Expanding the spectrum of CEP55‐associated disease to viable phenotypes

Homozygosity for nonsense variants in CEP55 has been associated with a lethal condition characterized by multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia, and hydranencephaly (MARCH syndrome) also known as Meckel‐like syndrome. Missense variants in CEP55 have not previously been reported in association with disease. Here we describe seven living individuals from five families with biallelic CEP55 variants. Four unrelated individuals with microcephaly, speech delays, and bilateral toe syndactyly all have a common CEP55 variant c.70G>A p.(Glu24Lys) in trans with nonsense variants. Three siblings are homozygous for a consensus splice site variant near the end of the gene. These affected girls all have severely delayed development, microcephaly, and varying degrees of lissencephaly/pachygyria. Here we compare our seven patients with three previously reported families with a prenatal lethal phenotype (MARCH syndrome/Meckel‐like syndrome) due to homozygous CEP55 nonsense variants. Our series suggests that individuals with compound heterozygosity for nonsense and missense variants in CEP55 have a different viable phenotype. We show that homozygosity for a splice variant near the end of the CEP55 gene is also compatible with life.     

Impaired p53/CEP‐1 is associated with lifespan extension through an age‐related imbalance in the energy metabolism of C. elegans

In the nematode Caenorhabditis elegans, the mammalian tumor suppressor p53 ortholog CEP‐1 mediates the stress response, activates germ line apoptosis and regulates meiotic chromosome segregation. A reduction in its expression, which frequently occurs in mammalian cancer cells, extends lifespan and induces an adaptive response in C. elegans. However, these effects do not involve an increase in oxidative stress resistance. Here, we showed that intermittent exposure to hyperoxia, which induces oxidative stress resistance and lowers the production of ROS derived from mitochondrial respiration in C. elegans, slightly improved the lifespan extension of cep‐1 mutant. Interestingly, ATP levels were increased without an increase in oxygen consumption in cep‐1 mutant during aging. In the wild‐type, lactate levels and consequentially the lactate/pyruvate ratio decreased during aging in adults. Furthermore, the expression levels of mitochondrial respiration‐related sco‐1, which is a target of p53/CEP‐1, as well as those of gluconeogenesis regulation and mammalian sirtuin ortholog genes, were also increased in the aged and adaptive conditioned wild‐type animals. In contrast, the lactate/pyruvate ratio increased in cells of the cep‐1 mutant and was amplified by intermittent hyperoxia. These results suggest that impaired p53/CEP‐1 leads to an imbalance in the age‐related energy metabolic alteration between mitochondrial oxidative phosphorylation and aerobic glycolysis and plays an important role in the extension of both intact and adaptive lifespans.     

The N-terminal region of centrosomal protein 290 (CEP290) restores vision in a zebrafish model of human blindness

The gene coding for centrosomal protein 290 (CEP290), a large multidomain protein, is the most frequently mutated gene underlying the non-syndromic blinding disorder Leber's congenital amaurosis (LCA). CEP290 has also been implicated in several cilia-related syndromic disorders including Meckel-Gruber syndrome, Joubert syndrome, Senor-Loken syndrome and Bardet-Biedl syndrome (BBS). In this study, we characterize the developmental and functional roles of cep290 in zebrafish. An antisense oligonucleotide [Morpholino (MO)], designed to generate an altered cep290 splice product that models the most common LCA mutation, was used for gene knockdown. We show that cep290 MO-injected embryos have reduced Kupffer's vesicle size and delays in melanosome transport, two phenotypes that are observed upon knockdown of bbs genes in zebrafish. Consistent with a role in cilia function, the cep290 MO-injected embryos exhibited a curved body axis. Patients with LCA caused by mutations in CEP290 have reduced visual perception, although they present with a fully laminated retina. Similarly, the histological examination of retinas from cep290 MO-injected zebrafish revealed no gross lamination defects, yet the embryos had a statistically significant reduction in visual function. Finally, we demonstrate that the vision impairment caused by the disruption of cep290 can be rescued by expressing only the N-terminal region of the human CEP290 protein. These data reveal that a specific region of the CEP290 protein is sufficient to restore visual function and this region may be a viable gene therapy target for LCA patients with mutations in CEP290.     

A nonsense mutation in CEP55 defines a new locus for a Meckel‐like syndrome,an autosomal recessive lethal fetal ciliopathy

Mutations in genes involved in the cilium‐centrosome complex are called ciliopathies. Meckel‐Gruber syndrome (MKS) is a ciliopathic lethal autosomal recessive syndrome characterized by genetically and clinically heterogeneous manifestations, including renal cystic dysplasia, occipital encephalocele and polydactyly. Several genes have previously been associated with MKS and MKS‐like phenotypes, but there are still genes remaining to be discovered. We have used whole‐exome sequencing (WES) to uncover the genetics of a suspected autosomal recessive Meckel syndrome phenotype in a family with 2 affected fetuses. RNA studies and histopathological analysis was performed for further delineation. WES lead to identification of a homozygous nonsense mutation c.256C>T (p.Arg86*) in CEP55 (centrosomal protein of 55 kDa) in the affected fetus. The variant has previously been identified in carriers in low frequencies, and segregated in the family. CEP55 is an important centrosomal protein required for the mid‐body formation at cytokinesis. Our results expand the list of centrosomal proteins implicated in human ciliopathies and provide evidence for an essential role of CEP55 during embryogenesis and development of disease.     

Dullard deficiency causes hemorrhage in the adult ovarian follicles

In mammals, the ovarian follicles are regulated at least in part by bone morphogenetic protein (BMP) family members. Dullard (also known as Ctdnep1) gene encodes a phosphatase that suppresses BMP signaling by inactivating or degrading BMP receptors. Here we report that the Col1a1‐Cre‐induced Dullard mutant mice displayed hemorrhagic ovarian cysts, with red blood cells accumulated in the follicles, resulting in infertility. Cells expressing Cre driven by Col1a1 2.3‐kb promoter and their descendants were found in granulosa cells in the ovary and in Sertoli cells in the testis. DullardmRNA was localized to granulosa cells in the ovary. Genes involved in steroid hormone genesis including Cyp11a1, Hsd3b1 and Star were reduced, whereas expression of Smad6 and Smad7, BMP‐inducible inhibitory Smads, was up‐regulated in the Dullard mutant ovaries. Tamoxifen‐inducible Dullard deletion in the whole body using Rosa26‐CreER mice also resulted in hemorrhagic ovarian cysts in 2 weeks, which was rescued by administration of LDN‐193189, a chemical inhibitor of BMP receptor kinase, suggesting that the hemorrhage in the Dullard‐deficient ovarian follicles might be caused by increased BMP signaling. Thus, we conclude that Dullard is essential for ovarian homeostasis at least in part via suppression of BMP signaling.     

Wingless signaling regulates winner/loser status in Minute cell competition

Cells heterozygously mutant for a ribosomal protein gene, called Minute/+ mutants, are eliminated from epithelium by cell competition when surrounded by wild‐type cells. Whereas several factors that regulate Minute cell competition have been identified, the mechanisms how winner/loser status is determined and thereby triggers cell competition are still elusive. To address this, we established two assay systems for Minute cell competition, namely (i) the CORE (competitive elimination of RpS3‐RNAi‐expressing cells) system in which RpS3‐RNAi‐expressing wing pouch cells are eliminated from wild‐type wing disc and (ii) the SURE (supercompetition of RpS3‐expressing clones in RpS3/+ tissue) system in which RpS3‐over‐expressing clones generated in RpS3/+ wing disc outcompete surrounding RpS3/+ cells. An ectopic over‐expression screen using the CORE system identified Wg signaling as a critical regulator of Minute cell competition. Activation of Wg signaling in loser cells suppressed their elimination, whereas down‐regulation of Wg signaling in loser cells enhanced their elimination. Furthermore, using the SURE system, we found that down‐regulation of Wg signaling in winner cells suppressed elimination of neighboring losers. Our observations suggest that cellular Wg signaling activity is crucial for determining winner/loser status and thereby triggering Minute cell competition.     

Trabecular architecture of the great ape and human femoral head

Studies of femoral trabecular structure have shown that the orientation and volume of bone are associated with variation in loading and could be informative about individual joint positioning during locomotion. In this study, we analyse for the first time trabecular bone patterns throughout the femoral head using a whole‐epiphysis approach to investigate how potential trabecular variation in humans and great apes relates to differences in locomotor modes. Trabecular architecture was analysed using microCT scans of Pan troglodytes (n = 20), Gorilla gorilla (n = 14), Pongo sp. (n = 5) and Homo sapiens (n = 12) in medtool 4.1. Our results revealed differences in bone volume fraction (BV/TV) distribution patterns, as well as overall trabecular parameters of the femoral head between great apes and humans. Pan and Gorilla showed two regions of high BV/TV in the femoral head, consistent with hip posture and loading during two discrete locomotor modes: knuckle‐walking and climbing. Most Pongo specimens also displayed two regions of high BV/TV, but these regions were less discrete and there was more variability across the sample. In contrast, Homo showed only one main region of high BV/TV in the femoral head and had the lowest BV/TV, as well as the most anisotropic trabeculae. The Homo trabecular structure is consistent with stereotypical loading with a more extended hip compared with great apes, which is characteristic of modern human bipedalism. Our results suggest that holistic evaluations of femoral head trabecular architecture can reveal previously undetected patterns linked to locomotor behaviour in extant apes and can provide further insight into hip joint loading in fossil hominins and other primates.     

Human papillomavirus E6 protein enriches the CD55(+) population in cervical cancer cells,promoting radioresistance and cancer aggressiveness

Accumulating evidence indicates that the human papillomavirus (HPV) E6 protein plays a crucial role in the development of cervical cancer. Subpopulations of cells that reside within tumours are responsible for tumour resistance to cancer therapy and recurrence. However, the identity of such cells residing in cervical cancer and their relationship with the HPV‐E6 protein have not been identified. Here, we isolated sphere‐forming cells, which showed self‐renewal ability, from primary cervical tumours. Gene expression profiling revealed that cluster of differentiation (CD) 55 was upregulated in primary cervical cancer sphere cells. Flow‐cytometric analysis detected abundant CD55(+) populations among a panel of HPV‐positive cervical cancer cell lines, whereas few CD55(+) cells were found in HPV‐negative cervical cancer and normal cervical epithelial cell lines. The CD55(+) subpopulation isolated from the C33A cell line showed significant sphere‐forming ability and enhanced tumourigenicity, cell migration, and radioresistance. In contrast, the suppression of CD55 in HPV‐positive CaSki cells inhibited tumourigenicity both in vitro and in vivo, and sensitized cells to radiation treatment. In addition, ectopic expression of the HPV‐E6 protein in HPV‐negative cervical cancer cells dramatically enriched the CD55(+) subpopulation. CRISPR/Cas9 knockout of CD55 in an HPV‐E6‐overexpressing stable clone abolished the tumourigenic effects of the HPV‐E6 protein. Taken together, our data suggest that HPV‐E6 protein expression enriches the CD55(+) population, which contributes to tumourigenicity and radioresistance in cervical cancer cells. Targeting CD55 via CRISPR/Cas9 may represent a novel avenue for developing new strategies and effective therapies for the treatment of cervical cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Dog saliva – an important source of dog allergens

Background

Allergy to dog (Canis familiaris) is a worldwide common cause of asthma and allergic rhinitis. However, dander extract in routine diagnostics is not an optimal predictor of IgE‐mediated dog allergy. Our objective was to evaluate saliva as an allergen source for improved diagnostics of allergy to dog.

Methods

IgE‐binding proteins in dog saliva and dander extract were analysed by immunoblot and mass spectrometry (LC‐MS/MS) using pooled or individual sera from dog‐allergic patients (n = 13). Sera from 59 patients IgE positive to dander and 55 patients IgE negative to dander but with symptoms to dog were analysed for IgE against saliva and dander by ELISA. Basophil stimulation with dog saliva and dander extract was measured by flow cytometry among three dog‐allergic patients. Additionally, IgE‐binding protein profiles of saliva from different breeds were investigated by immunoblot.

Results

Greater number and diversity of IgE‐binding proteins was found in saliva compared to dander extract and varied among dog breeds. In saliva, Can f 1, 2, 3 and 6 were identified but also four new saliva allergen candidates. The majority of the 59 dog dander–positive sera (n = 44) were IgE positive to dog saliva. Among patients IgE negative to dander, but with symptoms to dog, 20% were IgE positive to saliva. The biological activity of saliva was confirmed by basophil degranulation.

Conclusions

Dog saliva is an allergen source for improved diagnostics of dog allergy. The IgE‐binding protein profile of saliva from different dogs varies.

     

H‐ficolin binds Aspergillus fumigatus leading to activation of the lectin complement pathway and modulation of lung epithelial immune responses

Aspergillus fumigatus is an opportunistic fungal pathogen that typically infects the lungs of immunocompromised patients leading to a high mortality. H‐Ficolin, an innate immune opsonin, is produced by type II alveolar epithelial cells and could participate in lung defences against infections. Here, we used the human type II alveolar epithelial cell line, A549, to determine the involvement of H‐ficolin in fungal defence. Additionally, we investigated the presence of H‐ficolin in bronchoalveolar lavage fluid from transplant patients during pneumonia. H‐Ficolin exhibited demonstrable binding to A. fumigatus conidia via l ‐fucose, d ‐mannose and N‐acetylglucosamine residues in a calcium‐ and pH‐dependent manner. Moreover, recognition led to lectin complement pathway activation and enhanced fungal association with A549 cells. Following recognition, H‐ficolin opsonization manifested an increase in interleukin‐8 production from A549 cells, which involved activation of the intracellular signalling pathways mitogen‐activated protein kinase MAPK kinase 1/2, p38 MAPK and c‐Jun N‐terminal kinase. Finally, H‐ficolin concentrations were significantly higher in bronchoalveolar lavage fluid of patients with lung infections compared with control subjects (n = 16; P = 0·00726). Receiver operating characteristics curve analysis further highlighted the potential of H‐ficolin as a diagnostic marker for lung infection (area under the curve = 0·77; P < 0·0001). Hence, H‐ficolin participates in A. fumigatus defence through the activation of the lectin complement pathway, enhanced fungus–host interactions and modulated immune responses.     

Systemic inflammatory response and local cytokine expression in porcine models of endocarditis

The knowledge of systemic inflammation and local cytokine expression in porcine endocarditis models is limited, though it could provide valuable information about the pathogenesis and comparability to human endocarditis. Analyses of bacteriology and hematology were performed on blood samples from pigs with non‐bacterial thrombotic endocarditis (NBTE, n = 11), Staphylococcus aureus infective endocarditis (IE, n = 2), animals with S. aureus sepsis without endocarditis (n = 2) and controls (n = 2). Furthermore, immunohistochemistry was used to examine the local expression of IL‐1β and IL‐8. Bacterial blood cultures were continuously positive in IE pigs from inoculation to euthanasia, and negative in all other pigs at all times. The total white blood cell counts and total neutrophil counts were massively elevated in pigs with IE. Local IL‐1β and IL‐8 expression in IE pigs were moderate to high, and high, respectively. In addition, slight local expression of IL‐1β and IL‐8 was present in some NBTE pigs. In the IE model, both the systemic inflammatory response and the high local expression of IL‐8 were comparable to the human disease. Furthermore, the results indicate IL‐1β and IL‐8 as important contributors in the endocarditis pathogenesis.     

Comparative experimental subcutaneous glanders and melioidosis in the common marmoset (Callithrix jacchus)

Glanders and melioidosis are caused by two distinct Burkholderia species and have generally been considered to have similar disease progression. While both of these pathogens are HHS/CDC Tier 1 agents, natural infection with both these pathogens is primarily through skin inoculation. The common marmoset (Callithrix jacchus) was used to compare disease following experimental subcutaneous challenge. Acute, lethal disease was observed in marmosets following challenge with between 26 and 1.2 × 10⁸ cfu Burkholderia pseudomallei within 22–85 h. The reproducibility and progression of the disease were assessed following a challenge of 1 × 10² cfu of B. pseudomallei. Melioidosis was characterised by high levels of bacteraemia, focal microgranuloma progressing to non‐necrotic multifocal solid lesions in the livers and spleens and multi‐organ failure. Lethal disease was observed in 93% of animals challenged with Burkholderia mallei, occurring between 5 and 10.6 days. Following challenge with 1 × 10² cfu of B. mallei, glanders was characterised with lymphatic spread of the bacteria and non‐necrotic, multifocal solid lesions progressing to a multifocal lesion with severe necrosis and pneumonia. The experimental results confirmed that the disease pathology and presentation is strikingly different between the two pathogens. The marmoset provides a model of the human syndrome for both diseases facilitating the development of medical countermeasures.     

The spectrum of rare central nervous system (CNS) tumors with EWSR1‐non‐ETS fusions: experience from three pediatric institutions with review of the literature

The group of CNS mesenchymal (non‐meningothelial) and primary glial/neuronal tumors in association with EWSR1‐non‐ETS rearrangements comprises a growing spectrum of entities, mostly reported in isolation with incomplete molecular profiling. Archival files from three pediatric institutions were queried for unusual cases of pediatric (≤21 years) CNS EWSR1‐rearranged tumors confirmed by at least one molecular technique. Extra‐axial tumors and cases with a diagnosis of Ewing sarcoma (EWSR1‐ETS family fusions) were excluded. Additional studies, including anchored multiplex‐PCR with next‐generation sequencing and DNA methylation profiling, were performed as needed to determine fusion partner status and brain tumor methylation class, respectively. Five cases (median 17 years) were identified (M:F of 3:2). Location was parenchymal (n = 3) and undetermined (n = 2) with topographic distributions including posterior fossa (n = 1), frontal (n = 1), temporal (n = 1), parietal (n = 1) and occipital (n = 1) lobes. Final designation with fusion findings included desmoplastic small round cell tumor (EWSR1‐WT1; n = 1) and tumors of uncertain histogenesis (EWSR1‐CREM, n = 1; EWSR1‐CREB1, n = 1; EWSR1‐PLAGL1, n = 1; and EWSR1‐PATZ1, n = 1). Tumors showed a wide spectrum of morphology and biologic behavior. For EWSR1‐CREM, EWSR1‐PLAGL1 and EWSR1‐PATZ1 tumors, no significant methylation scores were reached in the known brain tumor classes. Available outcome (4/5) was reported as favorable (n = 2) and unfavorable (n = 2) with a median follow‐up of 30 months. In conclusion, we describe five primary EWSR1‐non‐ETS fused CNS tumors exhibiting morphologic and biologic heterogeneity and we highlight the clinical importance of determining specific fusion partners to improve diagnostic accuracy, treatment and monitoring. Larger prospective clinicopathological and molecular studies are needed to determine the prognostic implications of histotypes, anatomical location, fusion partners, breakpoints and methylation profiles in patients with these rare tumors.     

Tschimganine and its derivatives extend the chronological life span of yeast via activation of the Sty1 pathway

Most antiaging factors or life span extenders are associated with calorie restriction (CR). Very few of these factors function independently of, or additively with, CR. In this study, we focused on tschimganine, a compound that was reported to extend chronological life span (CLS). Although tschimganine led to the extension of CLS, it also inhibited yeast cell growth. We acquired a Schizosaccharomyces pombe mutant with a tolerance for tschimganine due to the gene crm1. The resulting Crm1 protein appears to export the stress‐activated protein kinase Sty1 from the nucleus to the cytosol even under stressful conditions. Furthermore, we synthesized two derivative compounds of tschimganine, α‐hibitakanine and β‐hibitakanine; these derivatives did not inhibit cell growth, as seen with tschimganine. α‐hibitakanine extended the CLS, not only in S. pombe but also in Saccharomyces cerevisiae, indicating the possibility that life span regulation by tschimganine derivative may be conserved across various yeast species. We found that the longevity induced by tschimganine was dependent on the Sty1 pathway. Based on our results, we propose that tschimganine and its derivatives extend CLS by activating the Sty1 pathway in fission yeast, and CR extends CLS via two distinct pathways, one Sty1‐dependent and the other Sty1‐independent. These findings provide the potential for creating an additive life span extension effect when combined with CR, as well as a better understanding of the mechanism of CLS.     

Identification of Toxoplasma gondii protein fractions induce immune response against melanoma in mice

Dendritic cells (DCs) play a crucial role in the initiation of adaptive immune responses against tumor cells. We recently found that protein components of Toxoplasma gondii (T. gondii) could mature DCs efficiently. Therefore, in this study, we aimed to find the most effective protein components of T. gondii which are able to mature DCs and consequently instruct immune responses in tumor‐bearing mice. Soluble tachyzoite antigens (STAgs) were fractionated by ammonium sulfate precipitation and subsequently by anion‐exchange HPLC. Immature DCs (iDCs) were treated by these protein fractions and were monitored for IL‐12p70 and IL‐10 production. Moreover, the capacity of mature DCs (mDCs) to induce lymphocyte proliferation was investigated. Ultimately, we analyzed the ability of mDCs in instructing immune responses in tumor‐bearing mice. We found that ammonium sulfate fraction one (A1) matured‐DCs produced higher IL‐12 level and IL‐12/IL‐10 ratio; therefore, this fraction was selected for further fractionation by anion‐exchange HPLC. The results showed that anion‐exchange HPLC fraction 14 (C14) matured‐DCs secrete higher levels of IL‐12p70 and IL‐12p70/IL‐10 ratio. Survival of the mice matured by A1 fraction increased significantly compared to other groups. Moreover, SDS‐PAGE electrophoresis showed that different obtained fractions have distinct proteins based on their size. These results demonstrate that two protein fractions of T. gondii are able to mature DCs more efficient.     

SPINK5 is associated with early‐onset and CHI3L1 with late‐onset atopic dermatitis

We have recently showed that filaggrin (FLG) mutations are associated only with early‐onset of AD, but not with late‐onset of AD. Consequently, other susceptibility genes should receive attention, especially in patients with late‐onset of AD. Our aim was to assess the associations between development of AD and the polymorphisms rs2303067 in SPINK5 and rs490928 in CHI3L1. A study population of 241 AD patients and 164 healthy controls was genotyped for two polymorphisms (rs2303067 in SPINK5 and rs490928 in CHI3L1). Rs2303067 in SPINK5 was significantly associated with early‐onset AD (≤8 years: p = .003; OR = 2.57) and was characterized by the need for hospitalization (p = .006; OR = 2.76), prolonged duration (≥10 years; p = .008; OR = 2.32) and more body parts affected (p = .015; OR = 2.01). In contrast, rs490928 in CHI3L1 was associated with late‐onset AD (>8 years: p = .048; OR = 1.65) and was characterized by no need for hospitalization (p = .049; OR = 1.59), shorter duration (<10 years; p = .017; OR = 1.94) and fewer body parts affected (p = .049; OR = 1.75). Our results confirmed that different AD phenotypes, specifically early‐ and late‐onset AD, have different genetic backgrounds. Early‐onset AD was associated with rs2303067 in SPINK5, which is involved in skin barrier functioning, and late‐onset was associated with rs4950928 in CHI3L1, which is involved in the immune response. Future studies should examine the early‐ versus late‐onset subgrouping more closely.     

An early function of the adenoviral E1B 55 kDa protein is required for the nuclear relocalization of the cellular p53 protein in adenovirus-infected normal human cells

It is well established that the human subgroup C adenovirus type 5 (Ad5) E1B 55 kDa protein can regulate the activity and concentration of the cellular tumor suppressor, p53. However, the contribution(s) of these functions of the E1B protein to viral reproduction remains unclear. To investigate this issue, we examined properties of p53 in normal human cells infected by E1B mutant viruses that display defective entry into the late phase or viral late mRNA export. The steady-state concentrations of p53 were significantly higher in cells infected by the E1B 55 kDa null mutant Hr6 or three mutants carrying small insertions in the E1B 55 kDa protein coding sequence than in Ad5-infected cells. Nevertheless, none of the mutants induced apoptosis in infected cells. Rather, the localization of p53 to E1B containing nuclear sites observed during infection by Ad5 was prevented by mutations that impair interaction of the E1B protein with p53 and/or with the E4 Orf6 protein. These results indicate that the E1B protein fulfills an early function that correlates efficient entry into the late phase with the localization of E1B and p53 in the nucleus of Ad5-infected normal human cells.