Grain chalkiness traits is affected by the synthesis and dynamic accumulation of the storage protein in rice.

BACKGROUOND: Grain chalkiness lowers the market value of rice. Alleviating grain chalkiness is the most challenging issue in many rice-producing areas of the world. Nitrogen (N) metabolism has received increasing attention as a result of its relationship with grain chalkiness, although little information is available on the mechanism of N-induced grain chalk. RESULTS: A highly chalky rice variety OM052 was used to explore the protein synthesis and its accumulation in the grain exposed to N topdressing (N+) at the panicle initiation stage and a control (N-). The results showed that chalky kernels were stimulated by the N+ treatment and more prone to occur on the top and primary rachis. The grain protein content was increased because of the increased average and maximum rates of protein accumulation during grain filling, which was related to the enhanced activities of glutamine synthetase, glutamate synthase, glutamic oxalo-acetic transaminase and glutamate pyruvate transaminase under the N+ treatment. The activities of these enzymes at 15 days after flowering (DAF) were notably positively correlated with grain chalky traits and protein content. CONCLUSION: N topdressing regulates the synthesis and accumulation of the protein by affecting the key enzymes, especially at 15 DAF, which is attributed to grain chalkiness in rice. © 2021 Society of Chemical Industry.

Automatic and Robust Infrared-Visible Image Sequence Registration via Spatio-Temporal Association.

To solve the problems of the large differences in gray value and inaccurate positioning of feature information during infrared-visible image registration, we propose an automatic and robust algorithm for registering planar infrared-visible image sequences through spatio-temporal association. In particular, we first create motion vector distribution descriptors which represent the temporal motion information of foreground contours in adjacent frames to complete coarse registration without feature extraction. Then, for precise registration, we extracted FAST corners of the foreground, which are described by the spatial location distribution of contour points based on connected blob detection, and match these corners using bidirectional optimal maximum strategy. Finally, a reservoir updated by Better-In, Worse-Out (BIWO) strategy is established to save matched point pairs and obtain the optimal global transformation matrix. Extensive evaluations on the LITIV dataset well demonstrate the effectiveness of the proposed algorithm. Particularly, our algorithm achieves lower registration overlapping errors than the other two state-of-the-arts.

The role and mechanism of action of miR­92a in endothelial cell autophagy.

Although microRNAs (miRNAs/miRs) serve a significant role in the autophagy of vascular endothelial cells (ECs), the effect of miR­92a on the autophagy of ECs is currently unclear. Therefore, the present study aimed to investigate the impact of miR­92a on autophagy in ECs and the underlying molecular processes that control this biological activity. Firstly, an autophagy model of EA.hy926 cells was generated via treatment with the autophagy inducer rapamycin (rapa­EA.hy926 cells). The expression levels of miR­92a were then detected by reverse transcription­quantitative PCR, and the effect of miR­92a expression on the autophagic activity of rapa­EA.hy926 cells was studied by overexpressing or inhibiting miR­92a. The level of autophagy was evaluated by western blot analysis, immunofluorescence staining and transmission electron microscopy. Dual­luciferase reporter assays were used to confirm the interaction between miR­92a and FOXO3. The results demonstrated that the expression levels of miR­92a were decreased in the rapa­EA.hy926 cell autophagy model. Furthermore, overexpression and inhibition of miR­92a revealed that upregulation of miR­92a in these cells inhibited autophagy, whereas miR­92a knockdown promoted it. It was also confirmed that miR­92a directly bound to the 3'­untranslated region of the autophagy­related gene FOXO3 and reduced its expression. In conclusion, the present study suggested that miR­92a inhibits autophagy activity in EA.hy926 cells by targeting FOXO3.

The effect of miR-223-3p on endothelial cells in coronary artery disease.

Endothelial cell damage and dysfunction are crucial factors in the development and early stages of coronary artery disease (CAD) and apoptosis plays a significant role in this process. In this study, We aimed to simulate the CAD vascular microenvironment by treating endothelial cells with tumor necrosis factor alpha (TNF-α) to construct an endothelial cell apoptosis model. Our findings revealed that the TNF-α model resulted in increased micro-RNA 223-3p (miR-223-3p) mRNA and Bax protein expression, decreased kruppel-like factor 15 (KLF15) and Bcl-2 protein expression, and decreased cell viability. More importantly, in the TNF-α-induced endothelial cell apoptosis model, transfection with the miR-223-3p inhibitor reversed the effects of TNF-α on Bcl-2, Bax expression. We transfected miRNA-223-3p mimics or inhibitors into endothelial cells and assessed miR-223-3p levels using RT-PCR. Cell viability was detected using CCK8. Western blot technology was used to detect the expression of Bcl-2, Bax, and KLF15. In summary, this study demonstrates the role and possible mechanism of miR-223-3p in endothelial cells during CAD, suggesting that miR-223-3p may serve as a promising therapeutic target in CAD by regulating KLF15.

Repurposing FDA-Approved Drugs for Temozolomide-Resistant IDH1 Mutant Glioma Using High-Throughput Miniaturized Screening on Droplet Microarray Chip.

To address the challenge of drug resistance and limited treatment options for recurrent gliomas with IDH1 mutations, a highly miniaturized screening of 2208 FDA-approved drugs is conducted using a high-throughput droplet microarray (DMA) platform. Two patient-derived temozolomide-resistant tumorspheres harboring endogenous IDH1 mutations (IDH1mut ) are utilized. Screening identifies over 20 drugs, including verteporfin (VP), that significantly affected tumorsphere formation and viability. Proteomics analysis reveals that nuclear pore complex may be a potential VP target, suggesting a new mechanism of action independent of its known effects on YAP1. Knockdown experiments exclude YAP1 as a drug target in tumorspheres. Pathway analysis shows that NUP107 is a potential upstream regulator associated with VP response. Analysis of publicly available genomic datasets shows a significant correlation between high NUP107 expression and decreased survival in IDH1mut astrocytoma, suggesting NUP107 may be a potential biomarker for VP response. This study demonstrates a miniaturized approach for cost-effective drug repurposing using 3D glioma models and identifies nuclear pore complex as a potential target for drug development. The findings provide preclinical evidence to support in vivo and clinical studies of VP and other identified compounds to treat IDH1mut gliomas, which may ultimately improve clinical outcomes for patients with this challenging disease.

TRIM67 drives tumorigenesis in oligodendrogliomas through Rho GTPase-dependent membrane blebbing.

BACKGROUND: IDH mutant gliomas are grouped into astrocytomas or oligodendrogliomas depending on the codeletion of chromosome arms 1p and 19q. Although the genomic alterations of IDH mutant gliomas have been well described, transcriptional changes unique to either tumor type have not been fully understood. Here, we identify Tripartite Motif Containing 67 (TRIM67), an E3 ubiquitin ligase with essential roles during neuronal development, as an oncogene distinctly upregulated in oligodendrogliomas. METHODS: We used several cell lines, including patient-derived oligodendroglioma tumorspheres, to knock down or overexpress TRIM67. We coupled high-throughput assays, including RNA sequencing, total lysate-mass spectrometry (MS), and coimmunoprecipitation (co-IP)-MS with functional assays including immunofluorescence (IF) staining, co-IP, and western blotting (WB) to assess the in vitro phenotype associated with TRIM67. Patient-derived oligodendroglioma tumorspheres were orthotopically implanted in mice to determine the effect of TRIM67 on tumor growth and survival. RESULTS: TRIM67 overexpression alters the abundance of cytoskeletal proteins and induces membrane bleb formation. TRIM67-associated blebbing was reverted with the nonmuscle class II myosin inhibitor blebbistatin and selective ROCK inhibitor fasudil. NOGO-A/Rho GTPase/ROCK2 signaling is altered upon TRIM67 ectopic expression, pointing to the underlying mechanism for TRIM67-induced blebbing. Phenotypically, TRIM67 expression resulted in higher cell motility and reduced cell adherence. In orthotopic implantation models of patient-derived oligodendrogliomas, TRIM67 accelerated tumor growth, reduced overall survival, and led to increased vimentin expression at the tumor margin. CONCLUSIONS: Taken together, our results demonstrate that upregulated TRIM67 induces blebbing-based rounded cell morphology through Rho GTPase/ROCK-mediated signaling thereby contributing to glioma pathogenesis.

Tumor heterogeneity and tumor-microglia interactions in primary and recurrent IDH1-mutant gliomas.

The isocitrate dehydrogenase (IDH) gene is recurrently mutated in adult diffuse gliomas. IDH-mutant gliomas are categorized into oligodendrogliomas and astrocytomas, each with unique pathological features. Here, we use single-nucleus RNA and ATAC sequencing to compare the molecular heterogeneity of these glioma subtypes. In addition to astrocyte-like, oligodendrocyte progenitor-like, and cycling tumor subpopulations, a tumor population enriched for ribosomal genes and translation elongation factors is primarily present in oligodendrogliomas. Longitudinal analysis of astrocytomas indicates that the proportion of tumor subpopulations remains stable in recurrent tumors. Analysis of tumor-associated microglia/macrophages (TAMs) reveals significant differences between oligodendrogliomas, with astrocytomas harboring inflammatory TAMs expressing phosphorylated STAT1, as confirmed by immunohistochemistry. Furthermore, inferred receptor-ligand interactions between tumor subpopulations and TAMs may contribute to TAM state diversity. Overall, our study sheds light on distinct tumor populations, TAM heterogeneity, TAM-tumor interactions in IDH-mutant glioma subtypes, and the relative stability of tumor subpopulations in recurrent astrocytomas.

[Investigation on the dietary iron intake of infants from four month-old to one year-old in Shunyi District of Beijing].

OBJECTIVE: To understand the present status of dietary iron intake of infants from 4 to 12 month-old in urban and rural areas of Shunyi District of Beijing, and to provide scientific basis for their supplementary foods. METHODS: Two hundred and sixty-six infants were selected and investigated with a 3-day (72h) dietary record questionary. RESULTS: The average dietary iron intake of 4 - 5 month-old infants has achieved to the Dietary Reference Intakes, and no significant difference was observed between the intakes in urban and rural areas (P > 0.05). The proportion of infants whose dietary iron intake was higher than the Adequate Intakes (AI) of iron was just 19.8% among the studied infants in rural and 36. 9% in urban areas (P < 0.01). There was no significant difference on breastfeeding rates in rural and urban groups (P > 0.05). The main dietary iron sources were from cereals, fruits, eggs, vegetables and meat. CONCLUSION: The dietary iron intake of 6 - 12 month-old infants in Shunyi was significantly insufficient. Encouraging mothers to go on fully breastfeeding for at least 6 months, as well as giving supplementary foods rich in iron at the right time according to the state of infants.

Two strategies to improve the supply of PKS extender units for ansamitocin P-3 biosynthesis by CRISPR-Cas9.

Ansamitocin P-3 (AP-3) produced by Actinosynnema pretiosum is a potent antitumor agent. However, lack of efficient genome editing tools greatly hinders the AP-3 overproduction in A. pretiosum. To solve this problem, a tailor-made pCRISPR-Cas9apre system was developed from pCRISPR-Cas9 for increasing the accessibility of A. pretiosum to genetic engineering, by optimizing cas9 for the host codon preference and replacing pSG5 with pIJ101 replicon. Using pCRISPR-Cas9apre, five large-size gene clusters for putative competition pathway were individually deleted with homology-directed repair (HDR) and their effects on AP-3 yield were investigated. Especially, inactivation of T1PKS-15 increased AP-3 production by 27%, which was most likely due to the improved intracellular triacylglycerol (TAG) pool for essential precursor supply of AP-3 biosynthesis. To enhance a "glycolate" extender unit, two combined bidirectional promoters (BDPs) ermEp-kasOp and j23119p-kasOp were knocked into asm12-asm13 spacer in the center region of gene cluster, respectively, by pCRISPR-Cas9apre. It is shown that in the two engineered strains BDP-ek and BDP-jk, the gene transcription levels of asm13-17 were significantly upregulated to improve the methoxymalonyl-acyl carrier protein (MM-ACP) biosynthetic pathway and part of the post-PKS pathway. The AP-3 yields of BDP-ek and BDP-jk were finally increased by 30% and 50% compared to the parent strain L40. Both CRISPR-Cas9-mediated engineering strategies employed in this study contributed to the availability of AP-3 PKS extender units and paved the way for further metabolic engineering of ansamitocin overproduction.

Global Regulator AdpA_1075 Regulates Morphological Differentiation and Ansamitocin Production in Actinosynnema pretiosum subsp. auranticum.

Actinosynnema pretiosum is a well-known producer of maytansinoid antibiotic ansamitocin P-3 (AP-3). Growth of A. pretiosum in submerged culture was characterized by the formation of complex mycelial particles strongly affecting AP-3 production. However, the genetic determinants involved in mycelial morphology are poorly understood in this genus. Herein a continuum of morphological types of a morphologically stable variant was observed during submerged cultures. Expression analysis revealed that the ssgA_6663 and ftsZ_5883 genes are involved in mycelial aggregation and entanglement. Combing morphology observation and morphology engineering, ssgA_6663 was identified to be responsible for the mycelial intertwining during liquid culture. However, down-regulation of ssgA_6663 transcription was caused by inactivation of adpA_1075, gene coding for an AdpA-like protein. Additionally, the overexpression of adpA_1075 led to an 85% increase in AP-3 production. Electrophoretic mobility shift assays (EMSA) revealed that AdpA_1075 may bind the promoter regions of asm28 gene in asm gene cluster as well as the promoter regions of ssgA_6663. These results confirm that adpA_1075 plays a positive role in AP-3 biosynthesis and morphological differentiation.

From Laboratory Studies to Clinical Trials: Temozolomide Use in IDH-Mutant Gliomas.

In this review, we discuss the use of the alkylating agent temozolomide (TMZ) in the treatment of IDH-mutant gliomas. We describe the challenges associated with TMZ in clinical (drug resistance and tumor recurrence) and preclinical settings (variabilities associated with in vitro models) in treating IDH-mutant glioma. Lastly, we summarize the emerging therapeutic targets that can potentially be used in combination with TMZ.

MiR-let-7i inhibits CD4+ T cell apoptosis in patients with acute coronary syndrome.

BACKGROUND: Abnormal CD4+ T cells appear in the peripheral blood of patients with acute coronary syndrome (ACS). Studies have confirmed that CD4+ T cells are resistant to apoptosis, but the specific mechanism has not been elucidated yet. OBJECTIVES: The microRNA (miR)-let-7i plays an important regulatory role in the cardiovascular system and is widely involved in cell proliferation and apoptosis. In this study, we aimed to investigate its functional and regulatory roles in CD4+ T cell apoptosis. MATERIAL AND METHODS: Apoptosis of CD4+ T cells was detected using TUNEL assay. Western blot analyses were used to detect the expression of Bcl-2 and Bax. Real-time polymerase chain reaction and western blot analyses were used to detect the expression of miR-let-7i, Fas and FasL. A miR-let-7i mimic or inhibitor was transfected into CD4+ T cells, and miR-let-7i activity was investigated using Cell Counting Kit-8 (CCK-8) and TUNEL assays. RESULTS: Apoptosis of CD4+ T cells in ACS patients was significantly decreased. Overexpression of miR-let-7i inhibited CD4+ T cell apoptosis and improved cell survival rates, while inhibition of miR-let-7i facilitated cell apoptosis. We also found that miR-let-7i negatively regulated Fas and FasL gene expression in CD4+ T cells. CONCLUSIONS: The present study identified that miR-let-7i significantly reduces Fas and FasL expression in ACS CD4+ T cells and inhibits apoptosis in these cells. Therefore, miR-let-7i may serve as a possible therapeutic target for the treatment of ACS.

Exploring the regulatory roles of circular RNAs in the pathogenesis of atherosclerosis.

Circular RNAs (circRNAs) are a class of noncoding RNAs with a covalently closed loop structure. Recent evidence has shown that circRNAs can regulate gene transcription, alternative splicing, microRNA (miRNA) "molecular sponges", RNA-binding proteins and protein translation. Atherosclerosis is one of the leading causes of death worldwide, and more studies have indicated that circRNAs are related to atherosclerosis pathogenesis, including vascular endothelial cells, vascular smooth muscle cells, inflammation and lipid metabolism. In this review, we systematically summarize the biogenesis, characteristics and functions of circRNAs with a focus on their roles in the pathogenesis of atherosclerosis.

iTRAQ-based quantitative proteomic analysis reveals the metabolic pathways of grain chalkiness in response to nitrogen topdressing in rice.

Grain chalkiness is a highly undesirable trait that adversely affects rice quality. This chalkiness is easily influenced by the application of chemical nitrogen (N) fertilizer at the late growth stage. However, on the molecular mechanism underlying grain chalkiness caused by late N fertilization is not fully clear. In this study, proteomic differences in expression were determined in developing grains exposed to N topdressing (108 kg N ha-1, N+) and a control (0 kg N ha-1, N0), using the rice variety OM052, which has a high level of chalkiness. A total of 198 differentially expressed proteins (DEPs) were detected between the N+ and N0 treatments, including 9 up-regulated proteins and 189 down-regulated proteins. Of these DEPs, approximately half were associated with carbohydrate metabolism (glycolysis, tricarboxylic acid cycle, pentose phosphate pathway, fermentation and starch metabolism) and N metabolism (protein synthesis, folding, degradation and storage, amino acid synthesis and catabolism). A detailed pathway dissection revealed that multiple metabolic pathways during the grain filling stage were involved in the N-induced grain chalkiness. Reduced abundances of proteins associated with respiratory metabolism and energy metabolism drastically impaired the biosynthesis and deposition of starch in the developmental endosperms, which might be a crucial trigger for the increase in grain chalkiness. The disturbed N metabolism and differential expression of storage proteins up-regulated during the grain filling stage are able to partially explain the occurrence of grain chalkiness in rice.

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq.

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused on bulk approaches that led to the molecular classification of brain tumors. Over the last five years, single cell sequencing approaches have highlighted several important features of gliomas. The majority of these studies have utilized fresh brain tumor specimens to isolate single cells using flow cytometry or antibody-based separation methods. Moving forward, the use of fresh-frozen tissue samples from biobanks will provide greater flexibility to single cell applications. Furthermore, as the single-cell field advances, the next challenge will be to generate multi-omics data from either a single cell or the same sample preparation to better unravel tumor complexity. Therefore, simple and flexible protocols that allow data generation for various methods such as single-nucleus RNA sequencing (snRNA-seq) and single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq) will be important for the field. Recent advances in the single cell field coupled with accessible microfluidic instruments such as the 10x genomics platform have facilitated single cell applications in research laboratories. To study brain tumor heterogeneity, we developed an enhanced protocol for the isolation of single nuclei from fresh frozen gliomas. This protocol merges existing single cell protocols and combines a homogenization step followed by filtration and buffer mediated gradient centrifugation. The resulting samples are pure single nuclei suspensions that can be used to generate single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.