Mouse Homologue Research Articles

High expression of SEZ6L2 as an independent prognostic Indicator in thyroid carcinoma.

Seizure-related 6 homolog (mouse)-like 2 (SEZ6L2) is a type 1 transmembrane protein that is primarily expressed in the brain . In recent reports, SEZ6L2 has been found to be overexpressed in some cancers and can drive the progression of tumors. However, its function and mechanism in thyroid cancer remain unclear. In this article ,we searched for the SEZ6L2 expressions in Pan-cancer on TCGA (The Cancer Genome Atlas) and evaluated these data using the TIMER2 method. Then, the immunohistochemical score (IHC score) of SEZ6L2 in cancer tissue was collected in human protein mapping (HPA) data. And we used CIBERSORT to assess the association between the levels of SEZ6L2 expression and the number of various immune cells in papillary thyroid carcinoma (PTC) tissue. Finally, Gene Expression Omnibus (GEO) analyses, real-time quantitative polymerase chain reaction (qRT-PCR) of tissues, and immunohistochemical staining were used to detect the result. The article illustrated that a large number of cancers had a higher expression of SEZ6L2 compared to the control tissues. And the immunohistochemical score (IHC score) of SEZ6L2 in cancer tissue was considerably elevated compared to that in normal tissues SEZ6L2 was elevated in thyroid carcinoma (THCA) tissue, besides, GEO analyses, qRT-PCR of tissues, and immunohistochemical staining were conducted to test the results. Finally, the Kaplan-Meier survival analysis illustrated that the increased expression of SEZ6L2 was correlated with a dismal prognosis-higher SEZ6L2 is associated with shorter survival. And the univariate analysis illustrated that T stage, SEZ6L2 and Pathologic stage were related to the overall survival (OS), multivariate analysis stated that elevated expression of SEZ6L2 was an independent risk factor that affected progression-free interval (PFI) (P<0.05). Consequently, we found that the expression of SEZ6L2 was correlated with tumor-infiltrating immune cells by TIMER. SEZ6L2 was upregulated in patients with THCA and that increased expression of SEZ6L2 was related with clinical progression and was regarded as an independent risk factor for PFI. In THCA patients, the expression of SEZ6L2 could be a significant prognostic factor, which is expected to be a prospective biomarker for THCA in the future.

POS-407 COMPLEMENT RECEPTOR 1 IS A POTENTIAL THERAPEUTIC TARGET OF INTERSTITIAL LESION IN IgA NEPHROPATHY

IgA nephropathy is one of leading causes of chronic kidney disease. Interstitial lesion in IgA nephropathy is correlated with unfavorable prognosis. Sensitive biomarkers are important for early detection and intervention targets of the disease. This study aims to identify Complement Receptor 1 (CR1) as a potential therapeutic target in IgA nephropathy patients with interstitial lesion. Renal tissue proteomics data is accessed from a local IgA nephropathy cohort with 59 IgA nephropathy patients and 19 healthy controls. CD21/35+B cells are separated with fluorescence activated cell sorting from mice spleen. Adaptive transfer is operated with a single injection from the renal vein immediately after urethral obstruction. Renal pathology is evaluated on pictures captured by a visual microscope. CR1 is identified as the most significantly downregulated complement component in IgA nephropathy patients. Clinical information characterization of IgA nephropathy patients is assessed with Pearson R correlation test to confirm their linear independence. Of the 28 complement components recognized, 24 protein expression is increased in IgA nephropathy patients and 4 is decreased compared to healthy control. CR1 is identified as the most significantly decreased complement component in IgA nephropathy patients. CR1 protein expression is inversely correlated with IgA nephropathy interstitial lesion but not with renal function progress. Further correlation analysis of clinical information reveals that CR1 protein expression is inversely correlated with IgA nephropathy interstitial lesion, as represented as Oxford Classification T score. However, CR1 is not significantly correlated with serum creatine level or renal function loss progress (calculated as average eGFR decline per month). CD21/35+ cells are increased in the experimental renal interstitial injury mice. To test the hypothesis CR1 may be associated with interstitial lesion, we study the CD21/35 (CD21/35 is the mouse homolog of CR1 molecule) expression in unilateral urethral obstruction mice. We find that CD45+bone marrow derived cells significantly infiltrated the obstructed kidney at day 12, of which the CD5+ CD21/35+cells significantly increased compared to control. Adaptive transfer of CD21/35+B cells protects the cortex structure in experimental renal interstitial injury. To test whether the CD21/35 molecule on immune cells are protective against interstitial lesion, we sorted CD21/35+B cells and CD21/35-B cells and test their respective protective potential through adaptive transfer into UUO mice. We find that the renal cortex width is significantly increased in CD21/35+group than that in CD21/35-group at day 12 after UUO. CR1 is inversely correlated with renal interstitial lesion in IgA nephropathy patients and is potentially protective in experimental renal interstitial injury.

Chinese herbal component, Praeruptorin E, enhances anti-asthma efficacy and prevents toxicity of aminophylline by targeting the NF-κB/PXR/CYP3A4 pathway

BackgroundAminophylline is widely used for the treatment of asthma, but the therapeutic dose is very close to the toxic dose, which makes this drug prone to accumulation poisoning. In the present study, we explored whether the Chinese herbal component, Praeruptorin E (PE), enhances anti-asthma efficacy and prevents the toxicity of aminophylline.MethodsFirst, an ovalbumin (OVA)-induced mouse model of asthma, immunohistochemistry, pathological staining, and bronchoalveolar lavage fluid (BALF) were used to detect the lung condition of asthmatic mice. The content of Th2 cytokines in serum was measured by enzyme-linked immunosorbent assay (ELISA), and the expression of related proteins was detected by Western blotting and immunofluorescence. Concentrations of theophylline and its metabolites in rat serum were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). siRNA transfection and chromatin immunoprecipitation (ChIP) were used to investigate the mechanism of PE.ResultsPE was found to synergize with aminophylline to reduce the infiltration of inflammatory cells, collagen deposition, and mucus hyperplasia in the lungs of asthmatic mice. It inhibited the expression of Th2 cytokines, interleukin (IL)-4, IL-5, and IL-13; promoted lung tissue repair; and reduced the toxic effect of aminophylline on the heart. Moreover, LC-MS/MS analysis showed that PE reduced the plasma concentration of the parent theophylline and its metabolite 1,3-dimethyluric acid (1,3-DMU). PE facilitated aminophylline’s suppression of nuclear factor-κB (NF-κB), and increased the expression of the xenobiotic nuclear receptor pregnane X receptor (PXR) and its primary target gene, CYP3A11 [this is the mouse homolog of cytochrome P450 3A (CYP3A)] in the asthmatic mouse liver and in the L-02 human fetal hepatocyte cell culture model. In addition, the ChIP assay revealed that PE attenuated the binding of NF-κB to the promoter region of the PXR gene and prevented the suppression of PXR gene expression by NF-κB.ConclusionsPE has a dual function in enhancing the immune regulation and anti-inflammatory effects of theophylline, as well as preventing theophylline toxicity by targeting the NF-κB/PXR/CYP3A4 axis. PE is a promising herbal medicine that will benefit asthmatics taking theophylline.

Impact of a SLC24A5 variant on the retinal pigment epithelium of a Japanese patient with oculocutaneous albinism type 6

Oculocutaneous albinism (OCA) 6 is a non-syndromic type of OCA that has distinct ocular symptoms and variable cutaneous hypopigmentation. The causative gene of OCA6 is SLC24A5, which encodes NCKX5, a K+ -dependent Na+ /Ca2+ exchanger 5. NCKX5 is involved in the maturation of melanosomes, but its function is still unclear. In this study, we characterized a Japanese patient with OCA6. Genetic analysis revealed compound heterozygous variants in SLC24A5, c.590+1dupG, and c.598G>A (p.G200R). To clarify the functional significance of the missense variant, we generated a knock-in (KI) mouse model carrying the mouse homolog of the G200R variant using the CRISPR/Cas9system. Chemical analysis showed decreased amounts of eumelanin in the hair and skin of KI mice, while levels of benzothiazine units in pheomelanin were significantly increased in their hair. Retinal pigment was also decreased in KI mice. Notably, a histopathologic study revealed a significant pigment loss in the retinal pigment epithelium (RPE) but not in the choroid. Immunohistochemically, the expression of NCKX5 in the RPE was decreased but was maintained in the choroid of KI mice. These findings could explain the difference in phenotypic severity between eye symptoms and hypopigmentation in the skin/hair.

Ankfn1-mutant vestibular defects require loss of both ancestral and derived paralogs for penetrance in zebrafish.

How and to what degree gene duplication events create regulatory innovation, redundancy, or neofunctionalization remain important questions in animal evolution and comparative genetics. Ankfn1 genes are single copy in most invertebrates, partially duplicated in jawed vertebrates, and only the derived copy retained in most mammals. Null mutations in the single mouse homolog have vestibular and neurological abnormalities. Null mutation of the single Drosophila homolog is typically lethal with severe sensorimotor deficits in rare survivors. The functions and potential redundancy of paralogs in species with two copies are not known. Here, we define a vestibular role for Ankfn1 homologs in zebrafish based on the simultaneous disruption of each locus. Zebrafish with both paralogs disrupted showed vestibular defects and early lethality from swim bladder inflation failure. One intact copy at either locus was sufficient to prevent major phenotypes. Our results show that vertebrate Ankfn1 genes are required for vestibular-related functions, with at least partial redundancy between ancestral and derived paralogs.

Mouse Homologue of Human HLA-DO Does Not Preempt Autoimmunity but Controls Murine Gammaherpesvirus MHV68.

H2-O (human HLA-DO) is a relatively conserved nonclassical MHC class II (MHCII)-like molecule. H2-O interaction with human HLA-DM edits the repertoire of peptides presented to TCRs by MHCII. It was long hypothesized that human HLA-DM inhibition by H2-O provides protection from autoimmunity by preventing binding of the high-affinity self-peptides to MHCII. The available evidence supporting this hypothesis, however, was inconclusive. A possibility still remained that the effect of H2-O deficiency on autoimmunity could be better revealed by using H2-O-deficient mice that were already genetically predisposed to autoimmunity. In this study, we generated and used autoimmunity-prone mouse models for systemic lupus erythematosus and organ-specific autoimmunity (type 1 diabetes and multiple sclerosis) to definitively test whether H2-O prevents autoimmune pathology. Whereas our data failed to support any significance of H2-O in protection from autoimmunity, we found that it was critical for controlling a γ-herpesvirus, MHV68. Thus, we propose that H2-O editing of the MHCII peptide repertoire may have evolved as a safeguard against specific highly prevalent viral pathogens.

ZDHHC19 localizes to the cell membrane of spermatids and is involved in spermatogenesis†.

Sperm is the ultimate executor of male reproductive function. Normal morphology, quantity, and motility of sperm ensure the normal reproductive process. Palmitoylation is a posttranslational modification mediated by palmitoyltransferases whereby palmitoyl is added to proteins. Seven palmitoyltransferases have been identified in Saccharomyces cerevisiae and 23 in humans (including ZDHHC1-9 and ZDHHC11-24), with corresponding homologs in mice. We identified two testis-specific palmitoyltransferases ZDHHC11 and ZDHHC19 in mice. The Zdhhc11 and Zdhhc19-knockout mouse models were constructed, and it was found that the Zdhhc11 knockout males were fertile, while Zdhhc19 knockout males were sterile. ZDHHC19 is located in the cell membrane of step 4-9 spermatids in the mouse testis, and phenotypic analysis showed that the testicular weight ratio in the Zdhhc19-/- mice decreased along with the number and motility of the sperm decreased, while sperm abnormalities increased, mainly due to the "folded" abnormal sperm caused by sperm membrane fusion, suggesting the involvement of ZDHHC19 in maintaining membrane stability in the male reproductive system. In addition, Zdhhc19-/- mice showed abnormal sperm morphologies and apoptosis during spermatogenesis, suggesting that spermatogenesis in the Zdhhc19-/- mice was abnormal. These results indicate that ZDHHC19 promotes membrane stability in male germ cells.

Molecular Cloning of Alternative Splicing Variants of the Porcine PML Gene and Its Expression Patterns During Japanese Encephalitis Virus Infection.

Promyelocytic leukemia (PML) protein is a crucial component of PML-nuclear bodies (PML-NBs). PML and PML-NBs are involved in the regulation of various cellular functions, including the antiviral immune response. The human PML gene can generate several different isoforms through alternative splicing. However, little is known about the porcine PML alternative splicing isoforms and their expression profiles during Japanese encephalitis virus (JEV) infection. In the present study, we cloned seven mature transcripts of porcine PML, all of which contained the same N-terminal sequence but differed in the C-terminal sequences due to alternative splicing. These seven transcripts encoded five proteins all of which had the RBCC motif and sumoylation sites. Amino acid sequence homology analysis showed that porcine PML-1 had relatively high levels of identity with human, cattle, and goat homologs (76.21, 77.17, and 77.05%, respectively), and low identity with the mouse homolog (61.78%). Immunofluorescence analysis showed that the typical PML-NBs could be observed after overexpression of the five PML isoforms in PK15 cells. Quantitative reverse transcription PCR (RT-qPCR) analysis showed significant upregulation of PML isoforms and PML-NB-associated genes (Daxx and SP100) at 36 and 48 h post-infection (hpi). Western blotting analysis indicated that the PML isoforms were upregulated during the late stage of infection. Moreover, the number of PML-NBs was increased after JEV infection. These results suggest that porcine PML isoforms may play essential roles in JEV infection.

Loss of zinc-finger protein 212 leads to Purkinje cell death and locomotive abnormalities with phospholipase D3 downregulation

Although Krüppel-associated box domain-containing zinc-finger proteins (K-ZNFs) may be associated with sophisticated gene regulation in higher organisms, the physiological functions of most K-ZNFs remain unknown. The Zfp212 protein was highly conserved in mammals and abundant in the brain; it was mainly expressed in the cerebellum (Cb). Zfp212 (mouse homolog of human ZNF212) knockout (Zfp212-KO) mice showed a reduction in survival rate compared to wild-type mice after 20 months of age. GABAergic Purkinje cell degeneration in the Cb and aberrant locomotion were observed in adult Zfp212-KO mice. To identify genes related to the ataxia-like phenotype of Zfp212-KO mice, 39 ataxia-associated genes in the Cb were monitored. Substantial alterations in the expression of ataxin 10, protein phosphatase 2 regulatory subunit beta, protein kinase C gamma, and phospholipase D3 (Pld3) were observed. Among them, Pld3 alone was tightly regulated by Flag-tagged ZNF212 overexpression or Zfp212 knockdown in the HT22 cell line. The Cyclic Amplification and Selection of Targets assay identified the TATTTC sequence as a recognition motif of ZNF212, and these motifs occurred in both human and mouse PLD3 gene promoters. Adeno-associated virus-mediated introduction of human ZNF212 into the Cb of 3-week-old Zfp212-KO mice prevented Purkinje cell death and motor behavioral deficits. We confirmed the reduction of Zfp212 and Pld3 in the Cb of an alcohol-induced cerebellar degeneration mouse model, suggesting that the ZNF212–PLD3 relationship is important for Purkinje cell survival.

Mouse Embryonic Fibroblasts Isolated From Nthl1 D227Y Knockin Mice Exhibit Defective DNA Repair and Increased Genome Instability

Oxidative DNA damage as a result of normal cellular metabolism, inflammation, or exposure to exogenous DNA damaging agents if left unrepaired, can result in genomic instability, a precursor to cancer and other diseases. Nth-like DNA glycosylase 1 (NTHL1) is an evolutionarily conserved bifunctional DNA glycosylase that primarily removes oxidized pyrimidine lesions. NTHL1 D239Y is a germline variant identified in both heterozygous and homozygous state in the human population. Here, we have generated a knockin mouse model carrying Nthl1 D227Y (mouse homologue of D239Y) using CRISPR-cas9 genome editing technology and investigated the cellular effects of the variant in the heterozygous (Y/+) and homozygous (Y/Y) state using murine embryonic fibroblasts. We identified a significant increase in double stranded breaks, genomic instability, replication stress and impaired proliferation in both the Nthl1 D227Y heterozygous Y/+ and homozygous mutant Y/Y MEFs. Importantly, we identified that the presence of the D227Y variant interferes with repair by the WT protein, possibly by binding and shielding the lesions. The cellular phenotypes observed in D227Y mutant MEFs suggest that both the heterozygous and homozygous carriers of this NTHL1 germline mutation may be at increased risk for the development of DNA damage-associated diseases, including cancer.

EXTH-45. MICROGLIA-SPECIFIC DISRUPTION OF SIALIC ACID-SIGLEC-9/E INTERACTIONS: A NOVEL IMMUNOTHERAPY AGAINST GLIOBLASTOMA?

Abstract Recently, ‘don’t eat me’-signals like CD47 have emerged as novel innate immune checkpoints, enabling cancer cells to evade clearance by phagocytes such as monocyte-derived macrophages (MDM) or microglia (MG). Here, we aim at defining the role of inhibitory Siglec-9 in human and its mouse homologue Siglec-E in MG-centered immunotherapy against GBM. TCGA RNA-sequencing data revealed a significant correlation between high expression of immunoinhibitory SIGLEC9 and poor survival in GBM patients (log-rank p = 0.02). Siglec-E blockade increased murine MG mediated GBM cell in vitro phagocytosis (normalized phagocytosis of 1.00 in isotype vs. 1.76 in anti-Siglec-E antibody, p &amp;lt; 0.001). By employing a CT-2A orthotopic GBM mouse model with MG-specific spatio-temporal deletion of Siglece (Sall1 CreERT2 x Siglece flox ), we observed high MG-proliferation upon Siglec-E knockdown (Ki-67+ MG 14.8% in Cre- vs. 34.9% in Cre+, p &amp;lt; 0.0001) accompanied by an enhanced microglial GBM-cell uptake (5.6% in Cre- vs. 12.3% in Cre+, p &amp;lt; 0.001). This beneficial response was counteracted by an accentuated influx of pro-tumorigenic MDM in the Cre+ group (CD163high CD86low MDM of total MDM 47.1% in Cre- vs. 65.3% in Cre+, p = 0.002), which prevented an efficient adaptive anti-tumor immune response and survival benefit. Currently, we are investigating the cross-talk between GBM-associated MG and MDM upon Siglec-E knockdown by scRNAseq of the tumor-infiltrating immune compartment, including TCR-clonotype tracking. By genetically targeting sialic acids, the ligand for Siglec receptors, on CT-2A cells (GNE-KO), we observed a strong innate and adaptive immune response with less exhausted tumor-infiltrating CD8+ T-cells (14.8% in WT vs. 5.9% in GNE-KO, p = 0.003), which resulted in a prolonged survival (30d in WT vs. 41d post-tumor-injection in GNE-KO, p = 0.03). These data identify the sialic-acid-Siglec-E pathway as an anti-phagocytic signal in a pre-clinical GBM model, and demonstrate its therapeutic potential in GBM immunotherapy.

The m6A \u201creader\u201d YTHDF1 promotes osteogenesis of bone marrow mesenchymal stem cells through translational control of ZNF839

N6-methyladenosine (m6A) is required for differentiation of human bone marrow mesenchymal stem cells (hBMSCs). However, its intrinsic mechanisms are largely unknown. To identify the possible role of m6A binding protein YTHDF1 in hBMSCs osteogenesis in vivo, we constructed Ythdf1 KO mice and showed that depletion of Ythdf1 would result in decreased bone mass in vivo. Both deletion of Ythdf1 in mouse BMSCs and shRNA-mediated knockdown of YTHDF1 in hBMSCs prevented osteogenic differentiation of cells in vitro. Using methylated RNA immunoprecipitation (Me-RIP) sequencing and RIP-sequencing, we found that ZNF839 (a zinc finger protein) served as a target of YTHDF1. We also verified its mouse homolog, Zfp839, was translationally regulated by Ythdf1 in an m6A-dependent manner. Zfp839 potentiated BMSC osteogenesis by interacting with and further enhancing the transcription activity of Runx2. These findings should improve our understanding of the mechanism of BMSC osteogenesis regulation and provide new ideas for the prevention and treatment of osteoporosis.

Circ-SIRT1 inhibits cardiac hypertrophy via activating SIRT1 to promote autophagy

Mounting studies have substantiated that abrogating autophagy contributes to cardiac hypertrophy (CH). Sirtuin 1 (SIRT1) has been reported to support autophagy and inhibit CH. However, the upstream regulation mechanism behind the regulation of SIRT1 level in CH remains unclear. Circular RNAs (circRNAs) are vital modulators in diverse human diseases including CH. This study intended to investigate the regulatory mechanism of circRNA on SIRT1 expression in CH. CH model was established by angiotensin II (Ang II) fusion or transverse aortic constriction (TAC) surgery and Ang II treatment on hiPSC-CMs and H9c2 cells in vitro. Our results showed that circ-SIRT1 (hsa_circ_0093884) expression was downregulated in Ang II-treated hiPSC-CMs, and confirmed that its conserved mouse homolog circ-Sirt1 (mmu_circ_0002354) was expressed at low levels in Ang II-treated H9c2 cells and TAC-induced mice model. Functionally, circ-SIRT1/circ-Sirt1 attenuated Ang II-induced CH and induced autophagy in hiPSC-CMs and H9c2 cardiomyocytes. Mechanistically, circ-SIRT1 could upregulate its host gene SIRT1 at the post-transcriptional level by sponging miR-3681-3p/miR-5195-3p and stabilized SIRT1 protein at the post-translational level by recruiting USP22 to induce deubiquitination on SIRT1 protein. Further, SIRT1 knockdown could rescue the effect of circ-SIRT1 upregulation on Ang II-induced CH and autophagy in vitro and in vivo. In conclusion, we first uncovered that circ-SIRT1 restrains CH via activating SIRT1 to promote autophagy, indicating circ-SIRT1 as a promising target to alleviate CH.

G protein–coupled receptor kinase 5 regulates thrombin signaling in platelets via PAR-1

AbstractThe interindividual variation in the functional response of platelets to activation by agonists is heritable. Genome-wide association studies (GWASs) of quantitative measures of platelet function have identified fewer than 20 distinctly associated variants, some with unknown mechanisms. Here, we report GWASs of pathway-specific functional responses to agonism by adenosine 5′-diphosphate, a glycoprotein VI–specific collagen mimetic, and thrombin receptor-agonist peptides, each specific to 1 of the G protein–coupled receptors PAR-1 and PAR-4, in subsets of 1562 individuals. We identified an association (P = 2.75 × 10−40) between a common intronic variant, rs10886430, in the G protein–coupled receptor kinase 5 gene (GRK5) and the sensitivity of platelets to activate through PAR-1. The variant resides in a megakaryocyte-specific enhancer that is bound by the transcription factors GATA1 and MEIS1. The minor allele (G) is associated with fewer GRK5 transcripts in platelets and the greater sensitivity of platelets to activate through PAR-1. We show that thrombin-mediated activation of human platelets causes binding of GRK5 to PAR-1 and that deletion of the mouse homolog Grk5 enhances thrombin-induced platelet activation sensitivity and increases platelet accumulation at the site of vascular injury. This corroborates evidence that the human G allele of rs10886430 is associated with a greater risk for cardiovascular disease. In summary, by combining the results of pathway-specific GWASs and expression quantitative trait locus studies in humans with the results from platelet function studies in Grk5−/− mice, we obtain evidence that GRK5 regulates the human platelet response to thrombin via the PAR-1 pathway.

Obesity and altered angiogenic-related gene expression in endometrial cancer

ObjectivesEvaluate association between obesity and angiogenic-related gene expression in endometrial cancer (EC). Evaluate interaction between diet and metformin on angiogenic-related gene expression. MethodsWe evaluated the association between 168 human angiogenic-related genes and body mass index (BMI) in the TCGA Uterine Corpus Endometrial Carcinoma cohort (endometrioid endometrial cancer (EEC) cohort n = 290, and copy number high cohort n = 55), an independent validation cohort from Gynecologic Cancer Center of Excellence (GYN-COE) (n = 62) and corresponding 185 homologous mouse genes in an LKB1fl/flp53fl/fl mouse model of EC (n = 20). Mice received 60% of calories from fat in a high-fat diet (HFD), mimicking diet-induced obesity, versus 10% of calories from fat in a low-fat diet (LFD). After tumor growth, HFD (n = 5) and LFD (n = 5) mice were treated with metformin (200 mg/kg/day) or control. Whole transcriptome analysis of mouse tumors was performed using RNA-Seq. ResultsAt a false-discovery rate of 10%, twenty-one angiogenic-related genes were differentially expressed with respect to BMI when adjusting for grade in the TCGA EEC cohort. Evaluation of these genes in the mouse model control group revealed association between increased Edil3 expression in HFD versus LFD mice (2.5-fold change (FC); unadjusted p = 0.03). An interaction was observed for expression of Edil3 between diet and metformin treatment (unadjusted p = 0.009). Association between BMI and increased expression of EDIL3 was validated in one of four EDIL3 probesets in the GYN-COE cohort (p = 0.0011, adjusted p = 0.0342). ConclusionsObesity may promote tumor progression via differential modulation of angiogenic pathways in EEC. Our exploratory findings demonstrated that EDIL3 may be a candidate gene of interest.

Discovery and Characterization of Novel Antagonists of the Proinflammatory Orphan Receptor GPR84.

GPR84 is a poorly characterized, nominally orphan, proinflammatory G protein-coupled receptor that can be activated by medium chain length fatty acids. It is attracting considerable interest as a potential therapeutic target for antagonist ligands in both inflammatory bowel diseases and idiopathic pulmonary fibrosis. Successful screening of more than 300 000 compounds from a small molecule library followed by detailed analysis of some 50 drug-like hits identified 3-((5,6-bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)methyl)-1H-indole as a high affinity and highly selective competitive antagonist of human GPR84. Tritiation of a di-iodinated form of the core structure produced [3H]3-((5,6-diphenyl-1,2,4-triazin-3-yl)methyl)-1H-indole, which allowed effective measurement of receptor levels in both transfected cell lines and lipopolysaccharide-treated THP-1 monocyte/macrophage cells. Although this compound series lacks significant affinity at mouse GPR84, homology modeling and molecular dynamics simulations provided a potential rationale for this difference, and alteration of two residues in mouse GPR84 to the equivalent amino acids in the human orthologue, predicted to open the antagonist binding pocket, validated this model. Sequence alignment of other species orthologues further predicted binding of the compounds as high affinity antagonists at macaque, pig, and dog GPR84 but not at the rat orthologue, and pharmacological experiments confirmed these predictions. These studies provide a new class of GPR84 antagonists that display species selectivity defined via receptor modeling and mutagenesis.

Autophagic elimination of ribosomes during spermiogenesis provides energy for flagellar motility.

How autophagy initiation is regulated and what the functional significance of this regulation is are unknown. Here, we characterized the role of yeast Vac8 in autophagy initiation through recruitment of PIK3C3-C1 to the phagophore assembly site (PAS). This recruitment is dependent on the palmitoylation of Vac8 and on its middle ARM domains for binding PIK3C3-C1. Vac8-mediated anchoring of PIK3C3-C1 promotes PtdIns3P generation at the PAS and recruitment of the PtdIns3P binding protein Atg18-Atg2. The mouse homolog of Vac8, ARMC3, is conserved and functions in autophagy in mouse testes. Mice lacking ARMC3 have normal viability but show complete male infertility. Proteomic analysis indicated that the autophagic degradation of cytosolic ribosomes was blocked in ARMC3-deficient spermatids, which caused low energy levels of mitochondria and motionless flagella. These studies uncovered a function of Vac8/ARMC3 in PtdIns3-kinase anchoring at the PAS and its physical significance in mammalian spermatogenesis with a germ tissue-specific autophagic function.

Histone acetylation at the sulfotransferase 1a1 gene is associated with its hepatic expression in normal aging.

Phase II drug metabolism is poorly studied in advanced age and older adults may exhibit significant variability in their expression of phase II enzymes. We hypothesized that age-related changes to epigenetic regulation of genes involved in phase II drug metabolism may contribute to these effects. We examined published epigenome-wide studies of human blood and identified the SULT1A1 and UGT1A6 genes as the top loci showing epigenetic changes with age. To assess possible functional alterations with age in the liver, we assayed DNA methylation (5mC) and histone acetylation changes around the mouse homologs Sult1a1 and Ugt1a6 in liver tissue from mice aged 4-32 months. Our sample shows a significant loss of 5mC at Sult1a1 (β = -1.08, 95% CI [-1.8, -0.2], SE = 0.38, P = 0.011), mirroring the loss of 5mC with age observed in human blood DNA at the same locus. We also detected increased histone 3 lysine 9 acetylation (H3K9ac) with age at Sult1a1 (β = 0.11, 95% CI [0.002, 0.22], SE = 0.05, P = 0.04), but no change to histone 3 lysine 27 acetylation (H3K27ac). Sult1a1 gene expression is significantly positively associated with H3K9ac levels, accounting for 23% of the variation in expression. We did not detect any significant effects at Ugt1a6. Sult1a1 expression is under epigenetic influence in normal aging and this influence is more pronounced for H3K9ac than DNA methylation or H3K27ac in this study. More generally, our findings support the relevance of epigenetics in regulating key drug-metabolizing pathways. In the future, epigenetic biomarkers could prove useful to inform dosing in older adults.

Molecular mechanism of interspecies differences in the binding affinity of TD139 to Galectin-3.

Galectin-3 (Gal-3), a β-galactoside-binding lectin, has been implicated in a plethora of pathological disorders including fibrosis, inflammation, cancer and metabolic diseases. TD139-a thio-digalactoside inhibitor developed by Galecto Biotech as a potential therapeutic for idiopathic pulmonary fibrosis-is the most advanced small-molecule Gal-3 inhibitor in clinical studies. It binds to human Gal-3 with high affinity but has lower affinity towards mouse and rat homologs, which is also manifested in the differential inhibition of Gal-3 function. Using biophysical methods and high-resolution X-ray co-crystal structures of TD139 and Gal-3 proteins, we demonstrate that a single amino acid change corresponding to A146 in human Gal-3 is sufficient for the observed reduction in the binding affinity of TD139 in rodents. Site-directed mutagenesis of A146V (in human Gal-3) and V160A (in mouse Gal-3) was sufficient to interchange the affinities, mainly by affecting the off rates of the inhibitor binding. In addition, molecular dynamics simulations of both wild-type and mutant structures revealed the sustained favorable noncovalent interactions between the fluorophenyl ring and the active site A146 (human Gal-3 and mouse V160A) that corroborate the finding from biophysical studies. Current findings have ramifications in the context of optimization of drug candidates against Gal-3.

REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

Persistent acinar to ductal metaplasia (ADM) is a recently recognized precursor of pancreatic ductal adenocarcinoma (PDAC). Here we show that the ADM area of human pancreas tissue adjacent to PDAC expresses significantly higher levels of regenerating protein 3A (REG3A). Exogenous REG3A and its mouse homolog REG3B induce ADM in the 3D culture of primary human and murine acinar cells, respectively. Both Reg3b transgenic mice and REG3B-treated mice with caerulein-induced pancreatitis develop and sustain ADM. Two out of five Reg3b transgenic mice with caerulein-induced pancreatitis show progression from ADM to pancreatic intraepithelial neoplasia (PanIN). Both in vitro and in vivo ADM models demonstrate activation of the RAS-RAF-MEK-ERK signaling pathway. Exostosin-like glycosyltransferase 3 (EXTL3) functions as the receptor for REG3B and mediates the activation of downstream signaling proteins. Our data indicates that REG3A/REG3B promotes persistent ADM through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway. Targeting REG3A/REG3B, its receptor EXTL3, or other downstream molecules could interrupt the ADM process and prevent early PDAC carcinogenesis.