A novel entity of massive multifocal osteolyses in the elderly.

Osteolyses are common findings in elderly patients and most frequently represent malignant or locally aggressive bone tumors, infection, inflammatory and endocrine disorders, histiocytoses, and rare diseases such as Gorham-Stout syndrome. We here report on a novel entity of massive multifocal osteolyses in both shoulders, the right hip and left knee joint and the dens of an 83-year-old patient not relatable to any previously known etiopathology of bone disorders. The soft tissue mass is of myxoid stroma with an unspecific granulomatous inflammatory process, aggressively destroying extensive cortical and cancellous bone segments and encroaching on articulating bones in diarthrodial large joints. Radiological, nuclear medical, serological, histological, and immunohistochemical analyses were incapable of further classifying the disease pattern within the existing scheme of pathology. Quantitative polymerase chain reaction and next generation sequencing revealed that mutations are not suggestive of any known hereditary or acquired bone disease. Possible treatment options include radionuclide therapy for pain palliation and percutaneous radiation to arrest bone resorption while surgical treatment is inevitable for pathological fractures. This case study shall increase the awareness of the musculoskeletal community and motivate to collect further information on this rare but mutilating disorder.

Dynamic and static circulating cancer microRNA biomarkers - a validation study.

For cancers and other pathologies, early diagnosis remains the most promising path to survival. Profiling of longitudinal cohorts facilitates insights into trajectories of biomarkers. We measured microRNA expression in 240 serum samples from patients with colon, lung, and breast cancer and from cancer-free controls. Each patient provided at least two serum samples, one prior to diagnosis and one following diagnosis. The median time interval between the samples was 11.6 years. Using computational models, we evaluated the circulating profiles of 21 microRNAs. The analysis yielded two sets of biomarkers, static ones that show an absolute difference between certain cancer types and controls and dynamic ones where the level over time provided higher diagnostic information content. In the first group, miR-99a-5p stands out for all three cancer types. In the second group, miR-155-5p allows to predict lung cancers and colon cancers. Classification in samples from cancer and non-cancer patients using gradient boosted trees reached an average accuracy of 79.9%. The results suggest that individual change over time or an absolute value at one time point may predict a disease with high specificity and sensitivity.

A Temporary Pause in the Replication Licensing Restriction Leads to Rereplication during Early Human Cell Differentiation.

Gene amplifications in amphibians and flies are known to occur during development and have been well characterized, unlike in mammalian cells, where they are predominantly investigated as an attribute of tumors. Recently, we first described gene amplifications in human and mouse neural stem cells, myoblasts, and mesenchymal stem cells during differentiation. The mechanism leading to gene amplifications in amphibians and flies depends on endocycles and multiple origin-firings. So far, there is no knowledge about a comparable mechanism in normal human cells. Here, we describe rereplication during the early myotube differentiation of human skeletal myoblast cells, using fiber combing and pulse-treatment with EdU (5'-Ethynyl-2'-deoxyuridine)/CldU (5-Chlor-2'-deoxyuridine) and IdU (5-Iodo-2'-deoxyuridine)/CldU. We found rereplication during a restricted time window between 2 h and 8 h after differentiation induction. Rereplication was detected in cells simultaneously with the amplification of the MDM2 gene. Our findings support rereplication as a mechanism enabling gene amplification in normal human cells.

Gene Amplification in Tumor Cells: Developed De Novo or Adopted from Stem Cells.

Gene amplifications have been known for several decades as physiological processes in amphibian and flies, e.g., during eggshell development in Drosophila and as part of pathological processes in humans, specifically in tumors and drug-resistant cells. The long-held belief that a physiological gene amplification does not occur in humans was, however, fundamental questioned by findings that showed gene amplification in human stem cells. We hypothesis that the physiological and the pathological, i.e., tumor associated processes of gene amplification share at their beginning the same underlying mechanism. Re-replication was reported both in the context of tumor related genome instability and during restricted time windows in Drosophila development causing the known developmental gene amplification in Drosophila. There is also growing evidence that gene amplification and re-replication were present in human stem cells. It appears likely that stem cells utilize a re-replication mechanism that has been developed early in evolution as a powerful tool to increase gene copy numbers very efficiently. Here, we show that, several decades ago, there was already evidence of gene amplification in non-tumor mammalian cells, but that was not recognized at the time and interpreted accordingly. We give an overview on gene amplifications during normal mammalian development, the possible mechanism that enable gene amplification and hypothesize how tumors adopted this capability for gene amplification.

Low miR-150-5p and miR-320b Expression Predicts Reduced Survival of COPD Patients.

Chronic obstructive pulmonary disease (COPD) is associated with an increased risk of death, reducing life expectancy on average between 5 and 7 years. The survival time after diagnosis, however, varies considerably as a result of the heterogeneity of COPD. Therefore, markers that predict individual survival of COPD patients are of great value. We analyzed baseline molecular profiles and collected 54 months of follow-up data of the cohort study "COPD and SYstemic consequences-COmorbidities NETwork" (COSYCONET). Genome-wide microRNA signatures from whole blood collected at time of the inclusion in the study were generated for 533 COPD patients including patients that deceased during the 54-month follow-up period (n = 53) and patients that survived this period (n = 480). We identified two blood-born microRNAs (miR-150-5p and miR-320b) that were highly predictive for survival of COPD patients. The expression change was then confirmed by RT-qPCR in 245 individuals. Ninety percent of patients with highest expression of miR-150-5p survived the 54-month period in contrast to only 50% of patients with lowest expression intensity. Moreover, the abundance of the oncogenic miR-150-5p in blood of COPD patients was predictive for the development of cancer. Thus, molecular profiles measured at the time of a COPD diagnosis have a high predictive power for the survival of patients.

Prospect and challenge of detecting dynamic gene copy number increases in stem cells by whole genome sequencing.

Gene amplification is an evolutionarily well-conserved and highly efficient mechanism to increase the amount of specific proteins. In humans, gene amplification is a hallmark of cancer and has recently been found during stem cell differentiation. Amplifications in stem cells are restricted to specific tissue areas and time windows, rendering their detection difficult. Here, we report on the performance of deep WGS sequencing (average 82-fold depth of coverage) on the BGISEQ with nanoball technology to detect amplifications in human mesenchymal and neural stem cells. As reference technology, we applied array-based comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), and qPCR. Using different in silico strategies for amplification detection, we analyzed the potential of WGS for amplification detection. Our results provide evidence that WGS accurately identifies changes of the copy number profiles in human stem cell differentiation. However, the identified changes are not in all cases consistent between WGS and aCGH. The results between WGS and the validation by qPCR were concordant in 83.3% of all tested 36 cases. In sum, both genome-wide techniques, aCGH and WGS, have unique advantages and specific challenges, calling for locus-specific confirmation by the low-throughput approaches qPCR or FISH. KEY MESSAGES: WGS allows for the identification of dynamic copy number changes in human stem cells. Less stringent threshold setting is crucial for detection of copy number increase. Broad knowledge of dynamic copy number is pivotal to estimate stem cell capabilities.

Gene amplification in mesenchymal stem cells and during differentiation towards adipocytes or osteoblasts.

Gene amplifications are an attribute of tumor cells and have for long time been overlooked in normal cells. A growing number of investigations describe gene amplifications in normal mammalian cells during development and differentiation. Possibly, tumor cells have rescued the gene amplification mechanism as a physiological attribute of stem cells. Here, we investigated human mesenchymal stem cells (hMSCs) for gene amplification using array-CGH, single cell fluorescence in situ hybridization and qPCR. Gene amplifications were detected in mesenchymal stem cells and in mesenchymal stem cells during differentiation towards adipocytes and osteoblasts. Undifferentiated hMSCs harbor 12 amplified chromosomal regions, hMSCs that differentiated towards adipocytes 18 amplified chromosome regions, and hMSCs that differentiate towards osteoblasts 19 amplified regions. Specifically, hMSCs that differentiated towards adipocytes or osteoblasts harbor CDK4 and MDM2 amplifications both of which frequently occur in osteosarcoma and liposarcoma that are both of same cell origin. Beside the amplifications, we identified 36 under-replicated regions in undifferentiated and in differentiating hMSC cells.

A high-resolution map of the human small non-coding transcriptome.

Motivation: Although the amount of small non-coding RNA-sequencing data is continuously increasing, it is still unclear to which extent small RNAs are represented in the human genome. Results: In this study we analyzed 303 billion sequencing reads from nearly 25 000 datasets to answer this question. We determined that 0.8% of the human genome are reliably covered by 874 123 regions with an average length of 31 nt. On the basis of these regions, we found that among the known small non-coding RNA classes, microRNAs were the most prevalent. In subsequent steps, we characterized variations of miRNAs and performed a staged validation of 11 877 candidate miRNAs. Of these, many were actually expressed and significantly dysregulated in lung cancer. Selected candidates were finally validated by northern blots. Although isolated miRNAs could still be present in the human genome, our presented set likely contains the largest fraction of human miRNAs. Contact: c.backes@mx.uni-saarland.de or andreas.keller@ccb.uni-saarland.de. Supplementary information: Supplementary data are available at Bioinformatics online.

Specific amplifications and copy number decreases during human neural stem cells differentiation towards astrocytes, neurons and oligodendrocytes.

There is growing evidence that gene amplifications are an attribute of normal cells during development and differentiation. During neural progenitor cell differentiation half of the genome is involved in amplification process. To answer the question how specific amplifications occur at different stages and in different lineages of differentiation we analyzed the genes CDK4, MDM2, EGFR, GINS2, GFAP, TP53, DDB1 and MDM4 in human neural stem cells that were induced to differentiate towards astrocytes, neurons and oligodendrocytes. We found specific amplification pattern for each of the eight analyzed genes both in undifferentiated neural stem and progenitor cells and in cells that were induced for differentiation. Different amplification patterns were also found between adherently grown neural stem cells and cells that were grown as spheres. The most frequently amplified genes were MDM2 and CDK4 with the latter amplified in all three lineages at all analyzed stages. Amplification of the analyzed genes was also found in four glioma stem-like cells. The combined amplification data of stem cells and of tumor stem cells can help to define cell populations at the origin of the tumor. Furthermore, we detected a decrease of gene copies at specific differentiation stages most frequently for MDM4. This study shows specific amplification pattern in defined stem cell populations within specific time windows during differentiation processes indicating that amplifications occur in an orderly sequence during the differentiation of human neural stem and progenitor cells.

Gene amplification during myogenic differentiation.

Gene amplifications are mostly an attribute of tumor cells and drug resistant cells. Recently, we provided evidence for gene amplifications during differentiation of human and mouse neural progenitor cells. Here, we report gene amplifications in differentiating mouse myoblasts (C2C12 cells) covering a period of 7 days including pre-fusion, fusion and post-fusion stages. After differentiation induction we found an increase in copy numbers of CDK4 gene at day 3, of NUP133 at days 4 and 7, and of MYO18B at day 4. The amplification process was accompanied by gamma-H2AX foci that are indicative of double stand breaks. Amplifications during the differentiating process were also found in primary human myoblasts with the gene CDK4 and NUP133 amplified both in human and mouse myoblasts. Amplifications of NUP133 and CDK4 were also identified in vivo on mouse transversal cryosections at stage E11.5. In the course of myoblast differentiation, we found amplifications in cytoplasm indicative of removal of amplified sequences from the nucleus. The data provide further evidence that amplification is a fundamental mechanism contributing to the differentiation process in mammalians.

Gene amplification during differentiation of mammalian neural stem cells in vitro and in vivo.

In development of amphibians and flies, gene amplification is one of mechanisms to increase gene expression. In mammalian cells, gene amplification seems to be restricted to tumorigenesis and acquiring of drug-resistance in cancer cells. Here, we report a complex gene amplification pattern in mouse neural progenitor cells during differentiation with approximately 10% of the genome involved. Half of the amplified mouse chromosome regions overlap with amplified regions previously reported in human neural progenitor cells, indicating conserved mechanisms during differentiation. Using fluorescence in situ hybridization, we verified the amplification in single cells of primary mouse mesencephalon E14 (embryonic stage) neurosphere cells during differentiation. In vivo we confirmed gene amplifications of the TRP53 gene in cryosections from mouse embryos at stage E11.5. Gene amplification is not only a cancer-related mechanism but is also conserved in evolution, occurring during differentiation of mammalian neural stem cells.

New insights into the genetics of glioblastoma multiforme by familial exome sequencing.

Glioblastoma multiforme (GBM) is the most aggressive and malignant subtype of human brain tumors. While a family clustering of GBM has long been acknowledged, relevant hereditary factors still remained elusive. Exome sequencing of families offers the option to discover respective genetic factors.We sequenced blood samples of one of the rare affected families: while both parents were healthy, both children were diagnosed with GBM. We report 85 homozygous non-synonymous single nucleotide variations (SNVs) in both siblings that were heterozygous in the parents. Beyond known key players for GBM such as ERBB2, PMS2, or CHI3L1, we identified over 50 genes that have not been associated to GBM so far. We also discovered three accumulative effects potentially adding to the tumorigenesis in the siblings: a clustering of multiple variants in single genes (e.g., PTPRB, CROCC), the aggregation of affected genes on specific molecular pathways (e.g., Focal adhesion or ECM receptor interaction) and genomic proximity (e.g., chr22.q12.2, chr1.p36.33). We found a striking accumulation of SNVs in specific genes for the daughter, who developed not only a GBM at the age of 12 years but was subsequently diagnosed with a pilocytic astrocytoma, a common acute lymphatic leukemia and a diffuse pontine glioma.The reported variants underline the relevance of genetic predisposition and cancer development in this family and demonstrate that GBM has a complex and heterogeneous genetic background. Sequencing of other affected families will help to further narrow down the driving genetic causes for this disease.

Glioma-amplified sequence KUB3 influences double-strand break repair after ionizing radiation.

Human glioblastomas are characterized by frequent DNA amplifications most often at chromosome regions 7p11.2 and 12q13-15. Although amplification is a well-known hallmark of glioblastoma genetics the function of most amplified genes in glioblastoma biology is not understood. Previously, we cloned Ku70-binding protein 3 (KUB3) from the amplified domain at 12q13-15. Here, we report that glioblastoma cell cultures with endogenous KUB3 gene amplification and with elevated KUB3 protein expression show an efficient double-strand break (DSB) repair after being irradiated with 1 Gy. A significantly less efficient DSB repair was found in glioma cell cultures without KUB3 amplification and expression. Furthermore, we found that a siRNA-mediated reduction of the endogenous KUB3 expression in glioblastoma cells resulted in a reduction of the repair efficiency. HeLa cells transfected with KUB3 showed an increased DSB repair in comparison to untreated HeLa cells. In addition, KUB3 seems to influence DSB efficiency via the DNA-PK-dependent repair pathway as shown by simultaneous inhibition of KUB3 and DNA-PK. The data provide the first evidence for a link between the level of KUB3 amplification and expression in glioma and DSB repair efficiency.

Thermal stability of anthocyanins and colourless phenolics in pomegranate (Punica granatum L.) juices and model solutions.

The present study aimed at a systematic assessment of the factors influencing the anthocyanin (AC) stability and colour retention of pomegranate juices and less complex model solutions with particular focus on the effects of colourless phenolic copigments (CP). The thermal stability of ACs in three pomegranate juices obtained from isolated arils and the entire fruit with and without previous steaming, in model solutions with AC:CP ratios ranging from 1:0 to 1:4 (m/m), and in two purified extracts from pomegranate juices characterised by different phenolic profiles, respectively, was investigated upon heating at 60, 70, 80 and 90°C for 15 min to 5h. The thermal impact on the AC and CP contents, and the formation of 5-hydroxymethylfurfural (HMF) and AC degradation products were monitored using HPLC-DAD-MS(n). Total phenolic contents, antioxidant capacity and colour properties were determined spectrophotometrically. Heating at 90°C for 5h resulted in total AC losses ranging from 76% to 87% of the initial AC levels in the juices, 78% in both extracts as well as 57% and ∼78% in the model solutions, showing the best stability at an AC:CP ratio of 1:2 and in juices having the highest initial AC contents, respectively. In contrast, the AC stability was independent of total phenolic contents, and low and high molecular pomegranate matrix components (such as organic acids and sugars). Good correlation of the AC contents with red colour (a(∗)) was observed for all samples at elevated temperatures (70-90°C). The stability of putative health-promoting polyphenols of pomegranate juices was not markedly affected by the thermal treatment. Unexpectedly, the HMF contents only slightly increased upon forced heating. Therefore, the visual appearance does not adequately reflect the quality and storage stability of pomegranate juices.

Genome-wide gene amplification during differentiation of neural progenitor cells in vitro.

DNA sequence amplification is a phenomenon that occurs predictably at defined stages during normal development in some organisms. Developmental gene amplification was first described in amphibians during gametogenesis and has not yet been described in humans. To date gene amplification in humans is a hallmark of many tumors. We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells. Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells. We identified several amplified genes in neural progenitor cells that are known to be amplified in malignant tumors. There is also a striking overlap of amplified chromosomal regions between differentiating neural progenitor cells and malignant tumor cells derived from astrocytes. Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

GAS41 amplification results in overexpression of a new spindle pole protein.

Amplification is a hallmark of many human tumors but the role of most amplified genes in human tumor development is not yet understood. Previously, we identified a frequently amplified gene in glioma termed glioma-amplified sequence 41 (GAS41). Using the TCGA data portal and performing experiments on HeLa and TX3868, we analyzed the role of GAS41 amplification on GAS41 overexpression and the effect on the cell cycle. Here we show that GAS41 amplification is associated with overexpression in the majority of cases. Both induced and endogenous overexpression of GAS41 leads to an increase in multipolar spindles. We showed that GAS41 is specifically associated with pericentrosome material. As result of an increased GAS41 expression we found bipolar spindles with misaligned chromosomes. This number was even increased by a combined overexpression of GAS41 and a reduced expression of NuMA. We propose that GAS41 amplification may have an effect on the highly altered karyotype of glioblastoma via its role during spindle pole formation.

Determination of lignans in edible and nonedible parts of pomegranate (Punica granatum L.) and products derived therefrom, particularly focusing on the quantitation of isolariciresinol using HPLC-DAD-ESI/MSn.

A method for the characterization and quantitation of phyto-estrogenic lignans from pomegranate (Punica granatum L.) fruits and fruit-derived products by HPLC-DAD-MS(n) was developed. For this purpose, edible and nonedible parts of pomegranate (aril, peel, mesocarp, seed, and twigs), commercial juices, juices produced on pilot-plant scale, and encapsulated dietary supplements were analyzed. In addition to the peel, mesocarp, and twigs, lignans were detected in two juices obtained from entire fruits, four commercial juices, and three encapsulated pomegranate extracts. Isolariciresinol was the predominant lignan with contents of 5.0, 10.5, and 45.8 mg/kg dry matter in processed pomegranate mesocarp, peel, and twigs, respectively. In contrast, due to their low amounts, quantitation of lignans in pomegranate products was impossible. Therefore, contrary to previous assumptions, lignans were found to be less relevant in pomegranate-derived products. However, the byproduct from pomegranate processing may be used for lignan extraction. The method presented allows one to differentiate between pomegranate-derived products obtained from fruits without peels or by dejuicing applying low pressures, which were devoid of lignans, and those obtained from entire fruits applying high pressures, thus containing lignans. Consequently, this study helps to optimize process technology aiming at the recovery of preparations with well-desired compositions, which may reduce the risk of a wide range of diseases, such as certain types of cancer.

Lack of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is accompanied by increased CK2α' levels.

DNA-PKcs is the catalytic subunit of DNA-dependent protein kinase, an enzyme necessary for non-homologous end-joining (NHEJ) and hence repair of DNA double strand breaks. Characterization of two isogenic cell lines, M059K and M059J, which are DNA-PKcs-proficient and -deficient, respectively, revealed that lack of DNA-PKcs is accompanied by an increase in the protein level of one of the catalytic isozymes of protein kinase CK2, i.e., CK2α' and a concomitant increase in CK2 activity. The increase was also detectable at the mRNA level as measured by quantitative real time PCR. However, no increase at the DNA level was observed either by comparative PCR or fluorescent in situ hybridization indicating that gene amplification is not involved. Interestingly, only CK2α' was increased and not the other two subunits of CK2, i.e., CK2ß or CK2α. In addition, the increase in CK2α' protein level was also observed in a DNA-PKcs-deficient mouse cell line.

Identification of novel SNPs in glioblastoma using targeted resequencing.

High-throughput sequencing opens avenues to find genetic variations that may be indicative of an increased risk for certain diseases. Linking these genomic data to other "omics" approaches bears the potential to deepen our understanding of pathogenic processes at the molecular level. To detect novel single nucleotide polymorphisms (SNPs) for glioblastoma multiforme (GBM), we used a combination of specific target selection and next generation sequencing (NGS). We generated a microarray covering the exonic regions of 132 GBM associated genes to enrich target sequences in two GBM tissues and corresponding leukocytes of the patients. Enriched target genes were sequenced with Illumina and the resulting reads were mapped to the human genome. With this approach we identified over 6000 SNPs, including over 1300 SNPs located in the targeted genes. Integrating the genome-wide association study (GWAS) catalog and known disease associated SNPs, we found that several of the detected SNPs were previously associated with smoking behavior, body mass index, breast cancer and high-grade glioma. Particularly, the breast cancer associated allele of rs660118 SNP in the gene SART1 showed a near doubled frequency in glioblastoma patients, as verified in an independent control cohort by Sanger sequencing. In addition, we identified SNPs in 20 of 21 GBM associated antigens providing further evidence that genetic variations are significantly associated with the immunogenicity of antigens.

Amplicons on chromosome 12q13-21 in glioblastoma recurrences.

There is limited knowledge on the in vivo behavior of amplified regions in human tumors. First evidence indicates that amplicon structures are largely maintained in recurrent tumors. Here, we investigated the fate of amplified regions in several independent cases of recurrent glioblastoma and the possible association of 12q13-21 amplifications and survival. We analyzed 12q13-21 amplicon numbers and sizes in glioblastoma and their recurrences by array-CGH. The majority of the 12q13-21 amplicons found in the original tumor are lost in the subsequent recurrence. Likewise, the majority of the amplicons found in the first recurrence are lost in the second recurrence. The remaining amplicons of recurrences often expanded or were maintained in size. Because of re-emergences and de novo appearances of amplicons, however, the overall number of amplicons did not decrease in the recurrences. Understanding genetic changes including gene amplifications in the development of tumor recurrences will contribute to rational therapeutic strategies for an improved patient survival. We recognized a significant longer survival time in glioblastoma patients that lack amplifications of either CDK4, CYP27B1, XRCC6BP1 (KUB3), or MDM2.