Mindbomb E3 Ubiquitin Protein Ligase Research Articles

SNORA73B promotes endometrial cancer progression through targeting MIB1 and regulating host gene RCC1 alternative splicing.

Endometrial cancer (EC) is a common gynaecological malignant tumour with unclear pathogenesis. Small nucleolar RNA (snoRNA) is involved in many biological processes, including those of cancers. Using the Cancer Genome Atlas (TCGA) database, the expression pattern of a snoRNA, SNORA73B, was analysed. The biological functions of SNORA73B were assessed by in vitro proliferation, apoptosis, migration, and invasion assays and in vivo by the xenograft model. RNA sequencing (RNA-seq) and RNA immunoprecipitation assays were performed to determine the relationship between SNORA73B and its target genes. High-performance liquid chromatography (HPLC) was performed to detect the pseudouridine content of the mindbomb E3 ubiquitin protein ligase 1 gene (MIB1). The stability of MIB1 mRNA was evaluated using a transcription inhibitor, actinomycin D. By performing co-immunoprecipitation assays, the change in the ubiquitin levels of the Jagged canonical Notch ligand 1 (Jag 1), caused by SNORA73B and MIB1, was identified. RNA-seq and qRT-PCR were performed to detect the alternative splicing of the regulator of the chromosome condensation 1 gene (RCC1). The TCGA database analysis showed that SNORA73B was highly expressed in EC. SNORA73B promoted cell proliferation, migration, and invasion and inhibited apoptosis. SNORA73B modified the pseudouridine content in MIB1 and increased the stability of MIB1 mRNA and protein; thus, it affected Jag 1 ubiquitination and further activated the Notch pathway. SNORA73B also affected the alternative splicing of RCC1, increasing the number of transcripts, RCC1-T2 and RCC1-T3, which promoted cell proliferation, migration, and invasion. SNORA73B can be a potential target for EC.

Lipotoxicity-induced circGlis3 impairs beta cell function and is transmitted by exosomes to promote islet endothelial cell dysfunction.

Lipotoxicity constitutes the major driving force for type 2 diabetes. Circular RNAs (circRNAs) play important roles in regulating beta cell function and exosomes are essential mediators of intercellular communication. The role of exosomal circRNAs in type 2 diabetes remains largely unknown. We aimed to examine whether lipotoxicity induces dysregulation of circRNAs in beta cell-derived exosomes and to determine the contribution of exosomal circRNAs to the development of type 2 diabetes. Exosomes were extracted from MIN6 cells treated with palmitate or BSA, and RNA sequencing was performed. CircGlis3 (Gli-similar 3) expression level was validated by qPCR. The impact of circGlis3 on beta cell function and the deleterious effects of exosomal circGlis3 on islet endothelial cells (islet ECs) were investigated in vitro and in vivo in human and mouse models by gain or loss of function assays. The molecular mechanism of circGlis3 was explored by RNA pull-down and immunoprecipitation assays. Beta cell-derived exosomal circGlis3 was significantly upregulated under lipotoxic conditions, and exosomal circGlis3 levels were also elevated in the serum of mouse models of diabetes and participants with type 2 diabetes. CircGlis3 participated in lipotoxicity-induced beta cell dysfunction in vitro and in vivo. Moreover, beta cell-derived exosomal circGlis3 could be transferred to islet ECs and reduce the cell viability, cell migration and angiogenesis of islet ECs. Mechanistically, circGlis3 promoted the degradation of glucocorticoid modulatory element-binding protein 1 (GMEB1) by facilitating the interaction between GMEB1 and mindbomb E3 ubiquitin protein ligase 2 (MIB2), thus suppressing the phosphorylation of heat shock protein 27 (HSP27). Our study points to the involvement of circGlis3 in diabetes development, and exosomal circGlis3 transfer as a communication mode between beta cells and islet ECs, suggesting that circGlis3 might be a potential biomarker and therapeutic target for type 2 diabetes. The RNA-sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) database, with accession number PRJNA689673. Mass spectrometry data are available via ProteomeXchange with identifier PXD024693.

MIB1‐mediated degradation of WRN promotes cellular senescence in response to camptothecin treatment

Werner syndrome protein (WRN) plays critical roles in DNA replication, recombination, and repair, as well as transcription and cellular senescence. Ubiquitination and degradation of WRN have been reported, however, the E3 ubiquitin ligase of WRN is little known. Here, we identify mindbomb E3 ubiquitin protein ligase 1 (MIB1) as a novel E3 ubiquitin ligase for WRN protein. MIB1 physically interacts with WRN in vitro and in vivo and induces ubiquitination and degradation of WRN in the ubiquitin-proteasome pathway. Camptothecin (CPT) enhances the interaction between MIB1 and WRN, and promotes WRN degradation in a MIB1-dependent manner. In addition, CPT-induced cellular senescence is facilitated by the expression of MIB1 and attenuated by WRN expression. Our results show that MIB1-mediated degradation of WRN promotes cellular senescence and reveal a novel model executed by MIB1 and WRN to regulate cellular senescence.

MicroRNA‑198 suppresses prostate tumorigenesis by targeting MIB1

MicroRNAs are small non-coding RNA molecules which act as modulators of gene function, and have been identified as playing important roles in cancer as both tumor suppressors and oncogenes. The present study aimed to examine the role of miR-198 in prostate cancer aggression by analyzing how it influences several hallmarks of cancer. Abundance of miR-198 in prostate cancer and association with clinical characteristics was analyzed using a CPC-Gene prostate cancer dataset. Overexpression of miR-198 was performed using transient transfection of miR-198 mimic prior to assaying proliferation, cell cycle, and colony formation in LNCaP and DU145 cell lines using standard protocols. In vivo tumor formation in athymic nude mice was examined using LNCaP xenografts with stable overexpression conferred using lentiviral miR-198 transduction. Protein and mRNA abundance of MIB1 was determined using western blotting and RT-qPCR respectively, while miR-198 binding to MIB1 was validated using a luciferase reporter assay. miR-198 abundance was lower in high Gleason grade prostate cancer relative to intermediate and low-grade cancer. Overexpression of miR-198 diminished proliferation of prostate cancer cell lines, increased G0/G1 cell cycle arrest, and significantly impaired colony formation. Elevated miR-198 abundance was also demonstrated to impair tumor formation in vivo using LNCaP xenografts. Mindbomb E3 ubiquitin protein ligase 1 (MIB1) was demonstrated to be directly targeted by miR-198, and knockdown of MIB1 recapitulated the effects of miR-198 on proliferation and colony formation. The present evidence supports miR-198 as an important tumor suppressor in prostate cancer, and demonstrates for the first time that it acts by targeting MIB1. The present study reinforces the importance and complexity of miRNA in regulating prostate cancer aggression.

Serous ovarian tumors: Immunohistochemical profiling as an aid to grading and understanding tumorigenesis

BackgroundThe aim of this study was to evaluate the expression of p53, p16, Wilms tumor gene (WT1), and Mindbomb E3 Ubiquitin Protein Ligase 1 (MIB-1) index by immunohistochemical (IHC) staining in benign, low-grade, and high-grade serous ovarian tumors. MethodsForty-one cases of ovarian serous tumors were included in the study (benign serous tumor [n = 10], low-grade ovarian serous carcinoma [n = 8], and high-grade ovarian serous carcinoma [n = 23]). Expression of p53, p16, WT1, and MIB-1 by IHC was evaluated statistically with the grade of tumor. Semiquantitative scoring system for percentage (0–5) and intensity (1–3) of staining pattern was used to bring about objectivity. Resultsp53, p16, and WT1 showed significantly higher staining scores in ovarian serous carcinoma group than in the benign group (p < 0.05). However, p16 score was not significant in benign versus low-grade tumors. In the carcinoma group, the high-grade serous tumors showed significantly higher staining scores of p53, p16, and WT1 than the low-grade serous tumors (p < 0.05). Papillary serous tumors had comparatively lower p53 and WT1 scores for the same grade of tumor. MIB-1 scores were not significant. Conclusionp53, p16, and WT1 are helpful for the subtyping of serous ovarian tumors as low grade and high grade. WT1 is helpful in establishing primary ovarian serous tumors. The combination of moderate-to-high p53 and WT1 scores provides a robust way of confirming high-grade tumors.

Glioblastoma with Oligodendroglioma Component (GBMO) in an adolescent: a case report

Background:Glioblastoma with oligodendroglioma component (GBMO) is a recently classified subtype of glioblastoma, which carries different clinical and prognostic outcomes, being frequently misdiagnosed. Both glioblastoma and GBMO are mainly seen in older ages, such as the 5th and 6th decades of life, being an extremely rare occurrence in children or adolescents and more frequent in male patients. &#x0D; Case report: A 15-year-old girl, presented with history of daily headache, not relieved by painkillers, vomiting, blurred vision and strabismus. Magnetic resonance imaging of the brain revealed expansive tumour on left temporo-occipital lobe. Patient was submitted to intracranial exeresis, along with histopathological examination: glial neoplasm with areas of pleomorphism, hyperchromatism, anaplasia, foci of oligodendroglial component, perinuclear halo and ramified capillaries, resembling oligodendroglioma, necrosis and intense mitotic activity. The immunohistochemical analysis revealed positive Glial Fibrillary Acidic Protein (GFAP), synaptophysin, Ki-67 (MindBomb E3 ubiquitin protein ligase 1 – MIB-1)and hyperexpression of Epidermal Growth Factor Receptor (EGFR), indicating GBMO. Subsequently, Fluorescence in situ Hybridization (FISH) showed 1p/19q codeletion and Isocitrate Dehydrogenase 1 (IDH 1) mutation, suggesting an oligodendroglioma component. Tumour resection was total and symptoms disappeared. Afterwards, she started adjuvant oral chemotherapy with temozolomide. Treatment was completed nine months after the diagnosis, with no greater symptoms or complications and complete remission. &#x0D; Conclusion: GBMO must be considered as a possible diagnosis when confronted with a malignant glioma with oligodendroglial tumour component, independent of age or genre. Necrosis upon histopathological examination has a strong relation to shorter median overall survival. IDH mutation and 1p/19q codeletion should be analyzed by immunohistochemistry. Total tumour resection, with adjuvant treatment (chemotherapy with temozolomide and radiotherapy), increases benefits and improves prognosis.

B-cell lymphoma 2 is associated with advanced tumor grade and clinical stage, and reduced overall survival in young Chinese patients with colorectal carcinoma.

The development of biomarkers that accurately and reliably detect colorectal cancer is a promising approach for colorectal cancer screening. Therefore, the objective of the present study was to evaluate the protein expression of α-methylacyl-CoA racemase (P504S/AMACR), tumor protein p53 (p53), B-cell lymphoma 2 (Bcl-2) and Ki-67/mindbomb E3 ubiquitin protein ligase 1 (MIB-1) in a population of Chinese patients with colorectal carcinoma. Colorectal tumors with matched normal tissue margins were collected from 148 surgical patients, and the demographic and clinical characteristics were collected. Immunohistochemical staining and western blot analysis of P504S/AMACR, p53, Bcl-2 and Ki-67/MIB-1 were conducted. Statistical analyses were used to compare protein expression in the colorectal tumors and matched normal tissue margins and to identify any associations between them and various clinicopathological parameters. Survival analyses were performed using the Kaplan-Meier method. In the present study, immunohistochemistry and western blot analysis revealed significantly higher expression of all four proteins in colorectal tumors compared with matched normal tissue margins (P<0.001). Spearman's rank correlation analysis revealed that Bcl-2 expression was negatively correlated with pathological grade and Tumor-Node-Metastasis (TNM) stage (−0.827 and −0.388, respectively; P<0.05). Bcl-2 expression was revealed to be a significant prognostic indicator of colorectal carcinoma [relative risk (95% CI), 0.703 (0.552–0.895); P<0.05]. The log-rank test revealed a significant association between low Bcl-2 expression and reduced overall survival (P=0.039), as well as a significant association between older age (>55 years) and reduced overall survival (P<0.001) in Chinese patients with colorectal carcinoma. In conclusion, low expression of Bcl-2 is significantly correlated with advanced pathological grade and TNM stage and is a prognostic indicator of reduced overall survival in young Chinese patients with colorectal carcinoma.

Orthopedia homeobox is preferentially expressed in typical carcinoids of the lung.

Twenty-seven percent of neuroendocrine tumors (NETs) are associated with distant metastases, and in some patients, the primary site is unknown. Orthopedia homeobox protein (OTP) has been described as a useful marker for lung carcinoids (LCs) and for separating low-grade typical carcinoids (TCs) from intermediate-grade atypical carcinoids (ACs) in resection specimens. This study evaluated OTP, thyroid transcription factor 1 (TTF-1), and Ki-67 expression in fine-needle aspiration (FNA) samples of various NETs. A search for NETs diagnosed via FNA with subsequent resection was performed. Cell block sections were stained for OTP, TTF-1, and mindbomb E3 ubiquitin protein ligase 1 (Mib-1). Nuclear expression for OTP and TTF-1 was considered positive. Nuclear Ki-67 staining was reported as a percentage. Results were correlated with the grade and primary site for resection specimens. Sixty-three FNA samples of NETs were identified: 14 liver samples, 14 pancreatic samples, 13 lymph node samples, 12 lung samples, 3 retroperitoneum samples, 2 small intestine samples, and 5 other samples. OTP was positive in 12 of 63 NETs (19%) from the following sites: lung (n = 8), liver (lung primary; n = 2), skin (n = 1), and lymph node (lung primary; n = 1). In well-differentiated NETs, only LCs were OTP-positive, whereas TTF-1 was positive in LCs and nonlung NETs (67% vs 7%). Within the LC category, OTP was positive in 100% of the TCs versus 17% of the ACs. OTP is specific for LCs because well-differentiated nonlung NETs are negative for OTP. OTP preferentially stains TCs over ACs. In well-differentiated NETs, OTP staining is highly specific for LCs, and in combination with a low Ki-67 index, it suggests a pulmonary TC. Cancer Cytopathol 2018;126:236-42. © 2018 American Cancer Society.

Detailed analysis of a superficial CD34-positive fibroblastic tumor: A case report and review of the literature.

Superficial cluster of differentiation (CD)34-positive fibroblastic tumor (SCPFT) is a rare mesenchymal neoplasm of borderline malignancy. It is characterized by a superficial location, marked cellular pleomorphism, an extremely low incidence of mitotic figures, and strong CD34 immunohistochemical positivity. As SCPFT is a recently described neoplasm, its characteristics are yet to be fully elucidated. To the best of our knowledge, no detailed studies regarding the imaging findings and cytogenetic analyses of SCPFTs exist. The present study describes a typical case of an 18-year-old man who developed an SCPFT measuring 87×70×80 mm in the subcutaneous adipose tissue of his right thigh. Computed tomography (CT) revealed a well-marginated tumor without calcification, and the enhancement on CT was weak. The tumor demonstrated abnormal uptake on 2-(18F) fluoro-2-deoxy-D-glucose positron emission tomography (PET), with a maximum standardized uptake value of 2.57. Magnetic resonance imaging (MRI) revealed a clearly defined tumor that exhibited homogeneous low signal intensity on T1-weighted imaging and high signal intensity on T2-weighted imaging, with small lobulated structures. Histopathologically, the tumor was composed of irregular spindle-to-oval-shaped cells with eosinophilic glassy cytoplasm and hyperchromatic, bizarre and pleomorphic nuclei that frequently exhibited intranuclear pseudoinclusions. Immunohistochemically, the tumor cells were diffusely and strongly positive for CD34. The Mindbomb E3 ubiquitin protein ligase 1 labeling index was 8.6%. Ultrastructurally, the tumor cells exhibited irregular or convoluted nuclei with abundant euchromatin-prominent nucleoli. The cytoplasmic organelles consisted of scattered, abundant rough endoplasmic reticulum, mitochondria, lysosomes, ribosomal rosettes and aggregated lipid globules. Of 18 metaphase cells identified, 2 demonstrated translocation between chromosomes 2 and 5 in cytogenetic studies. To the best of our knowledge, this is the first study describing imaging data (CT, MRI and PET-CT) and chromosomal aberrations for SCPFT.

The Retinal Pigment Epithelium Is a Notch Signaling Niche in the Mouse Retina

Notch signaling in neural progenitor cell is triggered by ligands expressed in adjacent cells. To identify the sources of active Notch ligands in the mouse retina, we negatively regulated Notch ligand activity in various neighbors of retinal progenitor cells (RPCs) by eliminating mindbomb E3 ubiquitin protein ligase 1 (Mib1). Mib1-deficient retinal cells failed to induce Notch activation in intra-lineage RPCs, which prematurely differentiated into neurons; however, Mib1 in post-mitotic retinal ganglion cells was not important. Interestingly, Mib1 in the retinal pigment epithelium (RPE) also contributed to Notch activationin adjacent RPCs by supporting the localizationofactive Notch ligands at RPE-RPC contacts. Combining this RPE-driven Notch signaling and intra-retinal Notch signaling, we propose a model in which one RPC daughter receives extra Notch signals from the RPE to become an RPC, whereas its sister cell receives only a subthreshold level of intra-retinal Notch signal and differentiates into a neuron.

Mindbomb 2 is dispensable for embryonic development and Notch signalling in zebrafish.

ABSTRACTThe Mindbomb E3 ubiquitin protein ligase (Mib) family of proteins, Mib1 and Mib2, are RING finger ubiquitin ligases that share specific substrates. Mib1 is known to play essential roles in Notch signalling by ubiquitinating Notch ligands in vivo. Conversely, the functions of Mib2 in vivo are not fully understood, although Mib2 ubiquitinates multiple substrates, including Notch ligands, in vitro. To determine the Notch-dependent and Notch-independent functions of Mib2 in vivo, we generated mutant alleles of zebrafish mib2 using transcription activator-like effector nucleases (TALENs). We found that mib2 homozygous mutants were viable and fertile. Notch-mediated functions, such as early neurogenesis, somitogenesis, and pigment cell development, were not affected in mib2 mutant embryos. The lack of Notch-deficient phenotypes in mib2 mutants was not due to compensation by a mib2 maternal gene product because mib2 maternal-zygotic mutants also did not exhibit a distinct phenotype. We also showed that Mib2 does not redundantly act with Mib1 because the genetic ablation of mib2 neither enhanced mibtfi91-null phenotypes nor did it alleviate antimorphic mibta52b phenotypes. Furthermore, the postulated Notch-independent roles of Mib2 in maintaining muscular integrity and N-methyl-D-aspartate receptor (NMDAR) activity were not evident: mib2 mutants did not show phenotypes different from that of the control embryos. These observations suggest that Mib2 is dispensable for embryonic development and does not have redundant functions with Mib1 in Notch signalling at least during early development stages in zebrafish.

The Prognostic Role of Ki-67/MIB-1 in Upper Urinary-Tract Urothelial Carcinomas: A Systematic Review and Meta-Analysis.

Upper urinary-tract urothelial carcinomas (UTUC) constitute 5% of urothelial malignancies. Prognostic biomarkers would allow lower risk surgical approaches for less aggressive UTUCs. One biomarker-Ki-67/mindbomb E3 ubiquitin protein ligase 1 (Ki-67/MIB-1)-shows promise in UTUC, but there have been conflicting findings regarding its prognostic role. The systematic review and meta-analysis aim to determine the prognostic value of Ki-67/MIB-1 in UTUC in terms of UTUC-specific mortality rate, 5-year disease-free survival, and 5-year overall survival (including disease-specific survival). A systematic review of the current literature produced 654 records. A total of 13 studies consisting of 1030 patients were finally included in the meta-analysis. Hazard ratios (HRs) with 95% confidence intervals (CI) were extracted or estimated. The individual HR estimates were combined into a pooled HR using a fixed-effects model that summed homogeneity of the individual true HRs. Patients with Ki-67/MIB-1 overexpression displayed significantly higher UTUC-specific mortality rate (pooled HR: 2.14, 95% CI: 1.73-2.64; p<0.00001), significantly reduced 5-year disease-free survival (pooled HR: 2.27, 95% CI: 1.79-2.92; p<0.00001), and significantly reduced 5-year overall survival (pooled HR=1.77; 95% CI: 1.39-2.23 p<0.00001). There was significant heterogeneity detected in the UTUC-specific mortality rate meta-analysis (I(2)=63%) and the 5-year disease-free survival meta-analysis (I(2)=65%), but there was no significant heterogeneity detected in the 5-year overall survival meta-analysis (I(2)=0%). Egger's testing showed that none of the outcomes were influenced by publication bias (p>0.05). Ki-67/MIB-1 overexpression shows promise as a prognostic biomarker for UTUC patients and requires further investigation.

Periosteal chondroma of the distal tibia: Computed tomography and magnetic resonance imaging characteristics and correlation with histological findings.

Periosteal chondroma is a rare benign hyaline cartilage neoplasm situated on the bone surface. This is the presentation of a unique case of periosteal chondroma arising in the left distal tibial metaphysis of a 25-year-old female patient with a history of antecedent trauma. The physical examination revealed swelling and tenderness in the anterolateral aspect of the left distal lower limb. Plain radiographs revealed a discernible soft tissue lesion with peripheral foci of mineralization. Computed tomography scans confirmed the presence of a surface-based mass with peripheral ossification and a thin rim of calcification. On magnetic resonance imaging, the well-circumscribed mass exhibited intermediate signal intensity on T1-weighted sequences and high signal intensity with foci of decreased signal intensity on T2-weighted sequences. Contrast-enhanced T1-weighted sequences revealed predominantly peripheral enhancement without intramedullary involvement. Following an open biopsy, marginal excision with curettage of the underlying bone cortex was performed. Histologically, the tumor consisted of mature hyaline cartilage arranged in distinct lobules. Foci of ossification with mature bone trabeculae forming a thin shell-like structure were identified in the periphery of the tumor. The mindbomb E3 ubiquitin protein ligase 1 labeling index was <1%. Based on these findings, the tumor was diagnosed as periosteal chondroma. There has been no evidence of local recurrence at 4 months following surgery. Despite its rarity, periosteal chondroma must be considered as a possible diagnosis when confronted with a surface-based, mineralized lesion in the metaphysis of long bones.

Axillary Lymph Node-to-Primary Tumor Standard Uptake Value Ratio on Preoperative (18)F-FDG PET/CT: A Prognostic Factor for Invasive Ductal Breast Cancer.

PurposeThis study assessed the axillary lymph node (ALN)-to-primary tumor maximum standard uptake value (SUVmax) ratio (ALN/T SUV ratio) in invasive ductal breast cancer (IDC) on preoperative 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) to determine the effectiveness in predicting recurrence-free survival (RFS).MethodsOne hundred nineteen IDC patients (mean age, 50.5±10.5 years) with pathologically proven ALN involvement without distant metastasis and preoperative FDG PET/CT were enrolled in the study. SUVmax values of the ALN and primary tumor were obtained on FDG PET/CT, and ALN/T SUV ratio was calculated. Several factors were evaluated for their effectiveness in predicting RFS. These included several parameters on FDG PET/CT as well as several clinicopathological parameters: pathologic tumor/node stage; nuclear and histological grade; hormonal state; status with respect to human epidermal growth factor receptor 2, mindbomb E3 ubiquitin protein ligase 1 (MIB-1), and p53; primary tumor size; and ALN size.ResultsAmong 119 patients with breast cancer, 17 patients (14.3%) experienced relapse during follow-up (mean follow-up, 28.4 months). The ALN/T SUV ratio of the group with disease recurrence was higher than that of the group without recurrence (0.97±1.60 and 0.45±0.40, respectively, p=0.005). Univariate analysis showed that the primary tumor SUVmax, ALN SUVmax, ALN/T SUV ratio, ALN status, nuclear and histological grade, estrogen receptor (ER) status, and MIB-1 status were predictors for RFS. Among these variables, ALN/T SUV ratio with hazard ratio of 4.20 (95% confidence interval [CI], 1.74-10.13) and ER status with hazard ratio of 4.33 (95% CI, 1.06-17.71) were predictors for RFS according to multivariate analysis (p=0.002 and p=0.042, respectively).ConclusionOur study demonstrated that ALN/T SUV ratio together with ER status was an independent factor for predicting relapse in IDC with metastatic ALN. ALN/T SUV ratio on preoperative FDG PET/CT may be a useful marker for selecting IDC patients that need adjunct treatment to prevent recurrence.

MicroRNA-320/RUNX2 axis regulates adipocytic differentiation of human mesenchymal (skeletal) stem cells.

The molecular mechanisms promoting lineage-specific commitment of human mesenchymal (skeletal or stromal) stem cells (hMSCs) into adipocytes (ADs) are not fully understood. Thus, we performed global microRNA (miRNA) and gene expression profiling during adipocytic differentiation of hMSC, and utilized bioinformatics as well as functional and biochemical assays, and identified several novel miRNAs differentially expressed during adipogenesis. Among these, miR-320 family (miR-320a, 320b, 320c, 320d and 320e) were ~2.2–3.0-fold upregulated. Overexpression of miR-320c in hMSC enhanced adipocytic differentiation and accelerated formation of mature ADs in ex vivo cultures. Integrated analysis of bioinformatics and global gene expression profiling in miR-320c overexpressing cells and during adipocytic differentiation of hMSC identified several biologically relevant gene targets for miR-320c including RUNX2, MIB1 (mindbomb E3 ubiquitin protein ligase 1), PAX6 (paired box 6), YWHAH and ZWILCH. siRNA-mediated silencing of those genes enhanced adipocytic differentiation of hMSC, thus corroborating an important role for those genes in miR-320c-mediated adipogenesis. Concordant with that, lentiviral-mediated stable expression of miR-320c at physiological levels (~1.5-fold) promoted adipocytic and suppressed osteogenic differentiation of hMSC. Luciferase assay validated RUNX2 (Runt-related transcription factor 2) as a bona fide target for miR-320 family. Therefore, our data suggest miR-320 family as possible molecular switch promoting adipocytic differentiation of hMSC. Targeting miR-320 may have therapeutic potential in vivo through regulation of bone marrow adipogenesis.

A 54-Year-Old Man Referred With Nonresolving Pneumonia

A 54-year-old man was referred with nonresolving pneumonia. He had been treated for community-acquired pneumonia 6 weeks earlier. He reported grade 2 dyspnea, malaise, and a nonproductive cough. He had also experienced three episodes of minimal hemoptysis but denied weight loss, fever, or any other constitutional symptoms. He was a nonsmoker and was being treated for dyslipidemia.

Lysine 63-linked TANK-binding kinase 1 ubiquitination by mindbomb E3 ubiquitin protein ligase 2 is mediated by the mitochondrial antiviral signaling protein.

Beta interferon (IFN-β) is involved in a wide range of cellular functions, and its secretion must be tightly controlled to inhibit viral spreading while minimizing cellular damage. Intracellular viral replication triggers cellular signaling cascades leading to the activation of the transcription factors NF-κB and interferon regulatory factor 3 (IRF3) and IRF7 (IRF3/7), which synergistically bind to the IFN-β gene promoter to induce its expression. The mitochondrial antiviral signaling protein (MAVS) is a governing adaptor protein that mediates signaling communications between virus-sensing proteins and transcription factors. The activity of MAVS in the regulation of IFN-β secretion is affected by many cellular factors. However, the mechanism of MAVS-mediated IRF3/7 activation is not completely understood. Here, we identified a highly conserved DLAIS motif at amino acid positions 438 to 442 of MAVS that is indispensable for IRF3/7 activation. Specifically, the L439S and A440R mutations suppress IRF3/7 activation. Pulldown experiments using wild-type and mutant MAVS showed that mindbomb E3 ubiquitin protein ligase 2 (MIB2) binds to the DLAIS motif. Furthermore, the DLAIS motif was found to be critical for MIB2 binding, the ligation of K63-linked ubiquitin to TANK-binding kinase 1, and phosphorylation-mediated IRF3/7 activation. Our results suggest that MIB2 plays a putative role in MAVS-mediated interferon signaling. Mitochondrial antiviral signaling protein (MAVS) mediates signaling from virus-sensing proteins to transcription factors for the induction of beta interferon. However, the mechanism underlying activation of MAVS-mediated interferon regulatory factors 3 and 7 (IRF3/7) is not completely understood. We found a highly conserved DLAIS motif in MAVS that is indispensable for IRF3/7 activation through TANK-binding kinase 1 (TBK1) and identified it as the binding site for mindbomb E3 ubiquitin protein ligase 2 (MIB2). The mutations that targeted the DLAIS motif abolished MIB2 binding, attenuated the K63-linked ubiquitination of TBK1, and decreased the phosphorylation-mediated activation of IRF3/7.

Adam10 mediates the choice between principal cells and intercalated cells in the kidney.

A disintegrin and metalloproteinase domain 10 (Adam10), a member of the ADAM family of cell membrane-anchored proteins, has been linked to the regulation of the Notch, EGF, E-cadherin, and other signaling pathways. However, it is unclear what role Adam10 has in the kidney in vivo. In this study, we showed that Adam10 deficiency in ureteric bud (UB) derivatives leads to a decrease in urinary concentrating ability, polyuria, and hydronephrosis in mice. Furthermore, Adam10 deficiency led to a reduction in the percentage of aquaporin 2 (Aqp2)(+) principal cells (PCs) in the collecting ducts that was accompanied by a proportional increase in the percentage of intercalated cells (ICs). This increase was more prominent in type A ICs than in type B ICs. Foxi1, a transcription factor important for the differentiation of ICs, was upregulated in the Adam10 mutants. The observed reduction of Notch activity in Adam10 mutant collecting duct epithelium and the similar reduction of PC/IC ratios in the collecting ducts in mice deficient for mindbomb E3 ubiquitin protein ligase 1, a key regulator of the Notch and Wnt/receptor-like tyrosine kinase signaling pathways, suggest that Adam10 regulates cell fate determination through the activation of Notch signaling, probably through the regulation of Foxi1 expression. However, phenotypic differences between the Adam10 mutants, the Mib1 mutants, and the Foxi1 mutants suggest that the functions of Adam10 in determining the fate of collecting duct cells are more complex than those of a simple upstream factor in a linear pathway involving Notch and Foxi1.

Cilia born out of shock and stress

Primary cilia are cell surface sensory organelles, whose dysfunction underlies various human genetic diseases collectively termed ciliopathies. A new study in The EMBO Journal by Villumsen et al now reveals how stress–response pathways converge to stimulate ciliogenesis by modulating protein composition of centriolar satellites. Better understanding of these mechanisms should bring us closer to identifying the cellular defects that underlie ciliopathies caused by mutations in centriolar satellite proteins. Centrioles are barrel‐shaped structures with two distinct identities. In proliferating cells centrioles provide structural support for the centrosome, a key microtubule‐organizing centre, whereas in quiescent cells centrioles are converted into basal bodies and promote the assembly of primary cilia. In centrosomes, centrioles are embedded in pericentriolar material (PCM), a dynamic structure responsible for microtubule nucleation. PCM proteins exhibit cell cycle‐dependent localisation, achieved at least in part by the regulation of their transport. Centriolar satellites, dense fibrous granules frequently clustered around the interphase centrosome, have been implicated in microtubule‐dependent protein transport to centrosomes (Kubo et al, 1999). In particular, PCM‐1, the core constituent of centriolar satellites, is required for centrosomal accumulation of several PCM components (Dammermann and Merdes, 2002). Although the proteomic composition of satellites is still elusive, the growing list of satellite proteins includes CEP131/AZI1 (Staples et al, 2012), CEP290 (Stowe et al, 2012), Bardet‐Biedl syndrome protein 4 (BBS4) and Oral facial digital syndrome protein (OFD1; Lopes et al, 2011). Mutations in OFD1, CEP290 and BBS4 cause ciliopathies (Kim et al, 2008), underscoring a functional link between satellites and ciliogenesis. So far, two roles have been proposed for satellites in cilia formation: …