Reciprocal regulation of m 6 A modification and miRNA production machineries via phase separation-dependent and -independent mechanisms.

Methyltransferase complex (MTC) deposits N 6-adenosine (m 6 A) onto RNA, whereas microprocessor produces miRNA. Whether and how these two distinct complexes cross-regulate each other has been poorly studied. Here we report that the MTC subunit B (MTB) tends to form insoluble condensates with poor activity, with its level monitored by 20S proteasome. Conversely, the microprocessor component SERRATE (SE) forms liquid-like condensates, which in turn promotes solubility and stability of MTB, leading to increased MTC activity. Consistently, the hypomorphic lines expressing SE variants, defective in MTC interaction or liquid-like phase behavior, exhibit reduced m 6 A level. Reciprocally, MTC can recruit microprocessor to MIRNA loci, prompting co-transcriptional cleavage of primary miRNA (pri-miRNAs) substrates. Additionally, pri-miRNAs carrying m 6 A modifications at their single-stranded basal regions are enriched by m 6 A readers, which retain microprocessor in the nucleoplasm for continuing processing. This reveals an unappreciated mechanism of phase separation in RNA modification and processing through MTC and microprocessor coordination.

RNA helicase Brr2a promotes miRNA biogenesis by properly remodelling secondary structure of pri-miRNAs.

RNA secondary structure (RSS) of primary microRNAs (pri-miRNAs) is a key determinant for miRNA production. Here we report that RNA helicase (RH) Brr2a, best known as a spliceosome component, modulates the structural complexity of pri-miRNAs to fine tune miRNA yield. Brr2a interacts with microprocessor component HYL1 and its loss reduces the levels of miRNAs derived from both intron-containing and intron-lacking pri-miRNAs. Brr2a binds to pri-miRNAs in vivo and in vitro. Furthermore, Brr2a hydrolyses ATP and the activity can be significantly enhanced by pri-miRNAs. Consequently, Brr2a unwinds pri-miRNAs in vitro. Moreover, Brr2a variants with compromised ATPase or RH activity are incapable of unwinding pri-miRNA, and their transgenic plants fail to restore miRNA levels in brr2a-2. Importantly, most of tested pri-miRNAs display distinct RSS, rendering them unsuitable for efficient processing in brr2a mutants vs Col-0. Collectively, this study reveals that Brr2a plays a non-canonical role in miRNA production beyond splicing regulation.

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.

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.

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.

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.

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.

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.

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.

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.

Degradation of SERRATE via ubiquitin-independent 20S proteasome to survey RNA metabolism.

SERRATE (SE) is a key factor in RNA metabolism. Here, we report that SE binds 20S core proteasome α subunit G1 (PAG1) among other components and is accumulated in their mutants. Purified PAG1-containing 20S proteasome degrades recombinant SE via an ATP- and ubiquitin-independent manner in vitro. Nevertheless, PAG1 is a positive regulator for SE in vivo, as pag1 shows comparable molecular and/or developmental defects relative to se. Furthermore, SE is poorly assembled into macromolecular complexes, exemplified by the microprocessor in pag1 compared with Col-0. SE overexpression triggered the destruction of both transgenic and endogenous protein, leading to similar phenotypes of se and SE overexpression lines. We therefore propose that PAG1 degrades the intrinsically disordered portion of SE to secure the functionality of folded SE that is assembled and protected in macromolecular complexes. This study provides insight into how the 20S proteasome regulates RNA metabolism through controlling its key factor in eukaryotes.

Regioselective electrosynthesis of tetra- and hexa-functionalized [60]fullerene derivatives with unprecedented addition patterns.

The efficient and regioselective electrosynthesis of tetra- and hexa-functionalized [60]fullerene derivatives with unprecedented addition patterns has been achieved. The tetra-functionalized [60]fullerene derivative with an intriguing 1,2,4,17-addition pattern is regioselectively obtained by cyclization reaction of the dianionic species generated electrochemically from a [60]fulleroindoline with 1,2-bis(bromomethyl)benzene at 0 °C, and can be converted to the more stable 1,2,3,4-adduct at 25 °C. Furthermore, the hexa-functionalized [60]fullerene derivative with the 1,2,3,4,9,10-addition pattern displaying a unique "S"-shaped configuration can be synthesized by protonation of the electrochemically generated dianion of the obtained tetra-functionalized 1,2,4,17-adduct. The structures of the tetra- and hexa-functionalized products have been determined by spectroscopic data and single-crystal X-ray analysis.

Successively Regioselective Electrosynthesis and Electron Transport Property of Stable Multiply Functionalized [60]Fullerene Derivatives.

With the recent advance in chemical modification of fullerenes, electrosynthesis has demonstrated increasing importance in regioselective synthesis of novel fullerene derivatives. Herein, we report successively regioselective synthesis of stable tetra- and hexafunctionalized [60]fullerene derivatives. The cycloaddition reaction of the electrochemically generated dianions from [60]fulleroindolines with phthaloyl chloride regioselectively affords 1,2,4,17-functionalized [60]fullerene derivatives with two attached ketone groups and a unique addition pattern, where the heterocycle is rearranged to a [5,6]-junction and the carbocycle is fused to an adjacent [6,6]-junction. This addition pattern is in sharp contrast with that of the previously reported biscycloadducts, where both cycles are appended to [6,6]-junctions. The obtained tetrafunctionalized compounds can be successively manipulated to 1,2,3,4,9,10-functionalized [60]fullerene derivatives with an intriguing "S"-shaped configuration via a novel electrochemical protonation. Importantly, the stability of tetrafunctionalized [60]fullerene products allows them to be applied in planar perovskite solar cells as efficient electron transport layers.

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.

[Environmental Response and Ecological Function Prediction of Aquatic Bacterial Communities in the Weihe River Basin].

The diversity of bacterial communities and their metabolic function in the waters of the Weihe River Basin are of great significance for water pollution remediation, ecological restoration, and water quality assessment. Illumina MiSeq high-throughput sequencing technology was applied to study the bacterial community distribution characteristics after the comprehensive treatment of the Shaanxi part of the Weihe River Basin. Furthermore, the correlation between the bacterial communities and environmental factors was analyzed by redundancy analysis (RDA), and the PICRUSt method was adopted to evaluate the ecological function of the bacterial communities. The results showed that Proteobacteria, Actinobacteria, Cyanobacteria, and Bacteroidetes were the main bacterial communities in the water, accounting for 85% of the total microbial community. Additionally, these bacteria showed a significant positive correlation (P=0.02, <0.05) with the TP, NO2--N, NO3--N, and TN. The bacteria community richness of water samples collected in Zaohe River was the lowest of all samples, while which in the downstream of the junction of Bahe river and Weihe river was the highest. In addition, water in the Weihe River Basin had great impact on the endocrine systems of aquatic organisms, and also had a possibility of infectious diseases for humans. Our research provides a theoretical basis for the safe and sustainable development of the water environment in the Weihe River Basin.