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Mutations in CDCA7 and HELLS that respectively encode a CXXC-type zinc finger protein and an SNF2 family chromatin remodeler cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome types 3 and 4. Here, we demonstrate that the classical nonhomologous end joining (C-NHEJ) proteins Ku80 and Ku70, as well as HELLS, coimmunoprecipitated with CDCA7. The coimmunoprecipitation of the repair proteins was sensitive to nuclease treatment and an ICF3 mutation in CDCA7 that impairs its chromatin binding. The functional importance of these interactions was strongly suggested by the compromised C-NHEJ activity and significant delay in Ku80 accumulation at DNA damage sites in CDCA7- and HELLS-deficient HEK293 cells. Consistent with the repair defect, these cells displayed increased apoptosis, abnormal chromosome segregation, aneuploidy, centrosome amplification, and significant accumulation of γH2AX signals. Although less prominent, cells with mutations in the other ICF genes DNMT3B and ZBTB24 (responsible for ICF types 1 and 2, respectively) showed similar defects. Importantly, lymphoblastoid cells from ICF patients shared the same changes detected in the mutant HEK293 cells to varying degrees. Although the C-NHEJ defect alone did not cause CG hypomethylation, CDCA7 and HELLS are involved in maintaining CG methylation at centromeric and pericentromeric repeats. The defect in C-NHEJ may account for some common features of ICF cells, including centromeric instability, abnormal chromosome segregation, and apoptosis.

Background The role and mechanism of centromeric protein N (CENP‐N), which has been associated with the development of various cancer types, are yet unclear in stomach adenocarcinoma (STAD). Methods Data from the Cancer Genome Atlas and Genotype‐Tissue Expression were used to determine whether CENP‐N expression was altered in STAD tumors compared to normal tissues. Xiantao was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis on CENP‐N. The relationship between CENP‐N expression and immune cell infiltration was assessed using TCGA database. The expression of CENP‐N in STAD and surrounding tissues was confirmed using immunohistochemical staining and the correlation between CENP‐N expression and clinicopathological characteristics was examined. The effects of CENP‐N knockdown by siRNA on proliferation were measured by CCK‐8 and EdU assays in AGS cells. Following siRNA transfection, flow cytometry was performed to evaluate cell cycle and apoptotic alterations in AGS cells. The effect of CENP‐N knockdown on the expression level of related proteins was detected by Westren blot. Results CENP‐N was highly expressed in STAD tissues, which was confirmed by our immunohistochemistry results. The degree of invasion, TNM stage, and lymph node metastases were all strongly associated with CENP‐N expression. CENP‐N was essential for the cell cycle, DNA replication, chromosomal segregation, and nuclear division; there was a positive correlation between CENP‐N expression and infiltrating Th2 and NK CD56dim cells and a negative correlation between CENP‐N expression and mast, pDC, NK, and B cell infiltration. When CENP‐N expression in AGS cells was knocked down, cell proliferation dramatically reduced (p < .05) and the percentage of cells in the S and G2‐M phases decreased significantly (p < .05). Silencing CENP‐N significantly promoted the apoptosis of AGS cells (p < .05). Mechanistic investigations showed that silencing CENP‐N expression may inhibit STAD proliferation through the Cyclin E1 and promote STAD apoptosis through the Bcl‐2/Bax. Conclusion According to our data, CENP‐N acts as an oncogene in STAD and may be a viable therapeutic target.

Centromere-associated protein-E (CENP-E) is a kinesin motor localizing at kinetochores. Although its mitotic functions have been well studied, it has been challenging to investigate direct consequences of CENP-E removal using conventional methods because CENP-E depletion resulted in mitotic arrest. In this study, we harnessed an auxin-inducible degron system to achieve acute degradation of CENP-E. We revealed a kinetochore-independent role for CENP-E that removes pericentriolar material 1 (PCM1) from centrosomes in late S/early G2 phase. After acute loss of CENP-E, centrosomal Polo-like kinase 1 (Plk1) localization is abrogated through accumulation of PCM1, resulting in aberrant phosphorylation and destabilization of centrosomes, which triggers shortened astral microtubules and oblique cell divisions. Furthermore, we also observed centrosome and cell division defects in cells from a microcephaly patient with mutations in CENPE. Orientation of cell division is deregulated in some microcephalic patients, and our unanticipated findings provide additional insights into how microcephaly can result from centrosomal defects.Owa and Dynlacht employ an auxin-inducible degron system to achieve acute degradation of CENP-E. As a result, the authors reveal a kinetochore-independent role for CENP-E that removes PCM1 from centrosomes in interphase, with implications in microcephaly.

BackgroundAs an important member of centromere family, centromere associated protein N (CENPN) was abnormally expressed in varied malignant tumors.ObjectiveThis paper aimed to analyze the expression and related mechanism of CENPN in lung adenocarcinoma (LUAD).MethodsThe expression of CENPN in LUAD was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) database. The mRNA expression, protein expression, cell viability, cell invasion, cell apoptosis, cell stem like characteristics were detected by RT-PCR, western blot, CCK8 assay, transwell assay, flow cytometry and spheroidization assay, respectively. Finally, the pathological changes of xenograft were estimated by H&E staining, and the expression of proteins was detected by immunohistochemistry.ResultsGEPIA analysis showed that the CENPN expression in LUAD was significantly higher than that in normal lung tissue, which was negatively correlated with the prognosis. These results were consistent with our clinical data. Besides, CENPN was highly expressed in LUAD cell lines. In addition, the upregulation of CENPN amplified the cell viability, stemness and invasive ability in PC9 cells. However, the knockdown of CENPN inhibited the cell activity, stemness, invasive ability with increased cell apoptosis in A549. Furthermore, CENPN could positively regulate the phosphorylation of PI3K and AKT. The PI3K inhibitor, 740Y-P, could reverse the effect of CENPN silencing on the expression of Ki-67, cleaved caspase 3, OCT4, and snail 1. Finally, the downregulation of CENPN restrained the growth of xenograft and inactivated the PI3K/AKT pathway.ConclusionCENPN was abnormally overexpressed in LUAD, and promoted tumor progression of LUAD by affecting PI3K/AKT pathway.

Anti-SS-A/Ro antibody (SS-A) and anti-SS-B/La antibody (SS-B) are important serologic markers in the diagnostic criteria for Primary Sjögren’s syndrome (SS). Although anti-centromere antibody (ACA)-positive SS is frequently experienced, ACA is not included in these criteria. The purpose of this study was to identify the clinical features of ACA-positive SS and discuss the usefulness of ACA in diagnosing SS. Forty-five patients with SS were divided into the following three groups: SS-A only-positive group ( n  = 17), SS-A and SS-B both-positive group ( n  = 18), and ACA only-positive group ( n  = 10). As a control, 54 patients without SS who were negative for antinuclear antibodies were also evaluated. The following items were compared among groups: Saxon’s test, unstimulated whole salivary flow (UWSF), salivary gland scintigraphy (SGS), histopathologic examination of the minor salivary glands, Schirmer’s test, and fluorescein staining of the cornea. In the ACA only-positive group, Saxon’s test was 0.21 ± 0.26 g/2 min (mean ± SD) and UWSF was 0.16 ± 0.25 ml/10 min (mean ± SD), showing a significant decrease in salivary secretion ( p  < 0.05; vs. non-SS). On SGS, accumulation and disappearance of 99m TcO 4 − were significantly decreased ( p  < 0.05; vs. non-SS). Histopathologic examination showed moderate or severe lymphocytic infiltration and tissue destruction in all cases, similar to that in the SS-A- and/or SS-B-positive groups. Schirmer’s test and fluorescein staining were positive in 60% and 80%, respectively. Impaired lacrimal secretion and keratoconjunctivitis sicca were similar to those in SS-A- and/or SS-B-positive groups. These results suggest that ACA is an autoantibody reflecting impairment in the salivary and lacrimal glands and may be a useful serologic marker for SS.

The survivin protein contains structural features of the inhibitor of apoptosis protein family. Previous studies have suggested that survivin is essential for cell survival because it counteracts an otherwise constitutive propensity to apoptosis during mitosis. In addition, survivin appears to be a component of the chromosomal passenger protein complex that participates in multiple facets of cell division. Here we report that euploid human cells do not die in the absence of survivin. Instead, depletion of survivin caused defects in cell division, followed by an arrest of DNA synthesis due to activation of a checkpoint involving the tumor suppressor protein p53. During anaphase mitosis in survivin-deficient cells, sister chromatids disjoined normally, but one or more of the sister chromatids frequently lagged behind the main mass of segregating chromosomes, probably because of merotelic kinetochore attachments. Survivin-deficient cells initiated but failed to complete cytokinesis, apparently because the spindle midzone and midbody microtublues were absent during late mitosis. The abnormalities of both chromosome segregation and cytokinesis could be attributed to a defect in the chromosomal passenger protein complex, with a consequent mislocalization of the kinesin-like motor protein MKLP-1 playing a more immediate role in the microtubule abnormalities. Depletion of another chromosomal passenger protein, aurora-B, recapitulated the survivin RNA interference phenotypes. We conclude that survivin can be essential for the proliferation of normal human cells by virtue of its contributions to accurate sister chromatid segregation and assembly/stabilization of microtubules in late mitosis. However, the protein is not inevitably required for the survival of normal cells.

Background To clarify the clinicopathological characteristics of primary Sjögren's syndrome (pSS) with anti-centromere antibody (ACA). Methods Characteristics of 14 patients of pSS with ACA were evaluated. All patients were anti-SS-A/Ro and SS-B/La antibodies negative (ACA+ group) without sclerodactyly. The prevalence of Raynaud's phenomenon (RP), titer of IgG and focus score (FS) in the minor salivary glands (MSGs) were determined. Quantification analysis of Azan Mallory staining was performed to detect collagenous fiber. Forty eight patients in whom ACA was absent were chosen as the conventional (ACA-) pSS group. Results Prevalence of ACA+ SS patients was 14 out of 129 (10.85%) pSS patients. RP was observed in 61.5% of the patients with ACA. The level of IgG in the ACA+ group was significantly lower than that of the ACA- group (p = 0.018). Statistical difference was also found in the FS of MSGs from the ACA+ group (1.4 ± 1.0) as compared with the ACA- group (2.3 ± 1.6) (p = 0.035). In contrast, the amount of fibrous tissue was much higher in the ACA+ group (65052.2 ± 14520.6 μm 2 versus 26251.3 ± 14249.8 μm 2 ) (p = 1.3 × 10 -12 ). Conclusions Low cellular infiltration but with an increase in fibrous tissues may explain the clinical feature of a high prevalence of RP and normal IgG concentration in ACA+ pSS.

A new method that simplifies the evaluation of the traditional HER2 fluorescence in situ hybridization (FISH) evaluation in breast cancer was proposed. HER2 status was evaluated in digital images (DIs) captured from 423 invasive breast cancer stained sections. All centromeric/CEP17 and HER2 gene signals obtained from separated stacked DIs were manually counted on the screen. The global ratios were compared with the traditional FISH evaluation and the immunohistochemical status. The 2 FISH scores were convergent in 96.93% of cases, showing an “almost perfect” agreement with a weighted k of 0.956 (95% confidence interval, 0.928-0.985). The new method evaluates at least 3 times more nuclei than traditional methods and also has an almost perfect agreement with the immunohistochemical scores. The proposed enhanced method substantially improves HER2 FISH assessment in breast cancer biopsy specimens because the evaluation of HER2/CEP17 copy numbers is more representative, easier, and faster than the conventional method.

Autoimmune diseases are very serious and also invalidating illnesses. The benchmark procedure for their diagnosis is the indirect immunofluorescence (IIF) assay performed on the HEp-2 substrate. Medical doctors first determine the fluorescence intensity exhibited by HEp-2 wells and then report the staining pattern. Despite its pivotal role, IIF is affected by inter- and intra-laboratory variabilities demanding for the development of computer-aided-diagnosis tools supporting medical doctor decisions. With reference to staining pattern recognition, state-of-the-art approaches recognize five main patterns characterized by well-defined cell edges. These approaches are based on cell segmentation, a task that recent work suggests to be harder than the classification itself. In this paper, we extend the panel of detectable HEp-2 staining patterns, introducing the recognition of centromere and cytoplasmic patterns, which have a high specific match with certain autoimmune diseases, from other stainings. Since image segmentation algorithms fail on these samples, we developed a classification system integrating local descriptors and the bag of visual word approach, which represents image contents without the burden of segmentation. We tested our approach on a large dataset of HEp-2 images with high variability in both fluorescence intensity and staining patterns correctly recognizing the 97.12 % of samples. The system has also been validated in a daily routine fashion on 108 consecutive IIF analyses of hospital outpatients and inpatients, achieving an accuracy rate of 97.22 %.

It has been hypothesized that human clinical neocentromeres and evolutionary novel centromeres (ENC) represent two faces of the same phenomenon. However, there are only two reports of loci harboring both a novel centromere and a clinical neocentromere. We suggest that only the tip of the iceberg has been scratched because most neocentromerization events have a very low chance of being observed. In support of this view, we report here on a neocentromere at 9q33.1 that emerged in a ring chromosome of about 12 Mb. The ring was produced by a balanced rearrangement that was fortuitously discovered because of its malsegregation in the propositus. Chromatin-immunoprecipitation-on-chip experiments using anti-centromere protein (CENP)-A and anti-CENP-C antibodies strongly indicated that a novel centromeric domain was present in the ring, in a chromosomal domain where an ENC emerged in the ancestor to Old World monkeys.