Labile assembly of a tardigrade protein induces biostasis.

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins, which also function to prevent perturbations induced by drying in vitro and in heterologous systems. CAHS proteins have been shown to form gels both in vitro and in vivo, which has been speculated to be linked to their protective capacity. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of fibrillization and gelation for CAHS D similar to that of intermediate filament assembly. We show that in vitro, gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fibrillar networks during osmotic stress. Fibrillar networking of CAHS D improves survival of osmotically shocked cells. We observe two emergent properties associated with fibrillization; (i) prevention of cell volume change and (ii) reduction of metabolic activity during osmotic shock. We find that there is no significant correlation between maintenance of cell volume and survival, while there is a significant correlation between reduced metabolism and survival. Importantly, CAHS D's fibrillar network formation is reversible and metabolic rates return to control levels after CAHS fibers are resolved. This work provides insights into how tardigrades induce reversible biostasis through the self-assembly of labile CAHS gels.

Abnormal neovascular and proliferative conjunctival phenotype in limbal stem cell deficiency is associated with altered microRNA and gene expression modulated by PAX6 mutational status in congenital aniridia.

PURPOSE: To evaluate conjunctival cell microRNA (miRNAs) and mRNA expression in relation to observed phenotype of progressive limbal stem cell deficiency in a cohort of subjects with congenital aniridia with known genetic status. METHODS: Using impression cytology, bulbar conjunctival cells were sampled from 20 subjects with congenital aniridia and 20 age and sex-matched healthy control subjects. RNA was extracted and miRNA and mRNA analyses were performed using microarrays. Results were related to severity of keratopathy and genetic cause of aniridia. RESULTS: Of 2549 miRNAs, 21 were differentially expressed in aniridia relative to controls (fold change ≤ -1.5 or ≥ +1.5). Among these miR-204-5p, an inhibitor of corneal neovascularization, was downregulated 26.8-fold in severely vascularized corneas. At the mRNA level, 539 transcripts were differentially expressed (fold change ≤ -2 or ≥ +2), among these FOSB and FOS were upregulated 17.5 and 9.7-fold respectively, and JUN by 2.9-fold, all being components of the AP-1 transcription factor complex. Pathway analysis revealed enrichment of PI3K-Akt, MAPK, and Ras signaling pathways in aniridia. For several miRNAs and transcripts regulating retinoic acid metabolism, expression levels correlated with keratopathy severity and genetic status. CONCLUSION: Strong dysregulation of key factors at the miRNA and mRNA level suggests that the conjunctiva in aniridia is abnormally maintained in a pro-angiogenic and proliferative state, and these changes are expressed in a PAX6 mutation-dependent manner. Additionally, retinoic acid metabolism is disrupted in severe, but not mild forms of the limbal stem cell deficiency in aniridia.

Limited Long-Term Impact of Insect Venom Immunotherapy on the Micro-RNA Landscape in Whole Blood.

OBJECTIVE: To perform a genome-wide characterization of changes in microRNA (miRNA) expression during the course of venom immunotherapy (VIT). METHODS: miRNA was isolated from the whole-blood of 13 allergic patients and 14 controls, who experienced no allergic reaction upon stings by honeybees and wasps. We analyzed 2549 miRNAs from the whole blood of these patients prior to VIT and 12 months after the start of VIT. The results for differential expression obtained on a microarray platform were confirmed by quantitative real-time PCR. Out of the 13 patients, 8 had a negative allergic reaction with VIT, thus indicating that this approach was successful. RESULTS: By comparing time points before and 12 months after ultrarush VIT, correlation analysis and principal component analysis both indicated a limited effect of VIT on the overall miRNA expression pattern. Volcano plot analysis based on raw P values revealed few deregulated miRNAs, most of which were increasingly expressed after VIT as compared with before VIT. Based on the 50 most altered miRNAs, no clear clustering was observed before or after VIT. CONCLUSIONS: Our results indicate an overall reduced effect of VIT on the miRNA expression pattern in whole blood.

Limited long-term impact of insect venom immunotherapy on the micro-RNA landscape in whole blood

Objective: To perform a genome-wide characterization of changes in microRNA (miRNA) expression during the course of venom immunotherapy (VIT). Methods: miRNA was isolated from the whole-blood of 13 allergic patients and 14 controls, who experienced no allergic reaction upon stings by honeybees and wasps. We analyzed 2549 miRNAs from the whole blood of these patients prior to VIT and 12 months after the start of VIT. The results for differential expression obtained on a microarray platform were confirmed by quantitative real-time PCR. Out of the 13 patients, 8 had a negative allergic reaction with VIT, thus indicating that this approach was successful. Results: By comparing time points before and 12 months after ultrarush VIT, correlation analysis and principal component analysis both indicated a limited effect of VIT on the overall miRNA expression pattern. Volcano plot analysis based on raw P values revealed few deregulated miRNAs, most of which were increasingly expressed after VIT as compared with before VIT. Based on the 50 most altered miRNAs, no clear clustering was observed before or after VIT. Conclusions: Our results indicate an overall reduced effect of VIT on the miRNA expression pattern in whole blood

Objetivo: Realizar la caracterización genómica de los cambios en la expresión de microARN (miARN) en el curso de ITV (inmunoterapia con veneno). Métodos: Los microARNs se analizaron en la sangre total de 13 pacientes alérgicos y 14 controles sin reacción alérgica a las picaduras de abejas y avispas. Se analizaron 2549 miRNAs diferentes en la sangre total de estos pacientes antes de la ITV y 12 meses después del inicio de la ITV. Los resultados de expresión diferencial obtenidos en la plataforma de microarrays se confirmaron mediante PCR cuantitativa a tiempo real (qRT-PCR). De los 13 pacientes, se confirmó que ocho tenían una reacción alérgica negativa tras la ITV, lo que indicó una ITV exitosa. Resultados: Al comparar los resultados de microRNAs, previa IT y 12 meses después de la ITV, la correlación y el análisis de componentes principales indican un efecto limitado de la ITV en el patrón de expresión general de miARN. El análisis de Volcano basado en los valores de P crudos, reveló la existencia de pocos miRNAs desregulados estando la mayoría de ellos sobre-expresados tras la ITV en comparación con la previa. Utilizando los 50 miRNAs que más se alteraban, no se observó una agrupación clara en función del tiempo, es decir, pre y post-ITV. Conclusiones: Nuestros resultados indican que la ITV tiene poco efecto en el patrón de expresión de miARN en sangre completa

Genome-wide copy number profiling of mouse neural stem cells during differentiation.

There is growing evidence that gene amplifications were present in neural stem and progenitor cells during differentiation. We used array-CGH to discover copy number changes including gene amplifications and deletions during differentiation of mouse neural stem cells using TGF-ß and FCS for differentiation induction. Array data were deposited in GEO (Gene Expression Omnibus, NCBI) under accession number GSE35523. Here, we describe in detail the cell culture features and our TaqMan qPCR-experiments to validate the array-CGH analysis. Interpretation of array-CGH experiments regarding gene amplifications in mouse and further detailed analysis of amplified chromosome regions associated with these experiments were published by Fischer and colleagues in Oncotarget (Fischer et al., 2015). We provide additional information on deleted chromosome regions during differentiation and give an impressive overview on copy number changes during differentiation induction at a time line.

Genome-wide copy number profiling to detect gene amplifications in neural progenitor cells.

DNA sequence amplification occurs at defined stages during normal development in amphibians and flies and seems to be restricted in humans to drug-resistant and tumor cells only. We used array-CGH to discover copy number changes including gene amplifications and deletions during differentiation of human neural progenitor cells. Here, we describe cell culture features, DNA extraction, and comparative genomic hybridization (CGH) analysis tailored towards the identification of genomic copy number changes. Further detailed analysis of amplified chromosome regions associated with this experiment, was published by Fischer and colleagues in PLOS One in 2012 (Fischer et al., 2012). We provide detailed information on deleted chromosome regions during differentiation and give an overview on copy number changes during differentiation induction for two representative chromosome regions.

miRNA-145 is downregulated in atypical and anaplastic meningiomas and negatively regulates motility and proliferation of meningioma cells.

Meningiomas are frequent, mostly benign intracranial or spinal tumors. A small subset of meningiomas is characterized by histological features of atypia or anaplasia that are associated with more aggressive biological behavior resulting in increased morbidity and mortality. Infiltration into the adjacent brain tissue is a major factor linked to higher recurrence rates. The molecular mechanisms of progression, including brain invasion are still poorly understood. We have studied the role of micro-RNA 145 (miR-145) in meningiomas and detected significantly reduced miR-145 expression in atypical and anaplastic tumors as compared with benign meningiomas. Overexpression of miR-145 in IOMM-Lee meningioma cells resulted in reduced proliferation, increased sensitivity to apoptosis, reduced anchorage-independent growth and reduction of orthotopic tumor growth in nude mice as compared with control cells. Moreover, meningioma cells with high miR-145 levels had impaired migratory and invasive potential in vitro and in vivo. PCR-array studies of miR145-overexpressing cells suggested that collagen type V alpha (COL5A1) expression is downregulated by miR-145 overexpression. Accordingly, COL5A1 expression was significantly upregulated in atypical and anaplastic meningiomas. Collectively, our data indicate an important anti-migratory and anti-proliferative function of miR-145 in meningiomas.

Profiling of regulatory microRNA transcriptomes in various biological processes: a review.

A class of small, non-coding ribonucleic acids, termed microRNA (miRNA), has recently emerged as a new key player in the cellular control of gene expression. By either blocking translation or inducing target mRNA degradation, miRNA not only participates in regular biological processes within cells and tissues but is also involved in pathological processes. Many human malignancies have been linked to specific miRNA expression patterns, raising hopes for new approaches to therapy. While such human disease-related mechanisms have been widely discussed and frequently reviewed, miRNA's general significance in animals has been less in editorial focus, despite its obvious role in basic physiological processes, e.g. neurosensory maturation, development of fertility, and hibernation. Using selected examples, this review highlights our current knowledge of miRNA's potential and its promise as a new tool for gene regulation.

miRNA: small molecules as potential novel biomarkers in cancer.

Four different types of small RNAs functionally associated with gene silencing have been discovered in animals including small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). Experimental evidence suggests that miRNAs regulate the expression of more than 30% of protein-coding genes. These molecules can also act as oncogenes or tumor suppressors. Expression profiling has revealed characteristic miRNA signatures not only in human cancers but also in serum and blood cells of cancer patients. Numerous human miRNA genes map to chromosomal regions which are susceptible to amplification, deletion or translocation in the process of tumor development. Despite the pivotal role of miRNA in cancer precise mechanisms of action are yet to be elucidated. This review is focused on recent findings related to the emerging field of miRNA serving as novel potential biomarkers in cancer diagnosis, prognosis and possibly, therapies.

Combining gene expression signatures and autoantibody profiles in human meningioma.

Gene expression profiling has emerged as powerful technique for studying the mechanisms of tumor genesis and development. Seroreactivity profiling of tumor antigens is a more recent technique that further contributes to the understanding of tumors and that offers itself for noninvasive tumor diagnosis. We performed expression profiling of 55,000 transcripts and expressed-sequence-tags for 24 meningiomas and related these data to autoantibody profiles of more than 50 antigens immunogenic in the autologous patients. The expression values of antigens in WHO grade I meningioma were significantly higher if the patients' sera reacted with these antigens as confirmed by a two-tailed Wilcoxon-Mann-Whitney test. Specifically, KIAA1344 that was previously identified as frequent antigen marker in meningioma, showed increased expression if antigens against KIAA1344 were detected in autologous patients. Our study is the first to combine genome-wide expression signatures and comprehensive seroreactivity patterns toward a more complete view on tumor immunology, especially concerning the overall role of the level of gene expression on the immunogenicity of meningioma antigens.

Glioblastoma multiforme: the role of DSB repair between genotype and phenotype.

Glioblastoma is the most frequent primary brain tumor in adults. The average survival time of less than 1 year did not improve notably over the last three decades. The dismal prognosis of glioblastoma patients is largely due to the striking radioresistance of this tumor. Here, we attempt a combined view on the genetics, the repair mechanisms and the radioresistance of glioblastoma. Specifically, we address the role of DNA-PKcs and the novel potential end-joining factor KUB3 in maintaining the radioresistant phenotype, the interrelationship between genetic lesions and repair mechanisms, and new perspectives that emerge from the identification of glioblastoma stem cells.

Parvovirus B19 detected in Rosai-Dorfman disease in nodal and extranodal manifestations.

Sinus histiocytosis with massive lymphadenopathy (SHML), also designated as Rosai-Dorfman disease (RDD), is a rare benign reactive lymphoproliferative disorder. It is defined by a characteristic histopathology with sinus histiocytosis and haemophagocytosis known as emperipolesis. In histiocytes S100 is strongly expressed, whereas CD1a staining typically is negative. The disease mainly manifests at a single lymph node; however, multilocular and extranodal affection can occur. Causative infectious agents, and virus infections in particular, have repeatedly been suspected, although until now the origin of the disease has been unclear. Four cases of RDD (two nodal sites and two extranodal upper respiratory tract sites) were analysed for parvovirus B19 (B19) infection by immunohistochemistry to detect B19 capsid proteins VP1/VP2. In all the four cases, huge numbers of B19-positive cells were partly detected. The positive cells were identified either as lymphocytes or, in one extranodal case, also as respiratory epithelial cells. This is the first report of B19 infection in RDD tissue, indicating that B19 may be associated with the pathogenesis of SHML.

Altered expression of beta-catenin/E-cadherin in meningiomas.

AIMS: Meningiomas are generally slow-growing benign tumours representing approximately 20% of all primary intracranial tumours. The hallmark of tumorigenesis of meningiomas is the loss of chromosome 22, including loss of heterozygosity of the neurofibromatosis type 2 (NF2) gene. The NF2 encoded protein merlin appears to function as a tumour suppressor gene by controlling cadherin-mediated cell-cell adhesion. The E-cadherin cell adhesion system includes beta-catenin that indirectly connects cadherin to actin filaments. The aim of this study was to analyse the expression and the subcellular location of E-cadherin and beta-catenin in human meningiomas, including meningiomas of different histomorphological subtypes and different World Health Organization (WHO) grades. METHODS AND RESULTS: Immunohistochemical analysis revealed lack of E-cadherin expression at the cell membrane in 34% of meningiomas independent of their WHO grade. Loss of membranous beta-catenin occurred in 79% of meningiomas. An intense perinuclear granular immunoreactivity of beta-catenin without nuclear location was detected in the majority of meningiomas. Both immunofluorescence and Western blot analysis of fractionated meningioma cells located beta-catenin mostly on the Golgi apparatus and ER/Golgi intermediate compartment (ERGIC). Cytogenetic analysis of meningiomas showed no correlation between NF2 loss and the loss of the proper location of beta-catenin. CONCLUSIONS: The lack of membranous beta-catenin and/or membranous E-cadherin in meningiomas may indicate an altered interaction between meningioma cells independent of loss of NF2 and independent of the tumour grade.

Human endogenous retroviruses in the primate lineage and their influence on host genomes.

Primates emerged about 60 million years ago. Since that time various primate-targeting retroviruses have integrated in the germ line of primate species, and some drifted to fixation. After germ line fixation, continued activity of proviruses resulted in intragenomic spread of so-called endogenous retroviruses (ERVs). Variant ERVs emerged, amplified in the genome and profoundly altered genome structures and potentially functionality. Importantly, ERVs are genome modifiers of exogenous origin. The human genome contains about 8% of sequences of retroviral origin. The human ERVs (HERVs) comprise many distinct families that amplified to copy numbers of up to several thousand. We review here the evolution of several well-characterized HERV families in the human lineage since initial germ line fixation. It is apparent that endogenous retroviruses profoundly affected the genomes of species in the evolutionary lineage leading to Homo sapiens.

Glioma-expressed antigen 2 (GLEA2): a novel protein that can elicit immune responses in glioblastoma patients and some controls.

Glioma constitutes the most frequent brain tumour in man with glioblastoma as the most prevalent and malignant type. The average survival time of less than 16 months underlines the need for improvements in diagnosis and therapy. Here, we report the identification of a novel antigen termed glioma-expressed antigen 2 (GLEA2) causing a frequent immune response in glioma patients. Screening of 450 000 clones from a glioblastoma lambda zap expression library with autologous patient serum revealed a group of five serum-positive clones sharing a high sequence homology. Further sequence analysis showed a sequence homology to a hepatocellular carcinoma associated antigen 58 (HCA58). We localized the novel HCA homologous gene termed glioma-expressed antigen 2 (GLEA2) on chromosome 20 by somatic cell hybrid panel mapping. Using allogenic sera from 39 glioblastoma patients, we found an immune response against GLEA2 in 17 patients (43%). In addition, screening with allogenic sera from other glioma patients revealed GLEA2 directed antibodies in two out of five pilocytic astrocytomas and in one out of two astrocytomas. Unrelated tumour sera revealed no immune response and sera from healthy persons showed an immune response in two out of 14 cases (14%). Northern blot hybridization and RT-PCR showed ubiquitous GLEA2 gene expression in glioma and normal tissues. The novel HCA homologous gene, GLEA2, appears to induce a frequent immune response in glioma. In the light of the lack of useful glioma markers, it appears reasonable to consider GLEA2 as a potential future diagnostic marker.

Genetic imbalances in preinvasive tissue of hypopharynx provide evidence for cytogenetic heterogeneity.

Multiple chromosomal aberrations have been reported in head and neck squamous cell carcinoma (HNSCC). But less information is available on specific patterns of chromosomal amplifications which distinguish different areas of head and neck tumors. To elucidate genetic mechanisms causing the aggressive growth and high proliferation of hypopharyngeal squamous cell carcinoma (SCC), we performed reverse chromosome painting (RCP) on a total of eight hypopharyngeal SCC including invasive carcinoma and preinvasive tissue. Five hypopharyngeal invasive carcinomas showed amplifications on chromosome 3q. Furthermore, we detected gains on chromosomes 11q and 6p. Compared to the histologically classified preinvasive tissues, we found amplified alterations on chromosome 6p, 11q and 12q, but none of them showed gains on chromosome 3q. This observed heterogeneity in hypopharyngeal SCC might reflect a specific role of chromosome 3q as a late event in the highly invasive capacity of these SCC.

Translation initiation factor eIF-4G is immunogenic, overexpressed, and amplified in patients with squamous cell lung carcinoma.

BACKGROUND: Recently, the authors reported identification and cloning of several novel immunogenic antigens in squamous cell lung carcinoma. Of 14 corresponding genes, 9 mapped in an amplified chromosomal region with the gene for eIF-4G that was amplified most frequently. METHODS: Recombinant eIF-4G was expressed in E. coli and screened with sera from patients with squamous cell carcinoma of the lung and of the head and neck. Protein extracts from squamous cell carcinoma tissues were analyzed for eIF-4G expression by Western blot analysis. Mutation analysis was performed using an automatic DNA sequencer. RESULTS: The authors screened a spectrum of 33 heterologous sera from lung carcinoma patients and detected antibodies against eIF-4G in 5 of these sera (15%). They found no immune response to eIF-4G in 17 sera from squamous cell carcinoma tissues derived from the head and neck. In addition, no antibodies were found to eIF-4G in a group of 17 control sera from individuals without known tumor formation. Sequence analysis of the eIF-4G gave no indication of mutations. To analyze the expression of eIF-4G, a monoclonal antibody was used. Western blot analysis clearly showed overexpression in the tumor tissue compared with the corresponding normal lung tissues. CONCLUSIONS: The translation initiation factor eIF-4G is the first protein in which the gene shows amplification, has increased expression in a human tumor, and induces an immune response in patients. eIF-4G lends itself as a marker for the diagnosis of and possibly as a future therapeutic target in patients with squamous cell lung carcinoma.

HERV-K(OLD): ancestor sequences of the human endogenous retrovirus family HERV-K(HML-2).

Sequences homologous to the human endogenous retrovirus (HERV) family HERV-K(HML-2) are present in all Old World primate species. A previous study showed that a central region of the HERV-K(HML-2) gag genes in Hominoidea species displays a 96-bp deletion compared to the gag genes in lower Old World primates. The more ancient HERV-K(HML-2) sequences present in lower Old World primates were apparently not conserved during hominoid evolution, as opposed to the deletion variants. To further clarify the evolutionary origin of the HERV-K(HML-2) family, we screened GenBank with the 96-bp gag-sequence characteristic of lower Old World primates and identified, to date, 10 human sequence entries harboring either full-length or partially deleted proviral structures, probably representing remnants of a more ancient HERV-K(HML-2) variant. The high degree of mutations demonstrates the long-time presence of these HERV-K(OLD) proviruses in the genome. Nevertheless, they still belong to the HML-2 family as deduced from dot matrix and phylogenetic analyses. We estimate, based on the family ages of integrated Alu elements and on long terminal repeat (LTR) divergence data, that the average age of HERV-K(OLD) proviruses is ca. 28 million years, supporting an integration time before the evolutionary split of Hominoidea from lower Old World primates. Analysis of HERV-K(OLD) LTR sequences led to the distinction of two subgroups, both of which cluster with LTRs belonging to an evolutionarily older cluster. Taken together, our data give further insight into the evolutionary history of the HERV-K(HML-2) family during primate evolution.

KUB3 amplification and overexpression in human gliomas.

Gene amplification is known to occur frequently in human glioma. Recently we reported cloning of a novel gene termed glioma-amplified sequence 16 (GAS16) by microdissection-mediated cDNA capture. In this article, we demonstrate that GAS16 results from an alternative splicing process of the Ku70 binding protein 3 (KUB3) that is essential for DNA double-strand break repair. The alternative splice product was found in glioblastoma and in normal fetal brain. We determined the amplification frequency of KUB3 in glioma with different grading. We analyzed a total of 102 glioma primary tumors and found KUB3 to be amplified in 12/82 (14%) glioblastomas, 4/13 anaplastic astrocytomas (30%), and 2/4 astrocytomas, but in none of three pilocytic astrocytomas. Northern blot analysis of glioblastoma shows a strong correlation between KUB3 amplification and overexpression. Amplification of KUB3 appears to be independent of other genetic changes frequently associated with the development of gliomas, including EGFR amplification, LOH of TP53, and LOH of chromosome 10. The KUB3 amplification and overexpression may interfere with the function of KUB3 in the DNA-PK complex involved in the maintenance of genome stability and reduction of mutation frequency.

Expression, cellular distribution and protein binding of the glioma amplified sequence (GAS41), a highly conserved putative transcription factor.

The glioma amplified sequence 41 (GAS41) was previously isolated by microdissection mediated cDNA capture from the glioblastoma multiforme cell line TX3868 and shown to be frequently amplified in human gliomas. We determined the complete cDNA sequence of the GAS41 gene, demonstrated that the GAS41 protein is evolutionarily conserved, specifically at the N-terminus, and identified the yeast transcription factor tf2f domain within the GAS41 sequence. A human multiple-tissue Northern blot revealed ubiquitous expression of GAS41 with the highest expression in human brain. After generating polyclonal antibodies we found GAS41 protein expression in the nucleus of the TX3868 cell line by Western blot analysis and immunofluorescence microscopy. The nuclear localization was confirmed for several human tumors including gliomas of different grades of malignancy. In neuroblastoma however, GAS41 was found in the nucleoli but not in the nucleoplasm. Yeast two-hybrid screening of the TX3868 cell line identified the nuclear mitotic apparatus protein (NuMA), the KIAA1009 protein, and prefoldin subunit 1 (PFDN1) as potential interacting partners of GAS41. We generated a polyclonal antibody against the KIAA1009 protein and we demonstrated that the KIAA1009 protein is a nuclear protein, which appears to be co-localized with the GAS41 protein and NuMA.