709 Peptidyl IL-2/15Rβγc-restricted agonists, highly-attenuated and linked to anti-PD-1 antibodies to achieve selectivity and amplified potency in stimulating PD-1high lymphocytes

BackgroundRecent reports demonstrate that directing a “non-alpha” IL-2 mutant to a PD-1high CD8+ stem-like population induces proliferation, and differentiation into a highly functional cytotoxic phenotype. We previously reported small synthetic peptides, unrelated to IL-2 or IL-15, that bind IL-2/15Rβγc to induce receptor signaling. These peptides do not bind IL-2Rα and are therefore IL-2/15Rβγc-restricted agonists. We now describe fusion of potency-attenuated peptide agonists to an anti-PD-1 antibody (α-PD-1) to achieve selective targeting to PD-1high lymphocytes, and enhanced potency of IL-2R agonists acting in Cis with α-PD-1 binding.MethodsPeptidyl IL-2/15Rβγc agonists with attenuated potency due to weakened binding to either IL-2/15Rβ or γc were fused to the C-termini of both heavy chains of an α-PD-1 IgG and expressed in CHO cells. The fusion proteins retained PD-1 binding affinity comparable to the α-PD-1; and were evaluated for potency of IL-2Rβγc-dependent STAT5 phosphorylation in TF-1β cells (with undetectable PD-1 expression), and in TF-1β-derived lines expressing varying levels of PD-1. The fusion proteins were also assessed for Rβγc stimulation of CD8+ cells treated with anti-CD3 and anti-CD28 to induce elevated PD-1 expression.ResultsAn analysis of pembrolizumab (Pem) fused to MDK1169, a potent IL-2Rβγc agonist, showed a 15-fold increase in potency in TF-1β/PD-1+ cells. This served as an initial demonstration of the PD-1-directed, cis-acting mechanism; but the potency of MDK1169 in this construct (~500pM, EC50 pSTAT5 induction) is too high (relative to the affinity of Pem for PD-1) to achieve a more substantial selectivity for PD-1+ cells. To improve selectivity, fusions of α-PD-1 to peptide agonists with potencies as weak as 1uM on TF-1β cells were constructed. Some of these fusion proteins exhibited up to 100-fold increase in potency when tested on TF-1β/PD-1high compared to parental TF-1β cells; and addition of an excess of α-PD-1 blocked this gain in potency in the PD-1high cells. When tested on CD8+ cells activated to express elevated PD-1 levels, potency of the PD-1-directed agonists correlated with PD-1 expression.ConclusionsThe malleability of peptidyl agonists makes them useful for optimizing antibody-targeted cis-acting agonists designed to produce minimal activity on non-targeted cells and high potency at targeted cells. IL-2/15Rβγc agonists directed by PD-1 binding to a stem-like highly cytotoxic tumor infiltrating CD8+ population may have useful anti-tumor applications.

Empirical Analysis on Effectiveness and Long-Acting Mechanism of Regional Physical Education Teaching

The physical quality of students is an important goal of quality education, and the basis for the training of high-quality talents. Physical education (PE) is of great significance to enhance students’ physical fitness, and improve their awareness of lifelong physical exercise. Targeting several colleges in Guizhou Province, this paper empirically studies the effectiveness and long-acting mechanism of regional PE teaching through interview, questionnaire survey, and mathematical statistics. Firstly, the status quo of PE teaching in the colleges was surveyed. The survey results show the following problems in college PE teaching: the lack of systematic organization and management, the outdated and non-diverse forms of activities, the absence of a strong sports atmosphere, and the lack of interest among the students. To solve these problems, this paper constructs a long-acting mechanism for regional PE teaching, and verifies its implementation effect through an empirical study. The mechanism was found to enhance the students’ physical fitness, help them develop a good habit of physical exercise, and boost their awareness of lifelong physical exercise. This research greatly promotes the lifelong physical exercise of college students and the PE teaching quality in colleges and universities.

Long-term epigenetic memory of jasmonic acid-dependent immunity against herbivory

Stress exposure can have long-lasting impacts on ecologically relevant life-history traits in plants. Here, we have investigated the long-term impacts of the stress hormone jasmonic acid (JA) on the defence phenotype, transcriptome and DNA methylome of Arabidopsis thaliana. Three weeks after transient JA signalling activity in seedlings, 5-week-old plants retained induced resistance (IR) against herbivory but showed enhanced susceptibility to necrotrophic and biotrophic pathogens. Transcriptome analysis revealed that JA seedling treatment causes prolonged priming and/or up-regulation of JA-dependent defence genes but repression of ethylene- and salicylic acid-dependent genes. Long-term JA-IR against herbivory was dependent on MYC2/3/4 transcription factors and DNA (de)methylation pathways. Although DNA methylome analysis did not reveal consistent changes in DNA methylation near MYC2/3/4-controlled defence genes, plants from JA-treated seedlings showed enrichment of differentially methylated regions at ATREP2 transposable elements (TEs). Our study points to a trans-acting mechanism whereby hypomethylated TEs mediate long-lasting epigenetic memory of JA-dependent immunity.

The carP lncRNA Is a carS-Related Regulatory Element with Broad Effects on the Fusarium fujikuroi Transcriptome

Carotenoid biosynthesis in the fungus Fusarium fujikuroi is regulated by environmental factors, with light being the main stimulating signal. The CarS RING-finger protein plays an important role in the downregulation of structural genes of the carotenoid pathway. A recent transcriptomic analysis on the effect of carS mutation identified a gene for a long non-coding RNA (lncRNA) upstream of carS, called carP, the deletion of which results in increased carS mRNA levels and lack of carotenoid production. We have investigated the function of carP by studying the transcriptomic effect of its deletion and the phenotypes resulting from the reintroduction of carP to a deletion strain. The RNA-seq data showed that the loss of carP affected the mRNA levels of hundreds of genes, especially after illumination. Many of these changes appeared to be cascade effects as a result of changes in carS expression, as suggested by the comparison with differentially expressed genes in a carS mutant. Carotenoid production only recovered when carP was integrated upstream of carS, but not at other genomic locations, indicating a cis-acting mechanism on carS. However, some genes hardly affected by CarS were strongly upregulated in the carP mutant, indicating that carP may have other regulatory functions as an independent regulatory element.

Roles of Nicotinamide N-Methyltransferase in Obesity and Type 2 Diabetes

Type 2 diabetes (T2D) is thought to be a complication of metabolic syndrome caused by disorders of energy utilization and storage and characterized by insulin resistance or deficiency of insulin secretion. Though the mechanism linking obesity to the development of T2D is complex and unintelligible, it is known that abnormal lipid metabolism and adipose tissue accumulation possibly play important roles in this process. Recently, nicotinamide N-methyltransferase (NNMT) has been emerging as a new mechanism-of-action target in treating obesity and associated T2D. Evidence has shown that NNMT is associated with obesity and T2D. NNMT inhibition or NNMT knockdown significantly increases energy expenditure, reduces body weight and white adipose mass, improves insulin sensitivity, and normalizes glucose tolerance and fasting blood glucose levels. Additionally, trials of oligonucleotide therapeutics and experiments with some small-molecule NNMT inhibitors in vitro and in preclinical animal models have validated NNMT as a promising therapeutic target to prevent or treat obesity and associated T2D. However, the exact mechanisms underlying these phenomena are not yet fully understood and clinical trials targeting NNMT have not been reported until now. Therefore, more researches are necessary to reveal the acting mechanism of NNMT in obesity and T2D and to develop therapeutics targeting NNMT.

Transcriptome-wide in vivo mapping of cleavage sites for the compact cyanobacterial ribonuclease E reveals insights into its function and substrate recognition

Ribonucleases are crucial enzymes in RNA metabolism and post-transcriptional regulatory processes in bacteria. Cyanobacteria encode the two essential ribonucleases RNase E and RNase J. Cyanobacterial RNase E is shorter than homologues in other groups of bacteria and lacks both the chloroplast-specific N-terminal extension as well as the C-terminal domain typical for RNase E of enterobacteria. In order to investigate the function of RNase E in the model cyanobacterium Synechocystis sp. PCC 6803, we engineered a temperature-sensitive RNase E mutant by introducing two site-specific mutations, I65F and spontaneously occurring V94A. This enabled us to perform RNA-seq after the transient inactivation of RNase E by a temperature shift (TIER-seq) and to map 1,472 RNase-E-dependent cleavage sites. We inferred a dominating cleavage signature consisting of an adenine at the -3 and a uridine at the +2 position within a single-stranded segment of the RNA. The data identified putative RNase-E-dependent instances of operon discoordination, mRNAs likely regulated jointly by RNase E and an sRNA, potential 3' end-derived sRNAs and a dual-acting mechanism for the glutamine riboswitch. Our findings substantiate the pivotal role of RNase E in post-transcriptional regulation and suggest the redundant or concerted action of RNase E and RNase J in cyanobacteria.

Deletion of a pseudogene within a fragile site triggers the oncogenic expression of the mitotic CCSER1 gene

The oncogenic role of common fragile sites (CFS), focal and pervasive gaps in the cancer genome arising from replicative stress, remains controversial. Exploiting the TCGA dataset, we found that in most CFS the genes residing within the associated focal deletions are down-regulated, including proteins involved in tumour immune recognition. In a subset of CFS, however, the residing genes are surprisingly overexpressed. Within the most frequent CFS in this group, FRA4F, which is deleted in up to 18% of cancer cases and harbours the CCSER1 gene, we identified a region which includes an intronic, antisense pseudogene, TMSB4XP8. TMSB4XP8 focal ablation or transcriptional silencing elicits the overexpression of CCSER1, through a cis-acting mechanism. CCSER1 overexpression increases proliferation and triggers centrosome amplifications, multinuclearity, and aberrant mitoses. Accordingly, FRA4F is associated in patient samples to mitotic genes deregulation and genomic instability. As a result, cells overexpressing CCSER1 become sensitive to the treatment with aurora kinase inhibitors. Our findings point to a novel tumourigenic mechanism where focal deletions increase the expression of a new class of “dormant” oncogenes.

The transcriptomic response to heat stress of a jujube (Ziziphus jujuba Mill.) cultivar is featured with changed expression of long noncoding RNAs

Long non-coding RNA (lncRNA) of plant species undergoes dynamic regulation and acts in developmental and stress regulation. Presently, there is little information regarding the identification of lncRNAs in jujube (Ziziphus jujuba Mill.), and it is uncertain whether the lncRNAs could respond to heat stress (HS) or not. In our previous study, a cultivar (Hqing1-HR) of Z. jujuba were treated by HS (45°C) for 0, 1, 3, 5 and 7 days, and it was found that HS globally changed the gene expression by RNA sequencing (RNA-seq) experiments and informatics analyses. In the current study, 8260 lncRNAs were identified successfully from the previous RNA-seq data, and it indicated that lncRNAs expression was also altered globally, suggesting that the lncRNAs might play vital roles in response to HS. Furthermore, bioinformatics analyses of potential target mRNAs of lncRNAs with cis-acting mechanism were performed, and it showed that multiple differentially expressed (DE) mRNAs co-located with DElncRNAs were highly enriched in pathways associated with response to stress and regulation of metabolic process. Taken together, these findings not only provide a comprehensive identification of lncRNAs but also useful clues for molecular mechanism response to HS in jujube.