Identification of Ubr1 as an amino acid sensor of steatosis in liver and muscle.

BACKGROUND: Malnutrition is implicated in human metabolic disorders, including hepatic steatosis and myosteatosis. The corresponding nutrient signals and sensors as well as signalling pathways have not yet been well studied. This study aimed to unravel the nutrient-sensing mechanisms in the pathogenesis of steatosis. METHODS: Plin2, a lipid droplet (LD) protein-inhibiting lipolysis, is associated with steatosis in liver and muscle. Taking advantage of the Gal4-UAS system, we used the Drosophila melanogaster wing imaginal disc as an in vivo model to study the regulation of Plin2 proteostasis and LD homeostasis. Drosophila Schneider 2 (S2) cells were used for western blotting, immunoprecipitation assays, amino acid-binding assays and ubiquitination assays to further investigate the regulatory mechanisms of Plin2 in response to nutrient signals. Mouse AML12 hepatocytes, human JHH-7 and SNU-475 hepatoma cells were used for immunofluorescence, western blotting and immunoprecipitation to demonstrate that the mode of Plin2 regulation is evolutionarily conserved. In addition, we purified proteins from HEK293 cells and reconstituted in vitro cell-free systems in amino acid-binding assays, pulldown assays and ubiquitination assays to directly demonstrate the molecular mechanism by which Ubr1 senses amino acids to regulate Plin2 proteostasis. RESULTS: As a lipolysis inhibitor, Plin2 was significantly elevated in liver (P < 0.05) and muscle (P < 0.05) in patients with steatosis. Consistently, we found that the ubiquitin moiety can be conjugated to any Lys residue in Plin2, ensuring robust clearance of Plin2 by protein degradation. We further demonstrated that the E3 ubiquitin ligase Ubr1 targets Plin2 for degradation in an amino acid-dependent manner. Ubr1 uses two canonical substrate-binding pockets, independent of each other, to bind basic and bulky hydrophobic amino acids, respectively. Mechanistically, amino acid binding allosterically activates Ubr1 by alleviating Ubr1's auto-inhibition. In the absence of amino acids, or when the amino acid-binding capacity of Ubr1 is diminished, Ubr1-mediated Plin2 degradation is inactivated, leading to steatosis. CONCLUSIONS: We identified Ubr1 as an amino acid sensor regulating Plin2 proteostasis, bridging the knowledge gap between steatosis and nutrient sensing. Our work may provide new strategies for the prevention and treatment of steatosis.

Wounds under diabetic milieu: The role of immune cellar components and signaling pathways.

A major challenge in the field of diabetic wound healing is to confirm the body's intrinsic mechanism that could sense the immune system damage promptly and protect the wound from non-healing. Accumulating literature indicates that macrophage, a contributor to prolonged inflammation occurring at the wound site, might play such a role in hindering wound healing. Likewise, other immune cell dysfunctions, such as persistent neutrophils and T cell infection, may also lead to persistent oxidative stress and inflammatory reaction during diabetic wound healing. In this article, we discuss recent advances in the immune cellular components in wounds under the diabetic milieu, and the role of key signaling mechanisms that compromise the function of immune cells leading to persistent wound non-healing.

The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity.

The RNA modification N6-methyladenosine (m6A) has critical roles in many biological processes1,2. However, the function of m6A in the early phase of mammalian development remains poorly understood. Here we show that the m6A reader YT521-B homology-domain-containing protein 1 (YTHDC1) is required for the maintenance of mouse embryonic stem (ES) cells in an m6A-dependent manner, and that its deletion initiates cellular reprogramming to a 2C-like state. Mechanistically, YTHDC1 binds to the transcripts of retrotransposons (such as intracisternal A particles, ERVK and LINE1) in mouse ES cells and its depletion results in the reactivation of these silenced retrotransposons, accompanied by a global decrease in SETDB1-mediated trimethylation at lysine 9 of histone H3 (H3K9me3). We further demonstrate that YTHDC1 and its target m6A RNAs act upstream of SETDB1 to repress retrotransposons and Dux, the master inducer of the two-cell stage (2C)-like program. This study reveals an essential role for m6A RNA and YTHDC1 in chromatin modification and retrotransposon repression.

MiR-940 promotes malignant progression of breast cancer by regulating FOXO3.

Breast cancer (BC) is a common cancer with poor survival. The present study aimed to explore the effect of miR-940 on the process of BC cells and its target gene FOXO3. The expression of miR-940 was assessed in BC tissues and cells using qRT-PCR. Furthermore, the correlation between miR-940 and prognosis of BC patients from the TCGA database was analyzed. CCK8 assays and colony formation assays were used to explore the effect of miR-940 on BC cell proliferation. The invasion abilities were detected by transwell assays. Luciferase reporter assay was performed to scrutinize the relationship between miR-940 and FOXO3. Finally, rescue experiments were performed through FOXO3 down-regulation and miR-940 inhibitors by using CCK8 assays, colony formation assays and transwell assays. miR-940 was significantly up-regulated in BC cells and tissues. In addition, the high level of miR-940 correlated with poor survival of BC patients (P=0.023). CCK8 assays, colony formation assays and transwell assays indicated that miR-940 promoted the proliferation and invasion abilities of BC cells. The luciferase reporter assay suggested that miR-940 directly targeted FOXO3. Moreover, we found that the effect of si-FOXO3 was rescued by miR-940 inhibitors in BC cells. miR-940 may promote the proliferation and invasion abilities of BC cells by targeting FOXO3. Our study suggested that miR-940 could be a novel molecular target for therapies against BC.

Identifying occult maternal malignancies from 1.93 million pregnant women undergoing noninvasive prenatal screening tests.

PURPOSE: Multiple chromosomal aneuploidies may be associated with maternal malignancies and can cause failure of noninvasive prenatal screening (NIPS) tests. However, multiple chromosomal aneuploidies show poor specificity and selectivity for diagnosing maternal malignancies. METHODS: This multicenter retrospective analysis evaluated 639 pregnant women who tested positive for multiple chromosomal aneuploidies on initial NIPS test between January 2016 and December 2017. Women were assessed using genome profiling of copy-number variations, which was translated to cancer risk using a novel bioinformatics algorithm called the cancer detection pipeline (CDP). Sensitivity, specificity, and positive predictive value (PPV) of diagnosing maternal malignancies were compared for multiple chromosomal aneuploidies, the CDP model, and the combination of CDP and plasma tumor markers. RESULTS: Of the 639 subjects, 41 maternal malignant cancer cases were diagnosed. Multiple chromosomal aneuploidies predicted maternal malignancies with a PPV of 7.6%. Application of the CDP algorithm to women with multiple chromosomal aneuploidies allowed 34 of the 41 (83%) cancer cases to be identified, while excluding 422 of 501 (84.2%) of the false positive cases. Combining the CDP with plasma tumor marker testing gave PPV of 75.0%. CONCLUSION: The CDP algorithm can diagnose occult maternal malignancies with a reasonable PPV in multiple chromosomal aneuploidies-positive pregnant women in NIPS tests. This performance can be further improved by incorporating findings for plasma tumor markers.

Copy number variation profile in noninvasive prenatal testing (NIPT) can identify co-existing maternal malignancies: Case reports and a literature review.

OBJECTIVE: The coexistence of maternal malignancy and pregnancy has received increasing attention in Noninvasive prenatal testing (NIPT) studies. Malignancy in pregnant women potentially affects the copy number variation (CNV) profile in NIPT results. Only one case of hematologic cancer has been reported in a Hong-Kong pregnant women, and solid tumors have never been reported in pregnant Chinese women. CASE REPORT: The patients with dysgerminoma and cervical cancer showed aberrant chromosomal aneuploidies in NIPT and concordant patterns of genome disruption in tumor tissues. The genomic aberrations in the gastric cancer patient had similar copy number variation pattern of gastric cancer. CONCLUSION: The findings in this study and the literature review further validate the effect of maternal malignancy on the copy number variation profile in NIPT data and strengthen the possibility of detecting malignant tumors with NIPT in the future.

Effect of an 88-amino-acid deletion in nsp2 of porcine reproductive and respiratory syndrome virus on virus replication and cytokine responses in vitro.

Previously, a spontaneous 88-amino-acid (aa) deletion in nsp2 was associated with cell-adaptation of porcine reproductive and respiratory syndrome virus (PRRSV) strain JXM100, which arose during passaging of the highly pathogenic PRRSV (HP-PRRSV) strain JX143 in MARC-145 cells. Here, to elucidate the biological role of this deletion, we specifically deleted the region of a cDNA clone of HP-PRRSV strain JX143 (pJX143) corresponding to these 88 amino acids. The effect of the deletion on virus replication in cultured cells and transcriptional activation of inflammatory cytokines and chemokines in pulmonary alveolar macrophages (PAMs) was examined. Mutant virus with the 88-aa deletion in nsp2 (rJX143-D88) had faster growth kinetics and produced larger plaques in MARC-145 cells than the parental virus (rJX143), suggesting that the deletion enhanced virus replication in MARC-145 cells. In contrast, the overall yield of rJX143 was almost 1 log higher than that of rJX143-D88, suggesting that the 88-aa deletion in nsp2 decreased the production of infectious viruses in PAMs. Infection with the mutant virus with the 88-aa deletion resulted in increased mRNA expression of type I interferon (IFN-α and IFN-ß) and chemokines genes. In addition, the mRNA expression of antiviral genes (ISG15, ISG54 and PKR) regulated by the IFN response was upregulated in PAMs infected with the mutant virus rJX143-D88. Our results demonstrate that virus-specific host immunity can be enhanced by modifying certain nsp2 epitope regions. These findings provide important insights for understanding virus pathogenesis and development of future vaccines.

Toxic responses of microorganisms to nickel exposure in farmland soil in the presence of earthworm (Eisenia fetida).

Nickel (Ni)-contamination impairs soil ecosystem, threatening human health. A laboratory simulation of Ni-polluted farmland soil study, in the presence or absence of earthworm, was carried out to investigate the toxic responses of soil microorganisms, including microbial biomass C (MBC), soil basal respiration (SBR), metabolic quotient (qCO2), urease (UA) and dehydrogenase activities (DHA). Additionally, the variations of Ni bioavailability were also explored. Results manifested that MBC and SBR were stimulated at 50 and 100 mg·kg-1 of Ni but inhibited by further increasing Ni level, showing a Hormesis effect. Earthworm input delayed the occurrence of a maximum SBR inhibition rate under the combined double-factors of time and dose. No specific effect of Ni concentration on the qCO2 was observed. UA was significantly suppressed at 800 mg·kg-1 Ni (P < 0.05 or 0.01), whereas DHA was more sensitive and significantly inhibited throughout all the treatments (P < 0.01), indicating a pronounced dose-response relationship. The addition of earthworm facilitated all the biomarkers above. The time-dependent of dose-effect relationship (TDR) on MBC and SBR inhibition rates suggested that the peak responsiveness of microorganisms to Ni stress were approximate on the 21st day. The bioavailable form of per unit Ni concentration declined with time expanded and concentration increased, and the changeable process of the relative amount of bioavailability was mainly controlled by a physicochemical reactions.

Cloning, functional characterization and nutritional regulation of Δ6 fatty acyl desaturase in the herbivorous euryhaline teleost Scatophagus argus.

Marine fish are generally unable or have low ability for the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, with some notable exceptions including the herbivorous marine teleost Siganus canaliculatus in which such a capability was recently demonstrated. To determine whether this is a unique feature of S. canaliculatus or whether it is common to the herbivorous marine teleosts, LC-PUFA biosynthetic pathways were investigated in the herbivorous euryhaline Scatophagus argus. A putative desaturase gene was cloned and functionally characterized, and tissue expression and nutritional regulation were investigated. The full-length cDNA was 1972 bp, containing a 1338 bp open-reading frame encoding a polypeptide of 445 amino acids, which possessed all the characteristic features of fatty acyl desaturase (Fad). Functional characterization by heterologous expression in yeast showed the protein product of the cDNA efficiently converted 18:3n-3 and 18:2n-6 to 18:4n-3 and 18:3n-6, respectively, indicating Δ6 desaturation activity. Quantitative real-time PCR showed that highest Δ6 fad mRNA expression was detected in liver followed by brain, with lower expression in other tissues including intestine, eye, muscle, adipose, heart kidney and gill, and lowest expression in stomach and spleen. The expression of Δ6 fad was significantly affected by dietary lipid and, especially, fatty acid composition, with highest expression of mRNA in liver of fish fed a diet with a ratio of 18:3n-3/18:2n-6 of 1.72:1. The results indicated that S. argus may have a different LC-PUFA biosynthetic system from S. canaliculatus despite possessing similar habitats and feeding habits suggesting that LC-PUFA biosynthesis may not be common to all marine herbivorous teleosts.