Intrinsically disordered proteins SAID1/2 condensate on SERRATE for dual inhibition of miRNA biogenesis in Arabidopsis.

Intrinsically disordered proteins (IDPs) SAID1/2 are hypothetic dentin sialophosphoprotein-like proteins, but their true functions are unknown. Here, we identified SAID1/2 as negative regulators of SERRATE (SE), a core factor in miRNA biogenesis complex (microprocessor). Loss-of-function double mutants of said1; said2 caused pleiotropic developmental defects and thousands of differentially expressed genes that partially overlapped with those in se. said1; said2 also displayed increased assembly of microprocessor and elevated accumulation of microRNAs (miRNAs). Mechanistically, SAID1/2 promote pre-mRNA processing 4 kinase A-mediated phosphorylation of SE, causing its degradation in vivo. Unexpectedly, SAID1/2 have strong binding affinity to hairpin-structured pri-miRNAs and can sequester them from SE. Moreover, SAID1/2 directly inhibit pri-miRNA processing by microprocessor in vitro. Whereas SAID1/2 did not impact SE subcellular compartmentation, the proteins themselves exhibited liquid-liquid phase condensation that is nucleated on SE. Thus, we propose that SAID1/2 reduce miRNA production through hijacking pri-miRNAs to prevent microprocessor activity while promoting SE phosphorylation and its destabilization in Arabidopsis.

A MYB4-MAN3-Mannose-MNB1 signaling cascade regulates cadmium tolerance in Arabidopsis.

Our previous studies showed that MAN3-mediated mannose plays an important role in plant responses to cadmium (Cd) stress. However, the underlying mechanisms and signaling pathways involved are poorly understood. In this study, we showed that an Arabidopsis MYB4-MAN3-Mannose-MNB1 signaling cascade is involved in the regulation of plant Cd tolerance. Loss-of-function of MNB1 (mannose-binding-lectin 1) led to decreased Cd accumulation and tolerance, whereas overexpression of MNB1 significantly enhanced Cd accumulation and tolerance. Consistently, expression of the genes involved in the GSH-dependent phytochelatin (PC) synthesis pathway (such as GSH1, GSH2, PCS1, and PCS2) was significantly reduced in the mnb1 mutants but markedly increased in the MNB1-OE lines in the absence or presence of Cd stress, which was positively correlated with Cd-activated PC synthesis. Moreover, we found that mannose is able to bind to the GNA-related domain of MNB1, and that mannose binding to the GNA-related domain of MNB1 is required for MAN3-mediated Cd tolerance in Arabidopsis. Further analysis showed that MYB4 directly binds to the promoter of MAN3 to positively regulate the transcript of MAN3 and thus Cd tolerance via the GSH-dependent PC synthesis pathway. Consistent with these findings, overexpression of MAN3 rescued the Cd-sensitive phenotype of the myb4 mutant but not the mnb1 mutant, whereas overexpression of MNB1 rescued the Cd-sensitive phenotype of the myb4 mutant. Taken together, our results provide compelling evidence that a MYB4-MAN3-Mannose-MNB1 signaling cascade regulates cadmium tolerance in Arabidopsis through the GSH-dependent PC synthesis pathway.

Application of Sigma metrics in the quality control strategies of immunology and protein analytes.

BACKGROUND: Six Sigma (6σ) is an efficient laboratory management method. We aimed to analyze the performance of immunology and protein analytes in terms of Six Sigma. METHODS: Assays were evaluated for these 10 immunology and protein analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C-reactive protein (CRP), and Cystatin C (Cys C). The Sigma values were evaluated based on bias, four different allowable total error (TEa) and coefficient of variation (CV) at QC materials levels 1 and 2 in 2020. Sigma Method Decision Charts were established. Improvement measures of analytes with poor performance were recommended according to the quality goal index (QGI), and appropriate quality control rules were given according to the Sigma values. RESULTS: While using the TEaNCCL , 90% analytes had a world-class performance with σ>6, Cys C showed marginal performance with σ<4. While using minimum, desirable, and optimal biological variation of TEa, only three (IgG, IgM, and CRP), one (CRP), and one (CRP) analytes reached 6σ level, respectively. Based on σNCCL that is calculated from TEaNCCL , Sigma Method Decision Charts were constructed. For Cys C, five multi-rules (13s /22s /R4s /41s /6X , N = 6, R = 1, Batch length: 45) were adopted for QC management. The remaining analytes required only one QC rule (13s , N = 2, R = 1, Batch length: 1000). Cys C need to improve precision (QGI = 0.12). CONCLUSIONS: The laboratories should choose appropriate TEa goals and make judicious use of Sigma metrics as a quality improvement tool.

Oncostatin M Is a Prognostic Biomarker and Inflammatory Mediator for Sepsis.

BACKGROUND: Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin-6 family. The role of OSM in sepsis remains unknown. METHODS: Serum OSM level was determined and analyzed in septic patients on the day of intensive care unit (ICU) admission. Furthermore, the effects of OSM on polymicrobial sepsis induced by cecal ligation and puncture (CLP) were assessed. RESULTS: On the day of ICU admission, septic patients had significantly higher serum OSM levels when compared with ICU patient controls and healthy volunteers, which were related to the severity of sepsis, including parameters such as the sequential (sepsis-related) organ failure assessment score, procalcitonin level, and white blood cell number. A high serum OSM level on ICU admission was associated with 28-day mortality in septic patients. In CLP-induced polymicrobial sepsis, anti-OSM antibody decreased tissue inflammation and injury, and thus improved survival, while local and systemic bacterial dissemination was almost constant. Complementarily, supplementation with recombinant OSM protein in septic mice increased tissue injury, amplified inflammation, and worsened mortality after CLP, while it did not affect bacterial dissemination in septic mice. CONCLUSIONS: Sepsis results in an increased production of OSM, which might be a potential prognostic biomarker and therapeutic target for sepsis.

Interleukin-17D Aggravates Sepsis by Inhibiting Macrophage Phagocytosis.

OBJECTIVES: Interleukin-17D has been shown to participate in the control of viral infections and cancer. Here we hypothesized that interleukin-17D may play a potential role in sepsis. DESIGN: Prospective randomized animal investigation and in vitro human blood studies. SETTING: Research laboratory from a university hospital. SUBJECTS: Female C57BL/6J mice, sepsis patients by Sepsis-3 definitions, ICU patient controls, and healthy individuals. INTERVENTIONS: Serum concentrations of interleukin-17D were measured and analyzed in human sepsis patients, patient controls, and healthy individuals. The contribution of interleukin-17D to sepsis-related survival, bacterial burden, and organ injury was assessed in a murine model of cecal ligation and puncture-induced polymicrobial sepsis by the use of anti-interleukin-17D antibody and recombinant interleukin-17D protein. The effects of interleukin-17D on bacterial phagocytosis by macrophages were also investigated using in vitro cell models. MEASUREMENTS AND MAIN RESULTS: On the day of ICU admission (day 0), septic patients had significantly higher serum concentrations of interleukin-17D than patient controls and healthy individuals. Serum interleukin-17D levels remained significantly elevated in septic patients from ICU admission to day 3 and correlated with Sequential (Sepsis-related) Organ Failure Assessment scores and documented bacteremia on day 0. Furthermore, nonsurvivors of septic patients displayed significantly higher interleukin-17D levels compared with survivors of septic patients on days 0 and 1 of ICU admission. In animal models of sepsis, treatment with anti-interleukin-17D antibody protected mice from cecal ligation and puncture-induced severe sepsis, which was associated with improved bacterial clearance and organ injury. Conversely, administration of recombinant interleukin-17D protein aggravated cecal ligation and puncture-induced nonsevere sepsis. Furthermore, we found that interleukin-17D impaired bacterial phagocytosis by macrophages. Phagocytosis inhibition by interleukin-17D involved its ability to down-regulate the activation of nuclear factor-κB signaling pathway in macrophages upon bacterial infection. CONCLUSIONS: This study indicates a previously undescribed role of interleukin-17D in sepsis and identifies a new target for antisepsis treatment.

Therapeutic Targeting of Apoptosis Inhibitor of Macrophage/CD5L in Sepsis.

The factors involved in disturbing host homeostasis during sepsis are largely unknown. We sought to determine the immunopathological role of apoptosis inhibitor of macrophage (AIM)/CD5L in sepsis. Here, we show that blockade of AIM led to significantly increased survival after experimental sepsis, and it decreased local and systemic inflammation, reduced tissue injury, and inhibited bacterial dissemination in the blood, in particular at later time points. Supplementation of recombinant AIM in sepsis resulted in increased tissue injury, amplified inflammation, increased bacteremia, and worsened mortality. Interestingly, the most important difference in the production of cytokines and chemokines after in vivo AIM blockade or AIM administration during sepsis was IL-10. In vitro, AIM enhanced IL-10 production from macrophages, neutrophils, or lymphocytes. In vivo, the beneficial effects of AIM blockade and the detrimental effects of AIM addition on experimental sepsis were ablated by treatment with recombinant IL-10 and neutralizing anti-IL-10 antibodies, respectively. This study is the first to identify AIM as an important mediator in disturbing host homeostasis in sepsis.

Progranulin aggravates pulmonary immunopathology during influenza virus infection.

Progranulin (PGRN) exerts multiple functions in various inflammatory diseases. However, the role of PGRN in the pathogenesis of virus infection is unknown. Here, we demonstrated that PGRN production was up-regulated in clinical and experimental influenza, which contributed to the deleterious inflammatory response after influenza virus infection in mice. PGRN-deficient mice were protected from influenza virus-induced lung injury and mortality. Decreased mortality was associated with significantly reduced influx of neutrophils and monocytes/macrophages, release of cytokines and chemokines, and permeability of the alveolar-epithelial barrier without affecting viral clearance. Our findings suggest that PGRN exacerbates pulmonary immunopathology during influenza virus infection.

The WRKY transcription factor, WRKY13, activates PDR8 expression to positively regulate cadmium tolerance in Arabidopsis.

Cadmium (Cd) extrusion is an important mechanism conferring Cd tolerance by decreasing its accumulation in plants. Previous studies have identified an Arabidopsis ABC transporter, PDR8, as a Cd extrusion pump conferring Cd tolerance. However, the regulation of PDR8 in response to Cd stress is still largely unknown. In this study, we identified an Arabidopsis cadmium-tolerant dominant mutant, designated xcd3-D, from the XVE-tagging T-DNA insertion lines by a gain-of-function genetic screen. The corresponding gene was cloned and shown to encode a nuclear WRKY transcription factor WRKY13. Expression of WRKY13 was induced by Cd stress. Overexpression of WRKY13 resulted in decreased Cd accumulation and enhanced Cd tolerance, whereas loss-of-function of WRKY13 led to increased Cd accumulation and sensitivity. Further analysis showed that WRKY13 activates the transcription of PDR8 by directly binding to its promoter. Genetic analysis indicated that WRKY13 acts upstream of PDR8 to positively regulate Cd tolerance. Our results provide evidence that WRKY13 directly targets PDR8 to positively regulate Cd tolerance in Arabidopsis.

Flagellin attenuates experimental sepsis in a macrophage-dependent manner.

BACKGROUND: Sepsis is the leading cause of death among critically ill patients, and no specific therapeutic agent is currently approved for the treatment of sepsis. METHODS: We assessed the effects of flagellin administration on survival, bacterial burden, and tissue injury after sepsis. In addition, we examined the effects on phagocytosis and bacterial killing in monocytes/macrophages. RESULTS: Therapeutic administration of flagellin increased bacterial clearance, decreased organ inflammation and injury, and reduced immune cell apoptosis after experimental sepsis, in a Toll-like receptor 5 (TLR5)-dependent manner. Macrophages, but not neutrophils, mediated the beneficial effects of flagellin on experimental sepsis, and flagellin induced macrophage polarization into M1 in septic mice. Flagellin treatment could directly enhance phagocytosis and bacterial killing of macrophages, but not neutrophils. Subsequent studies demonstrated that flagellin could promote phagosome formation and increase reactive oxygen species (ROS) levels in macrophages. Finally, we found that the expression of TLR5 was significantly elevated on the surface of circulating monocytes, but not neutrophils, from patients with sepsis. Higher expression levels of TLR5 on monocytes were associated with increased mortality, documented bacteremia, and higher Sequential Organ Failure Assessment scores of the septic patients. Moreover, flagellin treatment rescued the impaired phagocytosis and bacterial killing ability of monocytes/macrophages from patients who died of sepsis. CONCLUSIONS: These novel findings not only established the potential value of application of flagellin as an immunoadjuvant in treating sepsis, but also provided new insights into targeted therapeutic strategy on the basis of monocyte TLR5 expression in septic patients.

Interleukin 38 Protects Against Lethal Sepsis.

Background: Interleukin 38 (IL-38) is the most recently characterized cytokine of the interleukin 1 family. However, its role in sepsis remains unknown. Methods: Circulating IL-38 levels were measured in 2 cohorts of adult and pediatric patients with sepsis. Using 2 murine models of lipopolysaccharide (LPS)-induced endotoxemia and cecal ligation and puncture (CLP)-induced sepsis, the effects of IL-38 on survival, inflammation, tissue injury, and bacterial clearance were assessed. Results: Serum IL-38 concentrations were significantly elevated in adult and pediatric patients with sepsis relative to corresponding healthy adult and pediatric controls, respectively. An increased IL-38 level negatively correlated with the number of blood leukocytes and with the level of inflammatory cytokines, including interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in clinical sepsis. Anti-IL-38 antibody impaired survival and while recombinant IL-38 improved survival in the 2 murine models of LPS-induced endotoxemia and CLP-induced sepsis. IL-38 administration decreased the inflammatory response, as reflected by lower levels of cytokines and chemokines (including IL-6, TNF-α, interleukin 10, interleukin 17, interleukin 27, CXCL1, and CCL2), and less damage to tissues (including lung, liver, and kidney) in CLP-induced sepsis. Furthermore, IL-38 augmented bacterial clearance in CLP-induced polymicrobial sepsis. Conclusions: These findings suggest that IL-38 attenuates sepsis by decreasing inflammation and increasing bacterial clearance, thus providing a novel tool for antisepsis therapy.

Interleukin-34 Ameliorates Survival and Bacterial Clearance in Polymicrobial Sepsis.

OBJECTIVES: Sepsis is a devastating condition with a high mortality rate and limited treatments. Sepsis is characterized by a failed host immune response to contain the infection, resulting in organ dysfunction. Interleukin-34 is new cytokine involved in infection and immunity. Whether interleukin-34 is beneficial or deleterious to sepsis and the underlying mechanisms remains unknown. DESIGN: Prospective randomized animal investigation and in vitro studies. SETTING: Research laboratory at a university hospital. SUBJECTS: Wild-type C57BL/6 mice were used for in vivo studies, and septic human patients and healthy human subjects were used to obtain blood for in vitro studies. INTERVENTIONS: Interleukin-34 concentrations were measured in human sepsis patients and healthy individuals. The effects of interleukin-34 administration on survival, bacterial burden, organ injury, and inflammatory response were assessed in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. MEASUREMENTS AND MAIN RESULTS: Interleukin-34 levels were significantly elevated in human sepsis and cecal ligation and puncture-induced experimental sepsis. Interleukin-34 administration improved survival and bacterial clearance, although suppressed vascular leakage and organ injury after cecal ligation and puncture-induced polymicrobial sepsis. Neutralization of interleukin-34 increased mortality rate and decreased bacterial clearance in septic mice. An increased neutrophil and macrophage influx were developed in interleukin-34-treated mice at the site of infection, accompanied by elevated production of neutrophil chemokine chemokine (C-X-C motif) ligand 1 and macrophage chemokine C-C motif chemokine ligand 2 in the peritoneal cavity. Depletion of neutrophils or macrophages reversed interleukin-34-mediated protection against polymicrobial sepsis. CONCLUSIONS: We reported for the first time a potential therapeutic role for interleukin-34 in sepsis and suggested that interleukin-34 is a novel target for the development of therapeutic agents against sepsis.

Zinc-Finger Transcription Factor ZAT6 Positively Regulates Cadmium Tolerance through the Glutathione-Dependent Pathway in Arabidopsis.

Cadmium (Cd) is an environmental pollutant with high toxicity to animals and plants. It has been established that the glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway is one of the most important mechanisms contributing to Cd accumulation and tolerance in plants. However, the transcription factors involved in regulating GSH-dependent PC synthesis pathway remain largely unknown. Here, we identified an Arabidopsis (Arabidopsis thaliana) Cd-resistant mutant xcd2-D (XVE system-induced cadmium-tolerance2) using a forward genetics approach. The mutant gene underlying xcd2-D mutation was revealed to encode a known zinc-finger transcription factor, ZAT6. Transgenic plants overexpressing ZAT6 showed significant increase of Cd tolerance, whereas loss of function of ZAT6 led to decreased Cd tolerance. Increased Cd accumulation and tolerance in ZAT6-overexpressing lines was GSH dependent and associated with Cd-activated synthesis of PC, which was correlated with coordinated activation of PC-synthesis related gene expression. By contrast, loss of function of ZAT6 reduced Cd accumulation and tolerance, which was accompanied by abolished PC synthesis and gene expression. Further analysis revealed that ZAT6 positively regulates the transcription of GSH1, GSH2, PCS1, and PCS2, but ZAT6 is capable of specifically binding to GSH1 promoter in vivo. Consistently, overexpression of GSH1 has been shown to restore Cd sensitivity in the zat6-1 mutant, suggesting that GSH1 is a key target of ZAT6. Taken together, our data provide evidence that ZAT6 coordinately activates PC synthesis-related gene expression and directly targets GSH1 to positively regulate Cd accumulation and tolerance in Arabidopsis.

IL-36 induces cytokine IL-6 and chemokine CXCL8 expression in human lung tissue cells: Implications for pulmonary inflammatory responses.

IL-36α, IL-36ß and IL-36γ are cytokine members of IL-1 family. Although IL-36 expression was observed in human lung during pulmonary infections, it remains unknown whether IL-36 could act directly on lung tissue cells during pulmonary inflammatory responses. In this study, we showed that IL-36 receptor was expressed in human lung fibroblasts and bronchial epithelial cells. Correspondingly, IL-36α, IL-36ß or IL-36γ up-regulated gene expression of cytokine IL-6 and chemokine CXCL8 in human lung fibroblasts and bronchial epithelial cells, and promoted IL-6 and CXCL8 release from human lung fibroblasts and bronchial epithelial cells. The production of IL-6 and CXCL8 in these lung tissues cells induced by IL-36α, IL-36ß or IL-36γ was regulated by p38MAPK, ERK or Akt signaling pathways. Taken together, the above results suggest that IL-36-mediated IL-6 and CXCL8 production in human lung fibroblasts and bronchial epithelial cells may be involved in pulmonary inflammation especially caused by bacterial or viral infections.

Parallel degradome-seq and DMS-MaPseq substantially revise the miRNA biogenesis atlas in Arabidopsis.

MicroRNAs (miRNAs) are produced from highly structured primary transcripts (pri-miRNAs) and regulate numerous biological processes in eukaryotes. Due to the extreme heterogeneity of these structures, the initial processing sites of plant pri-miRNAs and the structural rules that determine their processing have been predicted for many miRNAs but remain elusive for others. Here we used semi-active DCL1 mutants and advanced degradome-sequencing strategies to accurately identify the initial processing sites for 147 of 326 previously annotated Arabidopsis miRNAs and to illustrate their associated pri-miRNA cleavage patterns. Elucidating the in vivo RNA secondary structures of 73 pri-miRNAs revealed that about 95% of them differ from in silico predictions, and that the revised structures offer clearer interpretation of the processing sites and patterns. Finally, DCL1 partners Serrate and HYL1 could synergistically and independently impact processing patterns and in vivo RNA secondary structures of pri-miRNAs. Together, our work sheds light on the precise processing mechanisms of plant pri-miRNAs.

Pirfenidone inhibits stromal collagen deposition and improves intra-tumoral delivery and antitumor efficacy of Pegylated liposomal doxorubicin.

The effectiveness of cancer nanotherapeutics is greatly restricted by the dense collagen network in solid tumors. Pirfenidone (PFD) is a clinically approved oral antifibrotic agent widely used to treat idiopathic pulmonary fibrosis. To investigate whether PFD can enhance the penetration and tumor delivery efficiency of Pegylated liposomal doxorubicin (PLD), colorectal cancer xenograft mice were administered PFD, PLD, or combined regimens. As expected, high-dose PFD (H-PFD, 270 mg/kg/day) combined with PLD (H-PFD + PLD) exhibited a significantly higher tumor inhibition rate than PLD monotherapy (75.09% vs. 60.87%). Similarly, the intra-tumoral doxorubicin level was markedly elevated using H-PFD pretreatment, which induced over 34% elevation compared to PLD treatment alone (3.37 ± 0.41 vs. 2.51 ± 0.19 µg/mL). Additionally, Masson's trichrome staining and immunohistochemistry results of the H-PFD + PLD group revealed an attenuation of collagen deposition in vivo, and the in vitro TGF-ß1, α-SMA, and collagen protein expression were inhibited using PFD treatment. In contrast, although low-dose PFD (60 mg/kg/day) did not present superior benefits in promoting PLD penetration into tumors, it did downregulate collagen expression in vivo. This study provides a new strategy for PFD combined with chemotherapeutic drugs to improve the antitumor efficacy of nanomedicines.

Electrochemically Promoted Benzylation of [60]Fullerooxazolidinone.

Benzylation of the electrochemically generated dianion from N-p-tolyl-[60]fullerooxazolidinone with benzyl bromide provides three products with different addition patterns. The product distribution can be dramatically altered by varying the reaction conditions. Based on spectral characterizations, these products have been assigned as mono-benzylated 1,4-adduct and bis-benzylated 1,2,3,16- and 1,4,9,25-adducts, respectively. The assigned 1,2,3,16-adduct has been further established by X-ray diffraction analysis. It is believed that the 1,4-adduct is obtained by decarboxylative benzylation of the dianionic species, while bis-benzylated 1,2,3,16- and 1,4,9,25-adducts are achieved via a rearrangement process. In addition, the electrochemical properties of these products have been studied.

PRP4KA phosphorylates SERRATE for degradation via 20S proteasome to fine-tune miRNA production in Arabidopsis.

Phosphorylation can quickly switch on/off protein functions. Here, we reported pre-mRNA processing 4 kinase A (PRP4KA), and its paralogs interact with Serrate (SE), a key factor in RNA processing. PRP4KA phosphorylates at least five residues of SE in vitro and in vivo. Hypophosphorylated, but not hyperphosphorylated, SE variants could readily rescue se phenotypes in vivo. Moreover, hypophosphorylated SE variants had stronger binding affinity to microprocessor component HYL1 and were more resistant to degradation by 20S proteasome than hyperphosphorylated counterparts. Knockdown of the kinases enhanced the accumulation of hypophosphorylated SE. However, the excessive SE interfered with the assembly and function of SE-scaffolded macromolecule complexes, causing the se-like defects in the mutant and wild-type backgrounds. Thus, phosphorylation of SE via PRP4KA can quickly clear accumulated SE to secure its proper amount. This study provides new insight into how protein phosphorylation regulates miRNA metabolism through controlling homeostasis of SE accumulation in plants.

In vitro Reconstitution Assays of Arabidopsis 20S Proteasome.

The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the ubiquitin-independent 20S proteasome. Emerging evidence indicates that plant IDP homeostasis may also be controlled by the 20S proteasome. Relatively little is known about the specific functions of the 20S proteasome and the regulatory mechanisms of IDP degradation in plants compared to other species because there is a lack of systematic protocols for in vitro assembly of this complex to perform in vitro degradation assays. Here, we present a detailed protocol of in vitro reconstitution assay of the 20S proteasome in Arabidopsis by modifying previously reported methods. The main strategy to obtain the 20S core proteasome here is to strip away the 19S regulatory subunits from the 26S proteasome. The protocol has two major parts: 1) Affinity purification of 20S proteasomes from stable transgenic lines expressing epitope-tagged PAG1, an essential component of the 20S proteasome (Procedures A-D) and 2) an in vitro 20S proteasome degradation assay (Procedure E). We anticipate that these protocols will provide simple and effective approaches to study in vitro degradation by the 20S proteasome and advance the study of protein metabolism in plants.

The epigenetic factor FVE orchestrates cytoplasmic SGS3-DRB4-DCL4 activities to promote transgene silencing in Arabidopsis.

Posttranscriptional gene silencing (PTGS) is a regulatory mechanism to suppress undesired transcripts. Here, we identified Flowering locus VE (FVE), a well-known epigenetic component, as a new player in cytoplasmic PTGS. Loss-of-function fve mutations substantially reduced the accumulation of transgene-derived small interfering RNAs (siRNAs). FVE interacts with suppressor of gene silencing 3 (SGS3), a master component in PTGS. FVE promotes SGS3 homodimerization that is essential for its function. FVE can bind to single-stranded RNA and double-stranded RNA (dsRNA) with moderate affinities, while its truncated form FVE-8 has a significantly increased binding affinity to dsRNA. These affinities affect the association and channeling of SGS3-RNA to downstream dsRNA binding protein 4 (DRB4)/Dicer-like protein 2/4 (DCL2/4) complexes. Hence, FVE, but not FVE-8, biochemically enhances the DRB4/DCL2/4 activity in vitro. We surmise that FVE promotes production of transgene-derived siRNAs through concertedly tuning SGS3-DRB4/DCL2/4 functions. Thus, this study revealed a noncanonical role of FVE in PTGS.

Timeliness, completeness, and timeliness-and-completeness of serial routine vaccinations among rural children in Southwest China: A multi-stage stratified cluster sampling survey.

INTRODUCTION: Vaccination coverage is widely used as metric of vaccination programme performance. However, VPDs outbreaks were reported in areas with high vaccination coverage. Timeliness and completeness have been considered more important assessment indicators of routine vaccination than overall vaccination coverage, but little is known in rural China. This study aimed to assess the timeliness and completeness of serial routine vaccinations among children in rural Southwest China. METHODS: A multi-stage stratified cluster survey was conducted among 1062 children aged 18-48 months in rural Guangxi. Vaccination status was obtained from child's vaccination certificate. We calculated timely vaccination coverage, complete vaccination coverage, timely-and-complete vaccination coverage and 95% CI for routine vaccination through weighted estimation analysis. Weighted Kaplan-Meier analyses were applied to estimate the median delay periods for each dose of serial routine vaccines, including one-dose BCG, three-dose HepB, three-dose OPV, four-dose DTP, two-dose MCV, two-dose JEV and two-dose MPV-A. Complete coverage, and timely-and-complete coverage for combined 5-vaccine series were calculated. RESULTS: For each dose of routine vaccines, overall vaccination coverages were over 90%, but timely vaccination coverage ranged from the lowest of 44.4% for JEV1 to the highest of 92.5% for MPV-A1. For multi-dose routine vaccines, complete vaccination coverages varied from the lowest of 92.9% for MCV to the highest of 100% for HepB, and timely-and-complete vaccination coverages were lower than 80%, ranging from the lowest of 30% for JEV to the highest of 77.2% for MPV-A. For combined 5-vaccine series, complete coverage was 77%, while timely-and-complete coverage was 12.1%. MPV-A1 had the longest median delay of 176 days, but BCG and HepB1 had the shortest of 1 day. CONCLUSIONS: The overall coverages of serial routine vaccinations were high, but the timeliness and completeness were poor. Relevant agencies of vaccination service should address timeliness-and-completeness into the assessment indicators of routine vaccination service quality.