expanding the substrate specificity of JMJ16 by preventing the FYR domain from binding to the catalytic domain. We propose that MMD1 and JMJ16 together in male meiocytes promote gene expression in an H3K9me3-dependent manner and thereby contribute to meiotic chromosome condensation.

Rapid construction of polyetheretherketone (PEEK) biological implants incorporated with brushite (CaHPO4·2H2O) and antibiotics for anti-infection and enhanced osseointegration.

Polyetheretherketone (PEEK) is an ideal implant material for orthopedic and dental application due to its high biocompatibility and mechanical property. However, biological inertness of PEEK hinders the effective clinical applications in treating bone defect, especially in the situation accompanied by bacterial infection. In this study, a layer-by-layer (LBL) deposition method with controlled cycles was developed to rapidly construct brushite (CaHPO4·2H2O) (CaP) layers containing gentamicin sulfate (GS) on PEEK to obtain CaP-and-GS modified PEEK, named as PEEK/CaP-GS. Different PEEK/CaP-GS, like PEEK/CaP-GS*3, PEEK/CaP-GS*6 and PEEK/CaP-GS*9 were conveniently prepared by repeating the LBL cycles to 3, 6 and 9 times, respectively. The morphology, structure and surface property of the fabricated PEEK/CaP-GS were carefully characterized. In vitro antibacterial experiments illustrated that all of the PEEK/CaP-GS samples had excellent and sustained antibacterial property. Cell proliferation experiments revealed the acceptable biocompatibility and cell osteogenic differentiation of PEEK/CaP-GS, especially in PEEK/CaP-GS*6. X-ray, µ-CT, and histological analysis showed that PEEK/CaP-GS exhibited in vivo antibacterial activity and osseointegration ability in the treatment of bone defect with infection. In both the in vitro and the in vivo experiments, PEEK/CaP-GS*6 prepared from the 6 LBL cycles exhibited the best antibacterial and osseointegration ability for bone healing. This work opens new avenue of the facile and effective modification of PEEK with special biological functions for clinical application, especially for the implants requiring excellent antibacterial and osseointegration ability.

Conservation and Divergence in the Meiocyte sRNAomes of Arabidopsis, Soybean, and Cucumber.

Meiosis is a critical process for sexual reproduction. During meiosis, genetic information on homologous chromosomes is shuffled through meiotic recombination to produce gametes with novel allelic combinations. Meiosis and recombination are orchestrated by several mechanisms including regulation by small RNAs (sRNAs). Our previous work in Arabidopsis (Arabidopsis thaliana) meiocytes showed that meiocyte-specific sRNAs (ms-sRNAs) have distinct characteristics, including positive association with the coding region of genes that are transcriptionally upregulated during meiosis. Here, we characterized the ms-sRNAs in two important crops, soybean (Glycine max) and cucumber (Cucumis sativus). Ms-sRNAs in soybean have the same features as those in Arabidopsis, suggesting that they may play a conserved role in eudicots. We also investigated the profiles of microRNAs (miRNAs) and phased secondary small interfering RNAs in the meiocytes of all three species. Two conserved miRNAs, miR390 and miR167, are highly abundant in the meiocytes of all three species. In addition, we identified three novel cucumber miRNAs. Intriguingly, our data show that the previously identified phased secondary small interfering RNA pathway involving soybean-specific miR4392 is more abundant in meiocytes. These results showcase the conservation and divergence of ms-sRNAs in flowering plants, and broaden our understanding of sRNA function in crop species.