The circadian clock provides organisms with the ability to adapt to daily and seasonal cycles. Eukaryotic clocks mostly rely on lineage-specific transcriptional-translational feedback loops (TTFLs). Posttranslational modifications are also crucial for clock functions in fungi and animals, but the posttranslational modifications that affect the plant clock are less understood. Here, using chemical biology strategies, we show that the Arabidopsis CASEIN KINASE 1 LIKE (CKL) family is involved in posttranslational modification in the plant clock. Chemical screening demonstrated that an animal CDC7/CDK9 inhibitor, PHA767491, lengthens the Arabidopsis circadian period. Affinity proteomics using a chemical probe revealed that PHA767491 binds to and inhibits multiple CKL proteins, rather than CDC7/CDK9 homologs. Simultaneous knockdown of Arabidopsis CKL-encoding genes lengthened the circadian period. CKL4 phosphorylated transcriptional repressors PSEUDO-RESPONSE REGULATOR 5 (PRR5) and TIMING OF CAB EXPRESSION 1 (TOC1) in the TTFL. PHA767491 treatment resulted in accumulation of PRR5 and TOC1, accompanied by decreasing expression of PRR5- and TOC1-target genes. A prr5 toc1 double mutant was hyposensitive to PHA767491-induced period lengthening. Together, our results reveal posttranslational modification of transcriptional repressors in plant clock TTFL by CK1 family proteins, which also modulate nonplant circadian clocks.

BackgroundEmerging evidence has suggested that circulating microRNAs (miRNAs) in body fluids have novel diagnostic and prognostic significance for patients with malignant diseases. The lack of useful biomarkers is a crucial problem of bone and soft tissue sarcomas; therefore, we investigated the circulating miRNA signature and its clinical relevance in osteosarcoma.MethodsGlobal miRNA profiling was performed using patient serum collected from a discovery cohort of osteosarcoma patients and controls and cell culture media. The secretion of the detected miRNAs from osteosarcoma cells and clinical relevance of serum miRNA levels were evaluated using in vitro and in vivo models and a validation patient cohort.ResultsDiscovery screening identified 236 serum miRNAs that were highly expressed in osteosarcoma patients compared with controls, and eight among these were also identified in the cell culture media. Upregulated expression levels of miR-17-5p and miR-25-3p were identified in osteosarcoma cells, and these were abundantly secreted into the culture media in tumor-derived exosomes. Serum miR-25-3p levels were significantly higher in osteosarcoma patients than in control individuals in the validation cohort, with favorable sensitivity and specificity compared with serum alkaline phosphatase. Furthermore, serum miR-25-3p levels at diagnosis were correlated with patient prognosis and reflected tumor burden in both in vivo models and patients; these associations were more sensitive than those of serum alkaline phosphatase.ConclusionsSerum-based circulating miR-25-3p may serve as a non-invasive blood-based biomarker for tumor monitoring and prognostic prediction in osteosarcoma patients.

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CD4+ CD25+ regulatory T cells (Treg), an essential subset for preventing autoimmune diseases, is implicated as a negative regulator in anti-tumor immunity. We found that metformin (Met) reduced tumor-infiltrating Treg (Ti-Treg), particularly the terminally-differentiated CD103+ KLRG1+ population, and also decreased effector molecules such as CTLA4 and IL-10. Met inhibits the differentiation of naïve CD4+ T cells into inducible Treg (iTreg) by reducing forkhead box P3 (Foxp3) protein, caused by mTORC1 activation that was determined by the elevation of phosphorylated S6 (pS6), a downstream molecule of mTORC1. Rapamycin and compound C, an inhibitor of AMP-activated protein kinase (AMPK) restored the iTreg generation, further indicating the involvement of mTORC1 and AMPK. The metabolic profile of iTreg, increased Glut1-expression, and reduced mitochondrial membrane-potential and ROS production of Ti-Treg aided in identifying enhanced glycolysis upon Met-treatment. The negative impact of Met on Ti-Treg may help generation of the sustained antitumor immunity.

CD11b + myeloid subpopulations, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), play crucial roles in the suppression of T-cell-mediated anti-tumor immunity. Regulation of these cell types is a primary goal for achieving efficient cancer immunotherapy. We found that metformin (Met) induces CD11b + -cell-mediated growth inhibition of a K7M2neo osteosarcoma independent of T cells, as growth inhibition of K7M2neo was still observed in wild-type (WT) mice depleted of T cells by antibodies and in SCID; this contrasted with the effect of Met on Meth A fibrosarcoma, which was entirely T-cell-dependent. Moreover, the inhibitory effect seen in SCID was abrogated by anti-CD11b antibody injection. PMN-MDSCs were significantly reduced in both spleens and tumors following Met treatment. In TAMs, production of IL-12 and TNF-α, but not IL-10, became apparent, and elevation of MHC class II with reduction of CD206 was observed, indicating a shift from an M2- to M1-like phenotype via Met administration. Metabolically, Met treatment decreased basal respiration and the oxygen consumption rate (OCR)/extracellular acidification rate (ECAR) ratio of CD11b + cells in tumors, but not in the spleen. In addition, decreased reactive oxygen species (ROS) production and proton leakage in MDSCs and TAMs were consistently observed in tumors. Uptake of both 2-deoxy-2- d -glucose (2-NBDG) and BODIPY® decreased in MDSCs, but only BODIPY® incorporation was decreased in TAMs. Overall, our results suggest that Met redirects the metabolism of CD11b + cells to lower oxidative phosphorylation (OXPHOS) while elevating glycolysis, thereby pushing the microenvironment to a state that inhibits the growth of certain tumors.

A recent study of exome analyses in 109 patients with undiagnosed diseases included a 5-year-old girl with intellectual disability and multiple congenital anomalies, who had an apparently de novo frameshift mutation in SON. However, the combination of the truncating mutation in SON and the phenotype has not been reproduced until date, and it remains unclear if this combination represents a distinctive disease entity. Here we report an additional male with intellectual disability, congenital heart disease, distinctive facial features with curly hair and protruding ears, and long slender extremities, and hyperextensible joints. Exome analysis showed that he had the same de novo frameshift mutation in SON in a heterozygous state. Along with the first and original description of the apparently de novo truncating mutation in SON mentioned above, we have established that haploinsufficiency of SON causes a new recognizable syndrome of intellectual disability. SON is located within 21q22.11, a critical region for Braddock-Carey syndrome, which is characterized by congenital thrombocytopenia, intellectual disability, micrognathia, and a distinctive facies. Therefore, we suggest that the intellectual disability observed in Braddock-Carey syndrome could be accounted for by haploinsufficiency of SON. Ó 2016 Wiley Periodicals, Inc.Key words: SON; intellectual disability; 21q22; Braddock-Carey syndrome; thrombocytopenia INTRODUCTIONCausative gene mutations in patients with intellectual disability/ autistic spectrum disorders with or without accompanying malformations are currently being investigated in various exome analyses [de Ligt et al., 2012]. In a recent study of exome analyses in 109 trios with undiagnosed diseases, the supplemental information included a 5-year-old girl with intellectual disability, seizures, minor dysmorphisms, brain white matter abnormalities, intestinal atresia, and ventricular septal defect [Zhu et al., 2015]. This patient had two apparently de novo candidate genetic mutations: a missense mutation in C5AR1 and a frameshift mutation in SON; which is a ubiquitously expressed and phylogenetically conserved gene that encodes a DNA-binding protein [Khan et al., 1994;Wynn et al., 2000]. Because typical newborns acquire at least one new de novo deleterious mutation per generation [Lynch, 2010], it remains unclear if the observation was a chance association or whether either/which of the two mutations was responsible for the phenotype. Documentation of the second, confirmatory patient with the combination of a phenotype similar to the first patient and a frameshift mutation in the same gene, SON, resolved the "n-of-1 2587problem," and our data established an existence of a new human disease entity caused by a mutation in SON. CLINICAL REPORTThe propositus was the first child of healthy unrelated parents. At delivery, the mother was 35 years old and the father was 30 years old. The pregnancy course was uneventful, except for mild maternal anemia. The propositus was born at 40 and 1/7 weeks of gestation by...

We previously documented a girl with macrothrombocytopenia and developmental delay who carried a de novo mutation in CDC42, which plays pivotal roles in the cell cycle and the formation of the actin cytoskeleton. The phenotype of mice lacking Cdc42 was strikingly similar to that of the reported patient, indicating that the mutation in CDC42 causes a new syndromic form of thrombocytopenia. We, herein, report another unrelated female patient with a similar phenotype and a de novo mutation in the same CDC42. The present observation provides further evidence supporting the notion that a mutation in CDC42 causes a recognizable syndromic form of thrombocytopenia. The cardinal features of this entity include macrothrombocytopenia, developmental delay, lymphedema in the lower extremities, camptodactyly, and distinctive facial features.

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